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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 -
Archives of Toxicology Jan 2022Duchenne muscular dystrophy (DMD) afflicts 1 in 5000 newborn males, leading to progressive muscle weakening and the loss of ambulation between the ages of 8 and 12.... (Review)
Review
Duchenne muscular dystrophy (DMD) afflicts 1 in 5000 newborn males, leading to progressive muscle weakening and the loss of ambulation between the ages of 8 and 12. Typically, DMD patients pass away from heart failure or respiratory failure. Currently, there is no cure, though exon-skipping therapy including eteplirsen (brand name Exondys 51), a synthetic antisense oligonucleotide designed to skip exon 51 of the dystrophin gene, is considered especially promising. Applicable to approximately 14% of DMD patients, a phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotide eteplirsen received accelerated approval by the US Food and Drug Administration (FDA) in 2016. Throughout clinical trials, eteplirsen has been well tolerated by patients with no serious drug-related adverse events. The most common events observed are balance disorder, vomiting, and skin rash. Despite its safety and promise of functional benefits, eteplirsen remains controversial due to its low production of dystrophin. In addition, unmodified PMOs have limited efficacy in the heart. To address these concerns of efficacy, eteplirsen has been conjugated to a proprietary cell-penetrating peptide; the conjugate is called SRP-5051. Compared to eteplirsen, SRP-5051 aims to better prompt exon-skipping and dystrophin production but may have greater toxicity concerns. This paper reviews and discusses the available information on the efficacy, safety, and tolerability data of eteplirsen and SRP-5051 from preclinical and clinical trials. Issues faced by eteplirsen and SRP-5051, including efficacy and safety, are identified. Lastly, the current state of eteplirsen and exon-skipping therapy in general as a strategy for the treatment of DMD are discussed.
Topics: Child; Exons; Humans; Infant, Newborn; Male; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense
PubMed: 34797383
DOI: 10.1007/s00204-021-03184-z -
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 -
Methods in Molecular Biology (Clifton,... 2023Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However,...
Antisense oligonucleotides (ASOs) have shown great therapeutic potential in the treatment of many neuromuscular diseases including myotonic dystrophy 1 (DM1). However, systemically delivered ASOs display poor biodistribution and display limited penetration into skeletal muscle. The conjugation of cell-penetrating peptides (CPPs) to phosphorodiamidate morpholino oligonucleotides (PMOs), a class of ASOs with a modified backbone, can be used to enhance ASO skeletal muscle penetration. Peptide-PMOs (P-PMOs) have been shown to be highly effective in correcting the DM1 skeletal muscle phenotype in both murine and cellular models of DM1 and at a molecular and functional level. Here we describe the synthesis and conjugation of P-PMOs and methods for analyzing their biodistribution and toxicity in the HSA-LR DM1 mouse model and their efficacy both in vitro and in vivo using FISH and RT-PCR splicing analysis.
Topics: Mice; Animals; Morpholinos; Myotonic Dystrophy; Tissue Distribution; Oligonucleotides, Antisense; Cell-Penetrating Peptides
PubMed: 36401033
DOI: 10.1007/978-1-0716-2772-3_13 -
Journal of Clinical Neuroscience :... Mar 2018Duchenne Muscular Dystrophy is a paediatric disorder resulting from a defective dystrophin gene. It causes progressive loss of muscle fibres, muscle weakness, and... (Meta-Analysis)
Meta-Analysis Review
BACKGROUND
Duchenne Muscular Dystrophy is a paediatric disorder resulting from a defective dystrophin gene. It causes progressive loss of muscle fibres, muscle weakness, and eventually loss of ambulation during adolescence, with death due to respiratory or cardiovascular complications soon afterwards. The drug eteplirsen has received support from medical experts and parents of affected children, but the FDA has delayed their decision for approval of this drug.
OBJECTIVE
This study analysed the results of previous studies to assess the safety and efficacy of the eteplirsen, and is the first pooled-analysis of its kind.
METHODS
A literature search of electronic databases was performed. Only human studies using eteplirsen were eligible.
RESULTS
A total of four relevant clinical studies were identified. A pooled-analysis was performed using data relating to percentage dystrophin-positive fibres obtained from muscle biopsy, and the six-minute walk test (6 MWT). The average increase in percentage dystrophin-positive fibres after treatment with eteplirsen was 24.23% (range -4 to 78; SD 24.44%). The average rate of decline in distance walked was 65metres (range -335 to 83; SD 100.08 m).
CONCLUSIONS
Whether or not this increase in percentage dystrophin-positive fibres and distance walked is clinically significant is unclear, and there is therefore a need for more clinical trials.
Topics: Adolescent; Child; Clinical Trials as Topic; Humans; Male; Morpholinos; Muscle Fibers, Skeletal; Muscle Weakness; Muscular Dystrophy, Duchenne; Walking
PubMed: 29254734
DOI: 10.1016/j.jocn.2017.10.082 -
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 -
Bioorganic Chemistry Sep 2021Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key enzyme involved in the metabolism of the mammalian phospholipid phosphatidylcholine into secondary...
Phosphatidylcholine-specific phospholipase C (PC-PLC) is a key enzyme involved in the metabolism of the mammalian phospholipid phosphatidylcholine into secondary messengers diacylglycerol (DAG) and phosphocholine. DAG and phosphocholine have been identified to amplify various cellular processes involved in oncogenesis such as proliferation, cell-cycle activation, differentiation and motility, therefore making PC-PLC a potential target for novel anti-cancer treatments. The current literature standard for PC-PLC inhibition, tricyclodecan-9-yl-potassium xanthate (D609), has been shown to arrest proliferation in multiple cancer cell lines, however, it is not drug-like resulting in low aqueous stability, making it a poor drug candidate. 2-Morpholinobenzoic acids have been shown to have improved PC-PLC inhibitory activity compared to D609, with molecular modelling identifying chelation of the carboxylic acid to catalytic Zn ions in the PC-PLC active site being a key interaction. In this study, the carboxylic acid motif was replaced with a hydroxamic acid to strengthen the Zn interaction. It was found that the hydroxamic acid derivatives displayed PC-PLC inhibitory activity similar, or better, than D609. Furthermore, these novel inhibitors had potent anti-proliferative activity in MDA-MB-231 and HCT-116 cancer cell lines, far greater than D609 and previous 2-morpholinobenzoic acids.
Topics: Antineoplastic Agents; Benzamides; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Development; Drug Screening Assays, Antitumor; Humans; Molecular Structure; Morpholinos; Structure-Activity Relationship
PubMed: 34328856
DOI: 10.1016/j.bioorg.2021.105152 -
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