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1.
Nat Immunol ; 17(8): 956-65, 2016 08.
Article in English | MEDLINE | ID: mdl-27376470

ABSTRACT

During T cell development, multipotent progenitors relinquish competence for other fates and commit to the T cell lineage by turning on Bcl11b, which encodes a transcription factor. To clarify lineage commitment mechanisms, we followed developing T cells at the single-cell level using Bcl11b knock-in fluorescent reporter mice. Notch signaling and Notch-activated transcription factors collaborate to activate Bcl11b expression irrespectively of Notch-dependent proliferation. These inputs work via three distinct, asynchronous mechanisms: an early locus 'poising' function dependent on TCF-1 and GATA-3, a stochastic-permissivity function dependent on Notch signaling, and a separate amplitude-control function dependent on Runx1, a factor already present in multipotent progenitors. Despite their necessity for Bcl11b expression, these inputs act in a stage-specific manner, providing a multitiered mechanism for developmental gene regulation.


Subject(s)
Core Binding Factor Alpha 2 Subunit/metabolism , GATA3 Transcription Factor/metabolism , Gene Expression Regulation, Developmental , Hepatocyte Nuclear Factor 1-alpha/metabolism , Lymphopoiesis/genetics , Receptors, Notch/metabolism , Repressor Proteins/metabolism , T-Lymphocytes/physiology , Tumor Suppressor Proteins/metabolism , Animals , Cell Differentiation/genetics , Cell Lineage/genetics , Cell Tracking , Cells, Cultured , Core Binding Factor Alpha 2 Subunit/genetics , GATA3 Transcription Factor/genetics , Hepatocyte Nuclear Factor 1-alpha/genetics , Mice , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Transgenic , Repressor Proteins/genetics , Signal Transduction , Single-Cell Analysis , Tumor Suppressor Proteins/genetics
2.
Mol Cell ; 77(5): 1032-1043.e4, 2020 03 05.
Article in English | MEDLINE | ID: mdl-31924447

ABSTRACT

An attractive approach to reduce gene expression is via the use of antisense oligonucleotides (ASOs) that harness the RNase H1 mechanism. Here we show that RNase H ASOs targeted to introns or exons robustly reduce the level of spliced RNA associated with chromatin. Surprisingly, intron-targeted ASOs reduce the level of pre-mRNA associated with chromatin to a greater extent than exon-targeted ASOs. This indicates that exon-targeted ASOs achieve full activity after the pre-mRNA has undergone splicing, but before the mRNA is released from chromatin. Even though RNase H ASOs can reduce the level of RNA associated with chromatin, the effect of ASO-directed RNA degradation on transcription has never been documented. Here we show that intron-targeted ASOs and, to a lesser extent, exon-targeted ASOs cause RNA polymerase II (Pol II) transcription termination in cultured cells and mice. Furthermore, ASO-directed transcription termination is mediated by the nuclear exonuclease XRN2.


Subject(s)
Chromatin/metabolism , Oligonucleotides, Antisense/metabolism , RNA Precursors/metabolism , RNA Stability , RNA, Messenger/metabolism , Ribonuclease H/metabolism , Transcription Termination, Genetic , Animals , Chromatin/genetics , Exons , Exoribonucleases/genetics , Exoribonucleases/metabolism , Female , HCT116 Cells , Humans , Introns , Mice, Inbred C57BL , Models, Genetic , Nedd4 Ubiquitin Protein Ligases/genetics , Nedd4 Ubiquitin Protein Ligases/metabolism , Oligonucleotides, Antisense/genetics , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA Precursors/genetics , RNA, Messenger/genetics , Ribonuclease H/genetics , Time Factors
3.
Genes Dev ; 29(8): 832-48, 2015 Apr 15.
Article in English | MEDLINE | ID: mdl-25846797

ABSTRACT

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.


