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Proceedings of the National Academy of... Aug 2023Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms....
Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca/Mg levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in oocytes. Downregulation of Unx1 mRNA with morpholine contains invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.
Topics: Protein Subunits; Phylogeny; Cell Membrane; Cytoplasm; Morpholinos
PubMed: 37487087
DOI: 10.1073/pnas.2307898120 -
Molecules (Basel, Switzerland) Jul 2023Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a... (Review)
Review
Phosphorodiamidate morpholinos (PMOs) are known as premier gene knockdown tools in developmental biology. PMOs are usually 25 nucleo-base-long morpholino subunits with a neutral phosphorodiamidate linkage. PMOs work via a steric blocking mechanism and are stable towards nucleases' inside cells. PMOs are usually synthesized using phosphoramidate P(V) chemistry. In this review, we will discuss the synthesis of PMOs, phosphoroamidate morpholinos (MO), and thiophosphoramidate morpholinos (TMO).
Topics: Morpholinos; Organophosphorus Compounds; Oligonucleotides, Antisense
PubMed: 37513252
DOI: 10.3390/molecules28145380 -
Journal of Medical Genetics Jun 2023is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy...
BACKGROUND
is thought to play an important role in cytoskeletal modification and development of the early nervous system. Previously, single-nucleotide variants (SNVs) or copy number variations (CNVs) in have been associated with the neurodevelopmental disorder Stocco dos Santos syndrome, but not with congenital anomalies of the urinary tract and the visceral or the cardiovascular system.
METHODS
Here, exome sequencing and CNV analyses besides expression studies in zebrafish and mouse and (KD) experiments using a splice blocking morpholino in zebrafish were performed to study the role of during embryonic development.
RESULTS
In this study, we identified putative disease-causing SNVs and CNVs in in six individuals from four families with congenital anomalies of the urinary tract and the anorectal, cardiovascular and central nervous systems (CNS). Embryonic mouse and zebrafish expression studies showed expression in the upper and lower urinary tract, the developing cloaca, the heart and the cerebral CNS. KD studies in zebrafish larvae revealed pronephric cysts, anomalies of the cloaca and the heart, decreased eye-to-head ratio and higher mortality compared with controls. These phenotypes could be rescued by co-injection of human wild-type mRNA and morpholino.
CONCLUSION
The identified SNVs and CNVs in affected individuals with congenital anomalies of the urinary tract, the anorectal, the cardiovascular and the central nervous systems, and subsequent embryonic mouse and zebrafish studies suggest as a developmental gene for different organ systems.
Topics: Pregnancy; Female; Humans; Animals; Mice; Zebrafish; DNA Copy Number Variations; Morpholinos; Urinary Tract; Central Nervous System; Cardiovascular System
PubMed: 36379543
DOI: 10.1136/jmg-2022-108738 -
PloS One 2015Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are...
Antisense oligonucleotides (ASOs) are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO)-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA) MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.
Topics: Animals; Embryonic Development; Feasibility Studies; Female; Gene Knockdown Techniques; Male; Morpholinos; Oligonucleotides, Antisense; RNA, Long Noncoding; RNA, Messenger; Transcription, Genetic; Zebrafish; Zebrafish Proteins; Zygote
PubMed: 26436892
DOI: 10.1371/journal.pone.0139504 -
Science (New York, N.Y.) Jan 2021
Topics: Dependovirus; Dynamin II; Exons; Gene Transfer Techniques; Genetic Therapy; Humans; Molecular Targeted Therapy; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; RNA Precursors; RNA Stability
PubMed: 33384365
DOI: 10.1126/science.aba4515 -
Nucleic Acids Research Nov 2022Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling...
Current therapies for Duchenne muscular dystrophy (DMD) use phosphorodiamidate morpholino oligomers (PMO) to induce exon skipping in the dystrophin pre-mRNA, enabling the translation of a shortened but functional dystrophin protein. This strategy has been hampered by insufficient delivery of PMO to cardiac and skeletal muscle. To overcome these limitations, we developed the FORCETM platform consisting of an antigen-binding fragment, which binds the transferrin receptor 1, conjugated to an oligonucleotide. We demonstrate that a single dose of the mouse-specific FORCE-M23D conjugate enhances muscle delivery of exon skipping PMO (M23D) in mdx mice, achieving dose-dependent and robust exon skipping and durable dystrophin restoration. FORCE-M23D-induced dystrophin expression reached peaks of 51%, 72%, 62%, 90% and 77%, of wild-type levels in quadriceps, tibialis anterior, gastrocnemius, diaphragm, and heart, respectively, with a single 30 mg/kg PMO-equivalent dose. The shortened dystrophin localized to the sarcolemma, indicating expression of a functional protein. Conversely, a single 30 mg/kg dose of unconjugated M23D displayed poor muscle delivery resulting in marginal levels of exon skipping and dystrophin expression. Importantly, FORCE-M23D treatment resulted in improved functional outcomes compared with administration of unconjugated M23D. Our results suggest that FORCE conjugates are a potentially effective approach for the treatment of DMD.
Topics: Animals; Mice; Dystrophin; Exons; Mice, Inbred mdx; Morpholinos; Muscle, Skeletal; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; Receptors, Transferrin
PubMed: 35944903
DOI: 10.1093/nar/gkac641 -
Chembiochem : a European Journal of... Nov 2022Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin,...
Caged morpholino oligonucleotides (cMOs) are synthetic tools that allow light-inducible gene silencing in live organisms. Previously reported cMOs have utilized hairpin, duplex, and cyclic structures, as well as caged nucleobases. While these antisense technologies enable efficient optical control of RNA splicing and translation, they can have limited dynamic range. A new caging strategy was developed where the two MO termini are conjugated to an internal position through a self-immolative trifunctional linker, thereby generating a bicyclic cMO that is conformationally resistant to RNA binding. The efficacy of this alternative cMO design has been demonstrated in zebrafish embryos and compared to linear MOs and monocyclic constructs.
