Biology subjects

O'Keefe, K.

Publications and source records attributed to O'Keefe, K..

8 recordsLinked to original sources

bioRxiv2025

CRISPR-engineered deletion of POGZ alters transcription factor binding at promoters of genes involved in synaptic signaling

One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDD) has been that loss-of-function (LoF) mutations in many genes that impact chromatin and transcriptional regulation confer substantial liability to NDD. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the direct or indirect regulatory targets of POGZ, or the mechanisms by which loss of this chromatin modifier alters early neuronal development and synaptic functions. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and biallelic POGZ deletions. In hiPSC-derived neural stem cells (NSC) and Neurogenin 2-induced neurons (iN), POGZ LoF altered the expression of genes associated with critical cellular processes and neuronal functions, including synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes in neuronal development associated with NDD and POGZ regulation, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iN. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with biallelic deletions, but not heterozygous lines, suggesting subtle effects in neuronal development associated with haploinsufficiency. Overall, these shared molecular consequences suggest key points of convergence that connect epigenetic regulation to neuronal function in the etiology of neurodevelopmental pathologies.

genomics↗

bioRxiv2025

Cold-water gut isolate from threespine stickleback (Gasterosteus aculeatus) reveals polypropylene surface oxidation and co-culture inhibition

Polyethylene terephthalate (PET) and polypropylene (PP), two of the most widely produced plastics in the United States, persist in cold-water environments where plastic-degrading microbes have been poorly characterized. Understanding how gut microbes interact and contribute to plastic degradation is essential for developing potential microbiome-based bioremediation strategies. We isolated 184 microbes from wild Alaskan threespine stickleback (Gasterosteus aculeatus) guts across six lakes and screened for plastic degrading potential using lipase/esterase assays and biofilm formation on PET and PP. During the screen for microbes with plastic degrading potential, we discovered that stickleback gut microbiota members enhance and suppressed the lipase, esterase, and biofilm activity of other microbes. Isolates with the highest plastic degrading potential were incubated in minimal media with PET or PP as the sole carbon source to determine whether plastic degradation potential is enhanced. Surface analysis identified a Pseudomonas trivialis strain that exhibited degradation of PP in monoculture; however, this activity was suppressed in the presence of another gut isolate, Pseudomonas germanica. These results demonstrate that microbes associated with the wild threespine stickleback gut microbiome possess plastic degradation potential and provide insights into how microbial interactions can either promote or inhibit bioremediation of plastic pollution in cold-water environments.

microbiology↗

bioRxiv2024

Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34+ cell therapy to induce fetal hemoglobin for sickle cell disease

Sickle cell disease (SCD) is a common severe blood disorder, caused by one major point mutation in the HBB gene. Current pharmacotherapies are only partially effective and potentially curative allogeneic hematopoietic stem cell transplantation (HSCT) is associated with immune toxicities. Genome editing of autologous patient hematopoietic stem cells (HSCs) to reactivate fetal hemoglobin (HbF) in erythroid progeny offers a potentially curative approach to treat SCD and circumvents some problems associated with allogeneic HSCT. Although the FDA has released guidelines for evaluating genome editing risks, it remains unclear how to best to assess the preclinical safety and efficacy of genome-edited cellular drug products to prepare for a clinical trial. Here we describe rigorous pre-clinical characterization and optimization of a therapeutic {gamma}-globin gene promoter editing strategy that supported an investigational new drug (IND) application cleared by the FDA. We compared targets in the {gamma}-globin promoter and BCL11A erythroid-specific enhancer, identified a lead candidate that potently induces HbF, and tested our approach in mobilized CD34+ HSPCs from normal donors and individuals with SCD. We observed efficient editing, induction of HbF to levels predicted to be therapeutic, and reduction of sickling in red blood cells derived from edited HSPCs. With single-cell western and RNA-seq analyses, we defined the heterogeneity and specificity of HbF induction and HBG1/HBG2 transcription. With CHANGE-seq for sensitive and unbiased genome-wide off-target discovery followed by multiplexed targeted sequencing, we did not detect off-target activity in edited HSPCs. Our study provides a blueprint for translating new discoveries on ex vivo genome editing of HSCs towards clinical trials for treating SCD and other blood disorders.

genomics↗

bioRxiv2022

Tissue and cell-type specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models