Subject(s)
Cell Differentiation , Gene Expression Regulation, Developmental , Proto-Oncogene Proteins/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Trans-Activators/metabolism , Animals , Cell Survival , Cells, Cultured , Mice, Inbred C57BL , Proto-Oncogene Proteins/genetics , Receptors, Notch/metabolism , Stem Cells , Trans-Activators/genetics , Transcriptome
4.
Am J Respir Cell Mol Biol ; 63(1): 46-56, 2020 07.
Article in English | MEDLINE | ID: mdl-32176858

ABSTRACT

Goblet cell metaplasia, excessive mucus production, and inadequate mucus clearance accompany and exacerbate multiple chronic respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Notch signaling plays a central role in controlling the fate of multiple cell types in the lung, including goblet cells. In the present study, we explored the therapeutic potential of modulating the Notch pathway in the adult murine lung using chemically modified antisense oligonucleotides (ASOs). To this end, we designed and characterized ASOs targeting the Notch receptors Notch1, Notch2, and Notch3 and the Notch ligands Jag1 (Jagged 1) and Jag2 (Jagged 2). Pulmonary delivery of ASOs in healthy mice or mice exposed to house dust mite, a commonly used mouse model of asthma, resulted in a significant reduction of the respective mRNAs in the lung. Furthermore, ASO-mediated knockdown of Jag1 or Notch2 in the lungs of healthy adult mice led to the downregulation of the club cell marker Scgb1a1 and the concomitant upregulation of the ciliated cell marker FoxJ1 (forkhead box J1). Similarly, ASO-mediated knockdown of Jag1 or Notch2 in the house dust mite disease model led to reduced goblet cell metaplasia and decreased mucus production. Because goblet cell metaplasia and excessive mucus secretion are a common basis for many lung pathologies, we propose that ASO-mediated inhibition of JAG1 could provide a novel therapeutic path for the treatment of multiple chronic respiratory diseases.


Subject(s)
Goblet Cells/drug effects , Goblet Cells/metabolism , Jagged-1 Protein/metabolism , Lung/drug effects , Metaplasia/drug therapy , Metaplasia/metabolism , Oligonucleotides, Antisense/pharmacology , Animals , Asthma/metabolism , Biomarkers/metabolism , Disease Models, Animal , Down-Regulation/drug effects , Forkhead Transcription Factors/metabolism , Lung/metabolism , Male , Mice , Mice, Inbred BALB C , Pyroglyphidae , Receptors, Notch/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effects
5.
BMC Genomics ; 20(1): 164, 2019 Feb 28.
Article in English | MEDLINE | ID: mdl-30819113

ABSTRACT

BACKGROUND: Microglia are multifunctional cells that are key players in brain development and homeostasis. Recent years have seen tremendous growth in our understanding of the role microglia play in neurodegeneration, CNS injury, and developmental disorders. Given that microglia show diverse functional phenotypes, there is a need for more precise tools to characterize microglial states. Here, we experimentally define gene modules as the foundation for describing microglial functional states. RESULTS: In an effort to develop a comprehensive classification scheme, we profiled transcriptomes of mouse microglia in a stimulus panel with 96 different conditions. Using the transcriptomic data, we generated fine-resolution gene modules that are robustly preserved across datasets. These modules served as the basis for a combinatorial code that we then used to characterize microglial activation under various inflammatory stimulus conditions. CONCLUSIONS: The microglial gene modules described here were robustly preserved, and could be applied to in vivo as well as in vitro conditions to dissociate the signaling pathways that distinguish acutely inflamed microglia from aged microglia. The microglial gene modules presented here are a novel resource for classifying and characterizing microglial states in health and disease.


Subject(s)
Cellular Senescence/genetics , Microglia/metabolism , Transcriptome , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cells, Cultured , Down-Regulation , Inflammation/genetics , Inflammation/metabolism , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Mice , Phenotype , Resveratrol/pharmacology , Signal Transduction , Toll-Like Receptor 2/metabolism , Transcription Factors/metabolism , Transcriptome/drug effects
6.
Haematologica ; 106(5): 1433-1442, 2019 May 01.
Article in English | MEDLINE | ID: mdl-32439726

ABSTRACT

ß-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for ß-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects ß-globin synthesis in ß-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the ß-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits ß-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between ß-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of ß-thalassemia caused by mutations leading to aberrant splicing.

7.
Nucleic Acids Res ; 45(16): 9528-9546, 2017 Sep 19.
Article in English | MEDLINE | ID: mdl-28934489

ABSTRACT

A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials.


