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.

Abstract Background Progressive supranuclear palsy (PSP) is a devastating neurodegenerative disorder with rapid progression to death. Multiple efforts to develop a disease‐modifying therapy for PSP have yet to succeed. We previously reported the discovery of three potential therapeutic targets for PSP. These three genes, DDR2, KANK2, and STOM, are consistently upregulated in the brains of PSP patients and mouse models of tauopathy. Reducing their expression in a drosophila model significantly rescued the tau‐induced eye degeneration and tau seeding activity. To translate these targets into the clinic, we are developing antisense oligonucleotides (ASO) as potential therapies. Method We screened candidate ASOs in neuroglioma cell line H4. ASO hits were validated for their potency and safety. We also used iPSC‐derived neurons from PSP patients to test the potency and potential toxicity of our ASOs. To investigate target engagement, therapeutic efficacy and preliminary pharmacodynamic properties, we treated iPSC‐derived midbrain organoids with the ASO leads. Lastly, we will assess the off‐target effects of the ASOs by investigating the levels of mRNA transcripts that share similar sequences as our targets using RNAseq. Result Our in vitro screening identified ASO leads that significantly reduce the target gene expressions at mRNA and protein levels in a dose‐dependent manner. The ASO hits also exhibit low cellular toxicity in the H4 cell line. Upon treating iPSC neurons with ASO hits, we were able to validate the dose‐dependent reduction of target mRNAs. Cell viability analysis indicated low toxicity, suggesting that these ASOs are potent and safe. In midbrain organoid model, DDR2‐targeting ASO showed time‐dependent target engagement and were prioritized for further development. Conclusion We developed candidate therapeutics for PSP using ASOs that silence novel therapeutic targets discovered in our systems based cross‐species screening. We showed that our ASOs have low toxicity and confirmed target engagement at both mRNA and protein levels in vitro. The results suggest a favorable preclinical profile of our ASO leads that warrants additional investigations. Importantly, PSP shares congruent pathophysiological changes with other tauopathies, such as Alzheimer’s disease. These common perturbed pathways indicate that our ASOs can be potentially repurposed for multiple neurodegenerative tauopathies.

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.

SUMMARY Tau aggregates propagate through the brain in a prion-like manner in Alzheimer’s disease (AD) and other tauopathies, but the molecular identity and functional partners of the seeding-competent Tau species remain poorly defined. Here, we present an unbiased proteomic profiling of a high-molecular-weight (HMW) Tau-seed isolated from AD patient brains. We contrast this interactome with that of a biochemically similar, seeding-incompetent HMW-Tau species from age-matched healthy controls. Despite comprising less than 5% of total Tau in the brain, Tau-seed associates with a distinct set of proteins enriched in synaptic, mitochondrial, and vesicle-trafficking functions. Cross-species functional screening in Drosophila and mouse models identifies interactors that modulate Tau toxicity and seeding. Spatially resolved analysis of postmortem AD brains reveals heterogenous co-deposition of these proteins with Tau aggregates, suggesting functionally distinct Tau-seed complexes. Together, this dataset provides a framework for understanding selective Tau-seed toxicity and identifies candidate regulators of Tau propagation with therapeutic potential.

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.

AbstractBackgroundTau aggregates, a hallmark of Alzheimer’s disease (AD) and other tauopathies, spread throughout the brain, contributing to neurodegeneration. How this propagation occurs remains elusive. Previous research suggests that tau‐seed interactors play a crucial role. Based on this, the study aimed to identify novel tau‐seed interactors in AD brains and validate their impact in vivo.MethodAD and control brain extracts were separated in fractions by Size Exclusion Chromatography. Fractions with the highest tau seeding activity, measured using a tai‐biosensor cell line, were analyzed by mass spectrometry to identify interacting proteins. Bioinformatic tools dissected enriched pathways, identifying interactors that were validated in a Drosophila tauopathy model by genetically interfering with their homologs and assessing tau accumulation and eye degeneration.ResultsTau seeding activity was concentrated in high molecular weight fractions containing only a small portion of total tau in the AD brains. Compared to controls, AD brains revealed a distinct interactome for tau‐seeds, enriched in proteins associated with synaptic and mitochondrial pathways. Notably, Drosophila screening confirmed that several novel interactors significantly reduced tau accumulation and eye degeneration, suggesting their potential therapeutic relevance.ConclusionThis study sheds light on tau propagation mechanisms in AD by identifying novel tau‐seed interactors. These interactors, particularly those involved in synaptic and mitochondrial pathways, offer promising targets for therapeutic interventions aimed at decreasing tau spread and potentially preventing neurodegeneration in tauopathies. The findings add to the growing evidence that targeting tau‐seed interactors, like previously identified BSN, could represent a novel strategy for treating these debilitating conditions.

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.

AbstractBackgroundGenetic variations have emerged as crucial players in the etiology of Alzheimer’s disease (AD), and they serve for a better understanding of the disease mechanisms; yet the specific roles of these genetic variants remain uncertain. Animal models with reminiscent disease pathology could uncover previously uncharacterized roles of these genes. Therefore, we generated zebrafish models for AD variants to analyze the in depth molecular and biological functions of these variants.MethodUsing CRISPR/Cas9, we generated a knockout model for abca7, orthologous to human ABCA7. We performed single cell transcriptomics and analyzed the altered genes and molecular pathways in zebrafish. We leveraged data from multiethnic AD cohorts at Mayo Clinic and Columbia University, to perform genetic association studies, co‐expression analyses, in silico interaction mapping, family based variant segregation analyses and epigenetic association studies, and the functional and histological studies in zebrafish.ResultThe abca7± zebrafish reduced astroglial proliferation, synaptic integrity, and microglial response after Aβ42 toxicity. We found that the abca7 loss‐of‐function (LOF) reduced neuropeptide Y (npy) expression as well as Brain‐derived neurotrophic factor (bdnf) and Nerve growth factor receptor (ngfr). Human brain analysis showed reduced NPY in AD, regulatory interaction between NPY and BDNF, genetic variants in NPY associated with AD, and segregation of variants in ABCA7, BDNF and NGFR in families. ABCA7 variants altered the epigenetic codes in NPY, BDNF, and NGFR promoter regions. Human results paralleled with zebrafish findings to indicate an evolutionarily conserved disease mechanism through ABCA7‐NPY signalling axis. NPY administration to zebrafish rescued the phenotypes in abca7 knockout, suggesting a true biological relevance.ConclusionOur results demonstrate a previously unknown link between ABCA7 and NPY in regulation of synaptic integrity and neurogenesis in AD. We propose that ABCA7‐dependent NPY is a resilience factor in vertebrate brains, and this reserve mechanism is impaired in AD.

ABSTRACTAlzheimer's disease (AD) is the most common neurodegenerative dementia with multi‐layered complexity in its molecular etiology. Multiple omics‐based approaches, such as genomics, epigenomics, transcriptomics, proteomics, metabolomics, and lipidomics are enabling researchers to dissect this molecular complexity, and to uncover a plethora of alterations yielding insights into the pathophysiology of this disease. These approaches reveal multi‐omics alterations essentially in all cell types of the brain, including glia. In this systematic review, we screen the literature for human studies implementing any omics approach within the last 10 years, to discover AD‐associated molecular perturbations in brain glial cells. The findings from over 200 AD‐related studies are reviewed under four different glial cell categories: microglia, oligodendrocytes, astrocytes and brain vascular cells. Under each category, we summarize the shared and unique molecular alterations identified in glial cells through complementary omics approaches. We discuss the implications of these findings for the development, progression and ultimately treatment of this complex disease as well as directions for future omics studies in glia cells.