Topics: Animals; Morpholinos; Zebrafish; Gene Silencing
PubMed: 36068175
DOI: 10.1002/cbic.202200374 -
American Journal of Physiology.... Jan 2023Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC)...
Primary sclerosing cholangitis (PSC) is characterized by increased ductular reaction (DR), liver fibrosis, hepatic total bile acid (TBA) levels, and mast cell (MC) infiltration. Apical sodium BA transporter (ASBT) expression increases in cholestasis, and ileal inhibition reduces PSC phenotypes. FVB/NJ and multidrug-resistant 2 knockout () mice were treated with control or ASBT Vivo-Morpholino (VM). We measured ) ASBT expression and MC presence in liver/ileum; ) liver damage/DR; ) hepatic fibrosis/inflammation; ) biliary inflammation/histamine serum content; and ) gut barrier integrity/hepatic bacterial translocation. TBA/BA composition was measured in cholangiocyte/hepatocyte supernatants, intestine, liver, serum, and feces. Shotgun analysis was performed to ascertain microbiome changes. In vitro, cholangiocytes were treated with BAs ± ASBT VM, and histamine content and farnesoid X receptor (FXR) signaling were determined. Treated cholangiocytes were cocultured with MCs, and FXR signaling, inflammation, and MC activation were measured. Human patients were evaluated for ASBT/MC expression and histamine/TBA content in bile. Control patient- and PSC patient-derived three-dimensional (3-D) organoids were generated; ASBT, chymase, histamine, and fibroblast growth factor-19 (FGF19) were evaluated. ASBT VM in mice decreased ) biliary ASBT expression, ) PSC phenotypes, ) hepatic TBA, and ) gut barrier integrity compared with control. We found alterations between wild-type (WT) and mouse microbiome, and ASBT/MC and bile histamine content increased in cholestatic patients. BA-stimulated cholangiocytes increased MC activation/FXR signaling via ASBT, and human PSC-derived 3-D organoids secrete histamine/FGF19. Inhibition of hepatic ASBT ameliorates cholestatic phenotypes by reducing cholehepatic BA signaling, biliary inflammation, and histamine levels. ASBT regulation of hepatic BA signaling offers a therapeutic avenue for PSC. We evaluated knockdown of the apical sodium bile acid transporter (ASBT) using Vivo-Morpholino in Mdr2KO mice. ASBT inhibition decreases primary sclerosing cholangitis (PSC) pathogenesis by reducing hepatic mast cell infiltration, altering bile acid species/cholehepatic shunt, and regulating gut inflammation/dysbiosis. Since a large cohort of PSC patients present with IBD, this study is clinically important. We validated findings in human PSC and PSC-IBD along with studies in novel human 3-D organoids formed from human PSC livers.
Topics: Humans; Animals; Mice; Cholangitis, Sclerosing; Bile Acids and Salts; Histamine; Morpholinos; Liver; Cholestasis; Liver Cirrhosis; Inflammation; Membrane Transport Proteins; Inflammatory Bowel Diseases
PubMed: 36410025
DOI: 10.1152/ajpgi.00112.2022 -
Molecular Cell Feb 2020Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to... (Review)
Review
Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.
Topics: Animals; CRISPR-Cas Systems; Clustered Regularly Interspaced Short Palindromic Repeats; Gene Editing; Genetic Engineering; Humans; Morpholinos; Protein Engineering; Protein Transport; Proteolysis
PubMed: 32004468
DOI: 10.1016/j.molcel.2020.01.010 -
Neuropsychopharmacology Reports Jun 2023The purpose of this study is to evaluate the safety and pharmacokinetics of the novel morpholino oligomer NS-089/NCNP-02 which can induce exon 44 skipping, in patients...
Systemic administration of the antisense oligonucleotide NS-089/NCNP-02 for skipping of exon 44 in patients with Duchenne muscular dystrophy: Study protocol for a phase I/II clinical trial.
AIM
The purpose of this study is to evaluate the safety and pharmacokinetics of the novel morpholino oligomer NS-089/NCNP-02 which can induce exon 44 skipping, in patients with DMD. Additionally, we aimed to identify markers predictive of therapeutic efficacy and determine the optimal dosing for future studies.
METHODS
This is an open-label, dose-escalation, two-center phase I/II trial in ambulant patients with DMD, presence of an out-of-frame deletion, and a mutation amenable to exon 44 skipping. Part 1 is a stepwise dose-finding stage (4 weeks) during which NS-089/NCNP-02 will be administered intravenously at four dose levels once weekly (1.62, 10, 40, and 80 mg/kg); Part 2 is a 24-week evaluation period based on the dosages determined during Part 1. The primary (safety) endpoints are the results of physical examinations, vital signs, 12-lead electrocardiogram and echocardiography tests, and adverse event reporting. Secondary endpoints include expression of dystrophin protein, motor function assessment, exon 44 skipping efficiency, plasma and urinary NS-089/NCNP-02 concentrations, and changes in blood creatine kinase levels.
DISCUSSION
Exon-skipping therapy using ASOs shows promise in selected patients, and this first-in-human study is expected to provide critical information for subsequent clinical development of NS-089/NCNP-02.
Topics: Humans; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; Morpholinos; Exons; Mutation; Clinical Trials, Phase II as Topic; Clinical Trials, Phase I as Topic
PubMed: 37326950
DOI: 10.1002/npr2.12335