Recurrent deletion and duplication of [~]743 kilobases of unique genomic sequence and segmental duplications at chromosome 16p11.2 underlie a reciprocal genomic disorder (RGD; OMIM 611913 and 614671) associated with neurodevelopmental and psychiatric phenotypes, including intellectual disability, autism spectrum disorder (ASD), and schizophrenia (SCZ). To define molecular alterations associated with the 16p11.2 RGD, we performed transcriptome analyses of mice with reciprocal copy number variants (CNVs) of the syntenic chromosome 7qF3 region and human neuronal models derived from isogenic human induced pluripotent stem cells (hiPSCs) carrying CRISPR-engineered CNVs at 16p11.2. Analysis of differentially expressed genes (DEGs) in mouse cortex, striatum, cerebellum and three non-brain tissues, as well as in human neural stem cells and induced glutamatergic neurons revealed that the strongest and most consistent effects occurred within the CNV sequence, with notable instances of differential expression of genes in the immediate vicinity that could reflect position effect. While differential expression of genes outside of chromosome 16p11.2 was largely region, tissue, and cell type-specific, a small but significant minority of such DEGs was shared between brain regions or human cell types. Gene Ontology (GO) enrichment analyses to identify cellular processes dysregulated due to these CNVs found support in select circumstances for terms related to energy metabolism, RNA metabolism, and translation but did not reveal a single universally affected process. Weighted gene co-expression network analysis identified modules that showed significant correlation with reciprocal or individual CNV genotype and better captured shared effects, indicating that energy metabolism, RNA metabolism, translation and protein targeting were disrupted across all three brain regions. The first two of these processes also emerged in the human neural stem cell (NSC) data. A subset of co-expression modules that correlated with CNV genotype revealed significant enrichments for known neurodevelopmental disorder genes, loss-of-function constrained genes, FMRP targets, and chromatin modifiers. Intriguingly, neuronal differentiation of the hiPSCs revealed that both the deletion and duplication CNV resulted in similar deficits in neurite extension and branching and alterations in electrical activity. Finally, generation of cerebral organoid derivatives indicated that the CNVs reciprocally altered the ratio of excitatory and inhibitory GABAergic neurons generated during in vitro neurodevelopment, consistent with a major mechanistic hypothesis for ASD. Collectively, our data suggest that the 16p11.2 RGD involves disruption of multiple biological processes, with a relative impact that is context-specific. Perturbation of individual and multiple genes within the CNV region will be required to dissect single-gene effects, uncover regulatory interactions, and define how each contributes to abnormal neurodevelopment.

genomics↗

bioRxiv2022

Transcriptional and functional consequences of alterations to MEF2C and its topological organization in neuronal models

Point mutations and structural variants directly disrupting the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs), while recent studies have also implicated altered noncoding regulation of MEF2C expression in NDDs. However, the impact of haploinsufficiency of MEF2C on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern regulation of MEF2C. To explore the transcriptional and functional changes associated with coding and noncoding structural variants, we generated an allelic series of 204 isogenic iPSC-derived neuronal cell lines harboring CRISPR-engineered mutations that directly delete predominant isoforms of MEF2C, as well as deletions to the boundaries of topologically associating domains (TADs) and chromatin loops encompassing MEF2C. We then performed systematic profiling of mutation-specific alterations to transcriptional signatures, regulatory interactions, chromatin contacts, and electrophysiological effects. Our analyses reveal that direct deletion of MEF2C causes differential expression of genes enriched for neurodevelopmental and synaptic-associated pathways, accompanied by a significant reduction in synaptic firing and synchrony in neurons. By contrast, we observe robust buffering against MEF2C regulatory disruption upon deletion of a distal 5q14.3 TAD and loop boundary; however, homozygous loss of proximal loop boundary resulted in significant down-regulation of MEF2C expression and significantly reduced electrophysiological activity that was comparable to direct MEF2C disruption. Collectively, our findings demonstrate the functional impact of MEF2C haploinsufficiency in human-derived neural models and highlight the complex interactions of gene regulation and chromatin topology that challenge a priori regulatory predictions of structural variant disruption to three-dimensional genome organization.

genomics↗

bioRxiv2021

Dystonia-specific mutations in THAP1 alter transcription of genes associated with neurodevelopment and myelin

Dystonia is a neurologic disorder associated with an increasingly large number of variants in many genes, resulting in characteristic disturbances in volitional movement. Dissecting the relationships between these mutations and their functional outcomes is a critical step in understanding the key pathways that drive dystonia pathogenesis. Here we established a pipeline for characterizing an allelic series of dystonia-specific mutations in isogenic induced pluripotent stem cells (iPSCs). We used this strategy to investigate the molecular consequences of variation in THAP1, which encodes a transcription factor that has been linked to neural differentiation. Multiple pathogenic mutations that have been associated with dystonia cluster within distinct THAP1 functional domains and are predicted to alter its DNA binding properties and/or protein interactions differently, yet the relative impact of these varied changes on molecular signatures and neural deficits is unclear. To determine the effects of these mutations on THAP1 transcriptional activity, we engineered an allelic series of eight mutations in a common iPSC background and differentiated these lines into a panel of near-isogenic neural stem cells (n = 94 lines). Transcriptome profiling of these neural derivatives followed by joint analysis of the most robust individual signatures across mutations identified a convergent pattern of dysregulated genes functionally related to neurodevelopment, lysosomal lipid metabolism, and myelin. Based on these observations, we examined mice bearing Thap1-disruptive alleles and detected significant changes in myelin gene expression and reduction of myelin structural integrity relative to tissue from control mice. These results suggest that deficits in neurodevelopment and myelination are common consequences of dystonia-associated THAP1 mutations and highlight the potential role of neuron-glial interactions in the pathogenesis of dystonia.