Subject(s)
5' Untranslated Regions , Oligonucleotides, Antisense/pharmacology , Protein Tyrosine Phosphatases/genetics , Proto-Oncogene Proteins/genetics , Receptors, LDL/genetics , Ribonuclease H/genetics , Animals , Humans , Lipoproteins, LDL/pharmacokinetics , Male , Mice, Inbred BALB C , Oligonucleotides, Antisense/chemistry , Open Reading Frames , Protein Biosynthesis , RNA, Messenger/chemistry , Receptors, LDL/metabolism , Ribonuclease H/metabolism
8.
Nucleic Acids Res ; 44(11): 5299-312, 2016 06 20.
Article in English | MEDLINE | ID: mdl-27131367

ABSTRACT

Viable constitutive and tamoxifen inducible liver-specific RNase H1 knockout mice that expressed no RNase H1 activity in hepatocytes showed increased R-loop levels and reduced mitochondrial encoded DNA and mRNA levels, suggesting impaired mitochondrial R-loop processing, transcription and mitochondrial DNA replication. These changes resulted in mitochondrial dysfunction with marked changes in mitochondrial fusion, fission, morphology and transcriptional changes reflective of mitochondrial damage and stress. Liver degeneration ensued, as indicated by apoptosis, fibrosis and increased transaminase levels. Antisense oligonucleotides (ASOs) designed to serve as substrates for RNase H1 were inactive in the hepatocytes from the RNase H1 knockout mice and in vivo, demonstrating that RNase H1 is necessary for the activity of DNA-like ASOs. During liver regeneration, a clone of hepatocytes that expressed RNase H1 developed and partially restored mitochondrial and liver function.


Subject(s)
Liver/metabolism , Mitochondria, Liver/genetics , Mitochondria, Liver/metabolism , Nucleic Acid Conformation , RNA/metabolism , Ribonuclease H/deficiency , Animals , Cluster Analysis , DNA Replication , DNA, Mitochondrial , Gene Expression Profiling , Gene Expression Regulation , Mice , Mice, Knockout , Organ Specificity/genetics , RNA/chemistry , RNA/genetics , Ribonuclease H/metabolism , Substrate Specificity
9.
J Immunol ; 193(7): 3470-91, 2014 Oct 01.
Article in English | MEDLINE | ID: mdl-25172496

ABSTRACT

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.


Subject(s)
GATA3 Transcription Factor/immunology , Precursor Cells, T-Lymphoid/immunology , Signal Transduction/immunology , Up-Regulation/immunology , Animals , Antineoplastic Agents, Hormonal/pharmacology , Cell Line , GATA3 Transcription Factor/genetics , Mice , Myeloid Cells/cytology , Myeloid Cells/immunology , Precursor Cells, T-Lymphoid/cytology , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/immunology , Receptors, Notch/genetics , Receptors, Notch/immunology , Repressor Proteins/genetics , Repressor Proteins/immunology , Signal Transduction/drug effects , Signal Transduction/genetics , Tamoxifen/pharmacology , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/immunology , Up-Regulation/drug effects , Up-Regulation/genetics
10.
Proc Natl Acad Sci U S A ; 109(5): 1548-53, 2012 Jan 31.
Article in English | MEDLINE | ID: mdl-22238426

ABSTRACT

Embryonic development is controlled by networks of interacting regulatory genes. The individual linkages of gene regulatory networks (GRNs) are customarily validated by functional cis-regulatory analysis, but an additional approach to validation is to rewire GRN circuitry to test experimentally predictions derived from network structure. Here we use this synthetic method to challenge specific predictions of the sea urchin embryo endomesoderm GRN. Expression vectors generated by in vitro recombination of exogenous sequences into BACs were used to cause elements of a nonskeletogenic mesoderm GRN to be deployed in skeletogenic cells and to detect their effects. The result of reengineering the regulatory circuitry in this way was to divert the developmental program of these cells from skeletogenesis to pigment cell formation, confirming a direct prediction of the GRN. In addition, the experiment revealed previously undetected cryptic repression functions.


