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Cell Stem Cell Mar 2023Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However,...
Adenosine deaminase acting on RNA1 (ADAR1) preserves genomic integrity by preventing retroviral integration and retrotransposition during stress responses. However, inflammatory-microenvironment-induced ADAR1p110 to p150 splice isoform switching drives cancer stem cell (CSC) generation and therapeutic resistance in 20 malignancies. Previously, predicting and preventing ADAR1p150-mediated malignant RNA editing represented a significant challenge. Thus, we developed lentiviral ADAR1 and splicing reporters for non-invasive detection of splicing-mediated ADAR1 adenosine-to-inosine (A-to-I) RNA editing activation; a quantitative ADAR1p150 intracellular flow cytometric assay; a selective small-molecule inhibitor of splicing-mediated ADAR1 activation, Rebecsinib, which inhibits leukemia stem cell (LSC) self-renewal and prolongs humanized LSC mouse model survival at doses that spare normal hematopoietic stem and progenitor cells (HSPCs); and pre-IND studies showing favorable Rebecsinib toxicokinetic and pharmacodynamic (TK/PD) properties. Together, these results lay the foundation for developing Rebecsinib as a clinical ADAR1p150 antagonist aimed at obviating malignant microenvironment-driven LSC generation.
Topics: Mice; Animals; Protein Isoforms; Hematopoietic Stem Cells; Adenosine Deaminase
PubMed: 36803553
DOI: 10.1016/j.stem.2023.01.008 -
International Journal of Biological... Dec 2019Adenosine deaminase is a critical enzyme in purine metabolism that regulates intra and extracellular adenosine concentrations by converting it to inosine. Adenosine is... (Review)
Review
Adenosine deaminase is a critical enzyme in purine metabolism that regulates intra and extracellular adenosine concentrations by converting it to inosine. Adenosine is an important purine that regulates numerous physiological functions by interacting with its receptors. Adenosine and consequently adenosine deaminase can have pro or anti-inflammatory effects on tissues depending on how much time has passed from the start of the injury. In addition, an increase in adenosine deaminase activity has been reported for various diseases and the significant effect of deaminase inhibition on the clinical course of different diseases has been reported. However, the use of inhibitors is limited to only a few medical indications. Data on the increase of adenosine deaminase activity in different diseases and the impact of its inhibition in various cases have been collected and are discussed in this review. Overall, the evidence shows that many studies have been done to introduce inhibitors, however, in vivo studies have been much less than in vitro, and often have not been expanded for clinical use.
Topics: Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Humans; Signal Transduction
PubMed: 31520704
DOI: 10.1016/j.ijbiomac.2019.09.078 -
Nature Reviews. Immunology Apr 2024Recent progress in human and mouse genetics has transformed our understanding of the molecular mechanisms by which recognition of self double-stranded RNA (self-dsRNA)... (Review)
Review
Recent progress in human and mouse genetics has transformed our understanding of the molecular mechanisms by which recognition of self double-stranded RNA (self-dsRNA) causes immunopathology. Novel mouse models recapitulate loss-of-function mutations in the RNA editing enzyme ADAR1 that are found in patients with Aicardi-Goutières syndrome (AGS) - a monogenic inflammatory disease associated with increased levels of type I interferon. Extensive analyses of the genotype-phenotype relationships in these mice have now firmly established a causal relationship between increased intracellular concentrations of endogenous immunostimulatory dsRNA and type I interferon-driven immunopathology. Activation of the dsRNA-specific immune sensor MDA5 perpetuates the overproduction of type I interferons, and chronic engagement of the interferon-inducible innate immune receptors PKR and ZBP1 by dsRNA drives immunopathology by activating an integrated stress response or by inducing excessive cell death. Biochemical and genetic data support a role for the p150 isoform of ADAR1 in the cytosol in suppressing the spontaneous, pathological response to self-dsRNA.
Topics: Humans; Mice; Animals; RNA, Double-Stranded; Interferon Type I; Adenosine Deaminase
PubMed: 37752355
DOI: 10.1038/s41577-023-00940-3 -
Redox Biology Nov 2023Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain...
Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain ischemia/reperfusion may reveal new therapeutic strategies. Adenosine deaminase acting on RNA (ADAR1), an RNA editing enzyme, is not normally expressed in astrocytes, but highly induced in astrocytes in ischemic stroke lesions. The expression of ADAR1 steeply increased from day 1 to day 7 after middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. ADAR1 deficiency markedly ameliorated the volume of the cerebral infarction and neurological deficits as shown by the rotarod and cylinder tests, which was due to the reduction of the numbers of activated astrocytes and microglia. Surprisingly, ADAR1 was mainly expressed in astrocytes while only marginally in microglia. In primary cultured astrocytes, ADAR1 promoted astrocyte proliferation via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, ADAR1 deficiency inhibited brain cell apoptosis in mice with MCAO as well as in activated astrocyte-conditioned medium-induced neurons in vitro. It appeared that ADAR1 induces neuron apoptosis by secretion of IL-1β, IL-6 and TNF-α from astrocytes through the production of reactive oxygen species. These results indicated that ADAR1 is a novel regulator promoting the proliferation of the activated astrocytes following ischemic stroke, which produce various inflammatory cytokines, leading to neuron apoptosis and worsened ischemic stroke outcome.
Topics: Mice; Animals; Astrocytes; Phosphatidylinositol 3-Kinases; Brain Ischemia; Infarction, Middle Cerebral Artery; Neurons; Brain Injuries; Ischemic Stroke; Apoptosis; Reperfusion Injury; Adenosine Deaminase
PubMed: 37801857
DOI: 10.1016/j.redox.2023.102903 -
Science (New York, N.Y.) Jul 2020CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA...
CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.
Topics: Adenine; Adenosine Deaminase; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Cryoelectron Microscopy; DNA; Deamination; Escherichia coli Proteins; Gene Editing
PubMed: 32732424
DOI: 10.1126/science.abb1390 -
Nature Biotechnology Sep 2019Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted...
Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
Topics: Adenosine Deaminase; Animals; Base Sequence; CRISPR-Cas Systems; Cell Line; Directed Molecular Evolution; Gene Editing; Gene Expression Regulation, Enzymologic; Gene Targeting; Humans; INDEL Mutation; Mice
PubMed: 31332326
DOI: 10.1038/s41587-019-0193-0 -
Frontiers in Immunology 2022
Topics: Humans; Adenosine Deaminase; Intercellular Signaling Peptides and Proteins; Vasculitis; Polyarteritis Nodosa
PubMed: 36569902
DOI: 10.3389/fimmu.2022.1108853 -
Nature Biotechnology Jun 2022Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target...
Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target editing. Here, we describe LEAPER 2.0, an updated version of LEAPER that uses covalently closed circular arRNAs, termed circ-arRNAs. We demonstrate on average ~3.1-fold higher editing efficiency than their linear counterparts when expressed in cells or delivered as in vitro-transcribed circular RNA oligonucleotides. To lower off-target editing we deleted pairings of uridines with off-target adenosines, which almost completely eliminated bystander off-target adenosine editing. Engineered circ-arRNAs enhanced the efficiency and fidelity of editing endogenous CTNNB1 and mutant TP53 transcripts in cell culture. Delivery of circ-arRNAs using adeno-associated virus in a mouse model of Hurler syndrome corrected the pathogenic point mutation and restored α-L-iduronidase catalytic activity, lowering glycosaminoglycan accumulation in the liver. LEAPER 2.0 provides a new design of arRNA that enables more precise, efficient RNA editing with broad applicability for therapy and basic research.
Topics: Adenosine; Adenosine Deaminase; Animals; Hydrolases; Mice; RNA; RNA Editing; RNA, Circular; RNA-Binding Proteins
PubMed: 35145313
DOI: 10.1038/s41587-021-01180-3 -
Immunity May 2023Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to...
Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.
Topics: RNA; Interleukin-6; Endothelial Cells; Cytokine Receptor gp130; Endothelium; Adenosine Deaminase
PubMed: 37100060
DOI: 10.1016/j.immuni.2023.03.021 -
Nature Biotechnology Sep 2019Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity,...
Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.
Topics: Adenosine Deaminase; Animals; Cell Line; Genetic Engineering; Humans; RNA; RNA Editing; RNA-Binding Proteins
PubMed: 31308540
DOI: 10.1038/s41587-019-0178-z