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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 -
PloS One 2017Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino... (Comparative Study)
Comparative Study
Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos.
Topics: Amino Acid Sequence; Animals; Animals, Genetically Modified; Base Sequence; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Hematopoietic System; In Situ Hybridization; Membrane Proteins; Morpholinos; Mutation; Phenotype; Phylogeny; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Zebrafish; Zebrafish Proteins
PubMed: 28192479
DOI: 10.1371/journal.pone.0172227 -
Biochemical and Biophysical Research... Apr 2021The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and...
The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.
Topics: Circular Dichroism; DNA; Models, Molecular; Molecular Conformation; Morpholinos; Peptides
PubMed: 33652207
DOI: 10.1016/j.bbrc.2021.02.087 -
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 -
EMBO Reports Jun 2022Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense...
Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.
Topics: Animals; Dystrophin; Genetic Therapy; Glycine; Humans; Metformin; Mice; Mice, Inbred mdx; Morpholinos; Muscle, Skeletal; Utrophin
PubMed: 35393769
DOI: 10.15252/embr.202153955 -
Developmental Biology Mar 2022In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides...
In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and -219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT, NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system, pigment cells and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small, it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively 'drop out' a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. To show specificity we have also rescued the knockout phenotype using miRNA mimics. MiRNA-219 and miR-196a KO's both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.
Topics: Animals; CRISPR-Cas Systems; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Gene Knockout Techniques; In Situ Hybridization; MicroRNAs; Morpholinos; Neural Crest; Neural Plate; Neurulation; Phenotype; RNA, Guide, CRISPR-Cas Systems; Transcription Factors; Transcriptome; Xenopus Proteins; Xenopus laevis
PubMed: 34968443
DOI: 10.1016/j.ydbio.2021.12.015 -
Molecular Therapy : the Journal of the... Mar 2022Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly...
Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy.
Topics: Animals; Cell-Penetrating Peptides; Disease Models, Animal; Humans; Morpholinos; Muscular Atrophy, Spinal; Oligonucleotides, Antisense; Phenotype
PubMed: 34808387
DOI: 10.1016/j.ymthe.2021.11.012 -
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 -
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 -
Current Protocols in Nucleic Acid... Mar 2017Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein...
Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis-nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos. © 2017 by John Wiley & Sons, Inc.
Topics: Cytosol; Electroporation; Endocytosis; Gene Expression Regulation; Gene Knockdown Techniques; Microinjections; Morpholinos; Peptides; RNA
PubMed: 28252184
DOI: 10.1002/cpnc.21