Subject(s)
Gene Regulatory Networks , Cell Lineage , Chromosomes, Artificial, Bacterial , Genetic Linkage
11.
Proc Natl Acad Sci U S A ; 109(35): 13972-7, 2012 Aug 28.
Article in English | MEDLINE | ID: mdl-22891353

ABSTRACT

Many cellular signaling events are regulated by tyrosine phosphorylation and mediated by the opposing actions of protein tyrosine kinases and phosphatases. Protein tyrosine phosphatases are emerging as drug targets, but poor cell permeability of inhibitors has limited the development of drugs targeting these enzymes [Tautz L, et al. (2006) Expert Opin Ther Targets 10:157-177]. Here we developed a method to monitor tyrosine phosphatase activity at the single-cell level and applied it to the identification of cell-permeable inhibitors. The method takes advantage of the fluorogenic properties of phosphorylated coumaryl amino propionic acid (pCAP), an analog of phosphotyrosine, which can be incorporated into peptides. Once delivered into cells, pCAP peptides were dephosphorylated by protein tyrosine phosphatases, and the resulting cell fluorescence could be monitored by flow cytometry and high-content imaging. The robustness and sensitivity of the assay was validated using peptides preferentially dephosphorylated by CD45 and T-cell tyrosine phosphatase and available inhibitors of these two enzymes. The assay was applied to high-throughput screening for inhibitors of CD45, an important target for autoimmunity and infectious diseases [Hermiston ML, et al. (2003) Annu Rev Immunol 21:107-137]. We identified four CD45 inhibitors that showed activity in T cells and macrophages. These results indicate that our assay can be applied to primary screening for inhibitors of CD45 and of other protein tyrosine phosphatases to increase the yield of biologically active inhibitors.


Subject(s)
Enzyme Inhibitors/metabolism , High-Throughput Screening Assays/methods , Leukocyte Common Antigens/antagonists & inhibitors , Leukocyte Common Antigens/metabolism , Anthrax/drug therapy , Anthrax/metabolism , Bacillus anthracis , Cytoprotection/drug effects , Drug Discovery , Enzyme Activation/drug effects , Flow Cytometry/methods , Humans , Jurkat Cells , Oligopeptides/metabolism , Phosphorylation/drug effects , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/metabolism , Signal Transduction/drug effects , Substrate Specificity , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology
12.
J Clin Invest ; 134(4)2024 Jan 09.
Article in English | MEDLINE | ID: mdl-38357922

ABSTRACT

Chronic and elevated levels of the antiviral cytokine IFN-α in the brain are neurotoxic. This is best observed in patients with genetic cerebral interferonopathies such as Aicardi-Goutières syndrome. Cerebral interferonopathies typically manifest in early childhood and lead to debilitating disease and premature death. There is no cure for these diseases with existing treatments largely aimed at managing symptoms. Thus, an effective therapeutic strategy is urgently needed. Here, we investigated the effect of antisense oligonucleotides targeting the murine IFN-α receptor (Ifnar1 ASOs) in a transgenic mouse model of cerebral interferonopathy. Intracerebroventricular injection of Ifnar1 ASOs into transgenic mice with brain-targeted chronic IFN-α production resulted in a blunted cerebral interferon signature, reduced neuroinflammation, restoration of blood-brain barrier integrity, absence of tissue destruction, and lessened neuronal damage. Remarkably, Ifnar1 ASO treatment was also effective when given after the onset of neuropathological changes, as it reversed such disease-related features. We conclude that ASOs targeting the IFN-α receptor halt and reverse progression of IFN-α-mediated neuroinflammation and neurotoxicity, opening what we believe to be a new and promising approach for the treatment of patients with cerebral interferonopathies.


Subject(s)
Interferon Type I , Nervous System Diseases , Child, Preschool , Humans , Mice , Animals , Neuroinflammatory Diseases , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Interferon-alpha/genetics , Mice, Transgenic
13.
Mol Ther Nucleic Acids ; 32: 289-301, 2023 Jun 13.
Article in English | MEDLINE | ID: mdl-37096163

ABSTRACT

Antisense oligonucleotides (ASOs) are short synthetic nucleic acids that recognize and bind to complementary RNA to modulate gene expression. It is well established that single-stranded, phosphorothioate-modified ASOs enter cells independent of carrier molecules, primarily via endocytic pathways, but that only a small portion of internalized ASO is released into the cytosol and/or nucleus, rendering the majority of ASO inaccessible to the targeted RNA. Identifying pathways that can increase the available ASO pool is valuable as a research tool and therapeutically. Here, we conducted a functional genomic screen for ASO activity by engineering GFP splice reporter cells and applying genome-wide CRISPR gene activation. The screen can identify factors that enhance ASO splice modulation activity. Characterization of hit genes uncovered GOLGA8, a largely uncharacterized protein, as a novel positive regulator enhancing ASO activity by ∼2-fold. Bulk ASO uptake is 2- to 5-fold higher in GOLGA8-overexpressing cells where GOLGA8 and ASOs are observed in the same intracellular compartments. We find GOLGA8 is highly localized to the trans-Golgi and readily detectable at the plasma membrane. Interestingly, overexpression of GOLGA8 increased activity for both splice modulation and RNase H1-dependent ASOs. Taken together, these results support a novel role for GOLGA8 in productive ASO uptake.