genetics↗

bioRxiv2020

Systematic evaluation of genome sequencing as a first-tier diagnostic test for prenatal and pediatric disorders

Current clinical guidelines recommend three genetic tests for the assessment of fetal structural anomalies: karyotype to detect microscopically-visible balanced and unbalanced chromosomal rearrangements, chromosomal microarray (CMA) to detect sub-microscopic copy number variants (CNVs), and exome sequencing (ES) to identify individual nucleotide changes in coding sequence. Advances in genome sequencing (GS) analysis suggest that it is poised to displace the sequential application of all three conventional tests to become a single diagnostic approach for the assessment of fetal structural anomalies. However, systematic benchmarking is required to assure that GS can capture the full mutational spectrum associated with fetal structural anomalies and to accurately quantify the added diagnostic yield of GS. We applied a novel GS analytic framework that included the discovery, filtration, and interpretation of nine classes of genomic variation to 7,195 individuals. We assessed the sensitivity of GS to detect diagnostic variants (pathogenic or likely pathogenic) from three standard-of-care tests using 1,612 autism spectrum disorder quartet families (ASD; n=6,448) with matched GS, ES, and CMA data, and validated these findings in 46 fetuses with a clinically reportable variant originally identified by karyotype, CMA, or ES. We then assessed the added diagnostic yield of GS in 249 trios (n=747) comprising a fetus with a structural anomaly detected by ultrasound and two unaffected parents that were pre-screened with a combination of all three standard-of-care tests. Across both cohorts, our GS analytic framework identified 98.2% of all diagnostic variants detected by standard-of-care tests, including 100% of those originally detected by CMA (n=88) and ES (n=61), as well as 78.6% (n=11/14) of the chromosomal rearrangements identified by karyotype. The diagnostic yield from GS was 7.8% across all 1,612 ASD probands, almost two-fold more than CMA (4.4%) and three-fold more than ES (3.0%). We also demonstrated that the yield of ES can approach that of GS when CNVs are captured with high sensitivity from exome data (7.4% vs. 7.8%, respectively). In 249 pre-screened fetuses with structural anomalies, GS provided an additional diagnostic yield of 0.4% beyond the combination of all three tests (karyotype, CMA, and ES). Applying our benchmarking results to existing data indicates that GS can achieve an overall diagnostic yield of 46.1% in unselected fetuses with fetal structural anomalies, providing an estimated 17.2% increase in diagnostic yield over karyotype, 14.1% over CMA, and 36.1% over ES when sequence variants are assessed, and 4.1% when CNVs are also identified from exome data. In this study we demonstrate that GS is sensitive to the detection of almost all pathogenic variation captured by karyotype, CMA, and ES, provides a superior diagnostic yield than any individual test by a wide margin, and contributes a modest increase in diagnostic yield beyond the combination of all three tests. We also outline several strategies to aid the interpretation of GS variants that are cryptic to conventional technologies, which we anticipate will be increasingly encountered as comprehensive variant identification from GS is performed. Taken together, these data suggest GS warrants consideration as a first-tier diagnostic approach for fetal structural anomalies.

genomics↗

bioRxiv2020

The DHODH Inhibitor PTC299 Arrests SARS-CoV-2 Replication and Suppresses Induction of Inflammatory Cytokines

The coronavirus disease 2019 (COVID-19) pandemic has created an urgent need for therapeutics that inhibit the SARS-CoV-2 virus and suppress the fulminant inflammation characteristic of advanced illness. Here, we describe the anti-COVID-19 potential of PTC299, an orally available compound that is a potent inhibitor of dihydroorotate dehydrogenase (DHODH), the rate-limiting enzyme of the de novo pyrimidine biosynthesis pathway. In tissue culture, PTC299 manifests robust, dose-dependent, and DHODH-dependent inhibition of SARS CoV-2 replication (EC50 range, 2.0 to 31.6 nM) with a selectivity index >3,800. PTC299 also blocked replication of other RNA viruses, including Ebola virus. Consistent with known DHODH requirements for immunomodulatory cytokine production, PTC299 inhibited the production of interleukin (IL)-6, IL-17A (also called IL-17), IL-17F, and vascular endothelial growth factor (VEGF) in tissue culture models. The combination of anti-SARS-CoV-2 activity, cytokine inhibitory activity, and previously established favorable pharmacokinetic and human safety profiles render PTC299 a promising therapeutic for COVID-19.

immunology↗