14.
Dev Biol ; 357(2): 505-17, 2011 Sep 15.
Article in English | MEDLINE | ID: mdl-21723273

ABSTRACT

Deployment of the gene-regulatory network (GRN) responsible for skeletogenesis in the embryo of the sea urchin Strongylocentrotus purpuratus is restricted to the large micromere lineage by a double negative regulatory gate. The gate consists of a GRN subcircuit composed of the pmar1 and hesC genes, which encode repressors and are wired in tandem, plus a set of target regulatory genes under hesC control. The skeletogenic cell state is specified initially by micromere-specific expression of these regulatory genes, viz. alx1, ets1, tbrain and tel, plus the gene encoding the Notch ligand Delta. Here we use a recently developed high throughput methodology for experimental cis-regulatory analysis to elucidate the genomic regulatory system controlling alx1 expression in time and embryonic space. The results entirely confirm the double negative gate control system at the cis-regulatory level, including definition of the functional HesC target sites, and add the crucial new information that the drivers of alx1 expression are initially Ets1, and then Alx1 itself plus Ets1. Cis-regulatory analysis demonstrates that these inputs quantitatively account for the magnitude of alx1 expression. Furthermore, the Alx1 gene product not only performs an auto-regulatory role, promoting a fast rise in alx1 expression, but also, when at high levels, it behaves as an auto-repressor. A synthetic experiment indicates that this behavior is probably due to dimerization. In summary, the results we report provide the sequence level basis for control of alx1 spatial expression by the double negative gate GRN architecture, and explain the rising, then falling temporal expression profile of the alx1 gene in terms of its auto-regulatory genetic wiring.


Subject(s)
Bone and Bones/embryology , Bone and Bones/metabolism , DNA-Binding Proteins/genetics , Gene Expression Regulation, Developmental , Regulatory Sequences, Nucleic Acid/genetics , Strongylocentrotus purpuratus/embryology , Strongylocentrotus purpuratus/genetics , Animals , Base Sequence , Chromosomes, Artificial, Bacterial/genetics , DNA, Intergenic/genetics , DNA-Binding Proteins/metabolism , Embryo, Nonmammalian/metabolism , Genes, Reporter , Genetic Loci/genetics , Green Fluorescent Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Phylogeny , Repressor Proteins/metabolism , Time Factors , Transcription, Genetic
15.
Nucleic Acid Ther ; 32(6): 473-485, 2022 12.
Article in English | MEDLINE | ID: mdl-36355073

ABSTRACT

Nucleic acid-based phosphorothioate containing antisense oligonucleotides (PS-ASOs) have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing the degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed human peripheral blood mononuclear cell (hPBMC)-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource intensive and easily affected by subject variability. This compelled us to develop a more convenient high-throughput assay. In this study, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPBMC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs through comparison to known benchmark PS-ASO controls that were previously shown to be safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO TLR9-dependent innate immune activation.


Subject(s)
Burkitt Lymphoma , Oligonucleotides, Antisense , Humans , Oligonucleotides, Antisense/genetics , Burkitt Lymphoma/genetics , Burkitt Lymphoma/therapy , Leukocytes, Mononuclear , Toll-Like Receptor 9/genetics
16.
Blood ; 113(9): 2079-87, 2009 Feb 26.
Article in English | MEDLINE | ID: mdl-19131548

ABSTRACT

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion death. We hypothesize that TRALI requires 2 events: (1) the clinical condition of the patient and (2) the infusion of antibodies against MHC class I antigens or the plasma from stored blood. A 2-event rat model was developed with saline (NS) or endotoxin (LPS) as the first event and the infusion of plasma from packed red blood cells (PRBCs) or antibodies (OX18 and OX27) against MHC class I antigens as the second event. ALI was determined by Evans blue dye leak from the plasma to the bronchoalveolar lavage fluid (BALF), protein and CINC-1 concentrations in the BALF, and the lung histology. NS-treated rats did not evidence ALI with any second events, and LPS did not cause ALI. LPS-treated animals demonstrated ALI in response to plasma from stored PRBCs, both prestorage leukoreduced and unmodified, and to OX18 and OX27, all in a concentration-dependent fashion. ALI was neutrophil (PMN) dependent, and OX18/OX27 localized to the PMN surface in vivo and primed the oxidase of rat PMNs. We conclude that TRALI is the result of 2 events with the second events consisting of the plasma from stored blood and antibodies that prime PMNs.


Subject(s)
Acute Lung Injury/etiology , Antibodies/adverse effects , Erythrocyte Transfusion/adverse effects , Erythrocytes/physiology , Histocompatibility Antigens Class I/immunology , Plasma/physiology , Acute Lung Injury/immunology , Acute Lung Injury/pathology , Animals , Blood Preservation/adverse effects , Bronchoalveolar Lavage Fluid/immunology , Disease Models, Animal , Humans , Male , Neutrophil Activation/immunology , Plasma/immunology , Rats , Rats, Sprague-Dawley
17.
Innovations (Phila) ; 16(1): 58-62, 2021.
Article in English | MEDLINE | ID: mdl-33124926

ABSTRACT

OBJECTIVE: Despite advancements in transcatheter aortic valve replacement (TAVR) technology, alternate access strategies are still required when transfemoral access is unsuitable. In these often anatomically complex group of patients, we sought to evaluate the safety and feasibility of suprasternal transinnominate (TI) artery access for TAVR. METHODS: At our institution, 652 patients underwent TAVR from November 2011 through February 2020. Of these, 23 patients underwent TI TAVR via a 5-cm suprasternal incision without special instrumentation. Outcomes of interest were technical considerations, postoperative complications, and perioperative recovery in relation to established access strategies. RESULTS: The mean Society of Thoracic Surgeons risk score was 8.6 ± 4.2 and the average age was 75 ± 8. All patients underwent TI TAVR using a self-expanding (12), or balloon-expandable (11) transcatheter heart valve. Average postoperative stay was 2 ± 0.7 days (range 2 to 4) with most 20/23 (87%) being discharged to home. There was no 30-day mortality or readmission. There was 1 access-site complication and 1 cerebrovascular accident within 30 days, both intraoperative, with excellent recovery. All patients had either trivial (19) or mild (4) aortic regurgitation on 30-day echocardiography. CONCLUSIONS: TAVR via suprasternal TI access is feasible, safe, provides satisfactory perioperative recovery and adds to the options when patients require alternate access. Further data would be optimal to validate this single-center experience.


Subject(s)
Aortic Valve Stenosis , Heart Valve Prosthesis , Transcatheter Aortic Valve Replacement , Aged , Aged, 80 and over , Aortic Valve/diagnostic imaging , Aortic Valve/surgery , Aortic Valve Stenosis/surgery , Arteries , Humans , Risk Factors , Treatment Outcome
18.
Nat Commun ; 12(1): 5180, 2021 08 30.
Article in English | MEDLINE | ID: mdl-34462437

ABSTRACT

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.


Subject(s)
Calcium-Binding Proteins/genetics , Cardiomyopathies/genetics , Cardiomyopathies/therapy , Genetic Therapy , Heart Failure/genetics , Heart Failure/therapy , Oligonucleotides, Antisense/genetics , Animals , Calcium/metabolism , Calcium-Binding Proteins/metabolism , Cardiomyopathies/metabolism , Female , Heart Failure/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/metabolism , Oligonucleotides, Antisense/metabolism , Rats , Rats, Inbred Lew
19.
J Appl Physiol (1985) ; 104(4): 1161-6, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18276905

ABSTRACT

Mesenteric lymph is the mechanistic link between splanchnic hypoperfusion and acute lung injury (ALI), but the culprit mediator(s) remains elusive. Previous work has shown that administration of a phospholipase A(2) (PLA(2)) inhibitor attenuated postshock ALI and also identified a non-ionic lipid within the postshock mesenteric lymph (PSML) responsible for polymorphonuclear neutrophil (PMN) priming. Consequently, we hypothesized that gut-derived leukotriene B(4) (LTB(4)) is a key mediator in the pathogenesis of ALI. Trauma/hemorrhagic shock (T/HS) was induced in male Sprague-Dawley rats and the mesenteric duct cannulated for lymph collection/diversion. PSML, arachidonic acid (AA), and a LTB(4) receptor antagonist were added to PMNs in vitro. LC/MS/MS was employed to identify bioactive lipids in PSML and the lungs. T/HS increased AA in PSML and increased LTB(4) and PMNs in the lung. Lymph diversion decreased lung LTB(4) by 75% and PMNs by 40%. PSML stimulated PMN priming (11.56 +/- 1.25 vs. 3.95 +/- 0.29 nmol O(2)(-)/min; 3.75 x 10(5) cells/ml; P < 0.01) that was attenuated by LTB(4) receptor blockade (2.64 +/- 0.58; P < 0.01). AA stimulated PMNs to produce LTB(4), and AA-induced PMN priming was attenuated by LTB(4) receptor antagonism. Collectively, these data indicate that splanchnic ischemia/reperfusion activates gut PLA(2)-mediated release of AA into the lymph where it is delivered to the lungs, provoking LTB(4) production and subsequent PMN-mediated lung injury.


Subject(s)
Arachidonic Acid/pharmacology , Leukotriene B4/biosynthesis , Lung/metabolism , Lymph/metabolism , Mesentery/metabolism , Animals , Leukotriene B4/antagonists & inhibitors , Lung/drug effects , Lung/pathology , Lung Diseases/metabolism , Lung Diseases/pathology , Lymph/drug effects , Male , Mass Spectrometry , Mesentery/drug effects , Mesentery/pathology , N-Formylmethionine Leucyl-Phenylalanine , Neutrophil Infiltration/physiology , Neutrophils/drug effects , Neutrophils/metabolism , Rats , Rats, Sprague-Dawley , Shock, Hemorrhagic/metabolism , Shock, Hemorrhagic/pathology , Superoxides/metabolism
20.
Genome Biol ; 19(1): 4, 2018 01 15.
Article in English | MEDLINE | ID: mdl-29334995

ABSTRACT

BACKGROUND: About 11% of all human genetic diseases are caused by nonsense mutations that generate premature translation termination codons (PTCs) in messenger RNAs (mRNA). PTCs not only lead to the production of truncated proteins, but also often result in  decreased mRNA abundance due to  nonsense-mediated mRNA decay (NMD). Although pharmacological inhibition of NMD could be an attractive therapeutic approach for the treatment of diseases caused by nonsense mutations, NMD also regulates the expression of 10-20% of the normal transcriptome. RESULTS: Here, we investigate whether NMD can be inhibited to stabilize mutant mRNAs, which may subsequently produce functional proteins, without having a major impact on the normal transcriptome. We develop antisense oligonucleotides (ASOs) to systematically deplete each component in the NMD pathway. We find that ASO-mediated depletion of each NMD factor elicits different magnitudes of NMD inhibition in vitro and are differentially tolerated in normal mice. Among all of the NMD factors, Upf3b depletion is well tolerated, consistent with previous reports that UPF3B is not essential for development and regulates only a subset of the endogenous NMD substrates. While minimally impacting the normal transcriptome, Upf3b-ASO treatment significantly stabilizes the PTC-containing dystrophin mRNA in mdx mice and coagulation factor IX mRNA in a hemophilia mouse model. Furthermore, when combined with reagents promoting translational read-through, Upf3b-ASO treatment leads to the production of functional factor IX protein in hemophilia mice. CONCLUSIONS: These data demonstrate that ASO-mediated reduction of the NMD factor Upf3b could be an effective and safe approach for the treatment of diseases caused by nonsense mutations.


Subject(s)
Codon, Nonsense , Nonsense Mediated mRNA Decay , Oligonucleotides, Antisense , RNA-Binding Proteins/antagonists & inhibitors , Animals , Cells, Cultured , Dystrophin/genetics , Factor IX/metabolism , Hemophilia B/genetics , Hemophilia B/metabolism , Hemophilia B/therapy , Liver/metabolism , Mice , RNA Stability , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Transcriptome
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