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EMBO Molecular Medicine Feb 2021Antisense oligonucleotide (AO)-mediated exon-skipping therapies show promise in Duchenne muscular dystrophy (DMD), a devastating muscular disease caused by...
Antisense oligonucleotide (AO)-mediated exon-skipping therapies show promise in Duchenne muscular dystrophy (DMD), a devastating muscular disease caused by frame-disrupting mutations in the DMD gene. However, insufficient systemic delivery remains a hurdle to clinical deployment. Here, we demonstrate that MOTS-c, a mitochondria-derived bioactive peptide, with an intrinsic muscle-targeting property, augmented glycolytic flux and energy production capacity of dystrophic muscles in vitro and in vivo, resulting in enhanced phosphorodiamidate morpholino oligomer (PMO) uptake and activity in mdx mice. Long-term repeated administration of MOTS-c (500 μg) and PMO at the dose of 12.5 mg/kg/week for 3 weeks followed by 12.5 mg/kg/month for 3 months (PMO-M) induced therapeutic levels of dystrophin expression in peripheral muscles, with up to 25-fold increase in diaphragm of mdx mice over PMO alone. PMO-M improved muscle function and pathologies in mdx mice without detectable toxicity. Our results demonstrate that MOTS-c enables enhanced PMO uptake and activity in dystrophic muscles by providing energy and may have therapeutic implications for exon-skipping therapeutics in DMD and other energy-deficient disorders.
Topics: Animals; Dystrophin; Genetic Therapy; Mice; Mice, Inbred mdx; Morpholinos; Oligonucleotides, Antisense
PubMed: 33337582
DOI: 10.15252/emmm.202012993 -
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 -
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 -
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 -
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 -
Scientific Reports Sep 2020Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9,...
Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.
Topics: 5' Untranslated Regions; Alleles; Animals; Binding Sites; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Genetic Techniques; Morpholinos; Mutation; Phenotype; RNA Splice Sites; Sensitivity and Specificity; Zebrafish; Zebrafish Proteins; Zygote
PubMed: 32958829
DOI: 10.1038/s41598-020-71708-1 -
Molecules (Basel, Switzerland) Aug 2019One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered...
One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.
Topics: Animals; Cell Line; Electroporation; Fibroblasts; Humans; Integrin alpha Chains; Lipids; Mice; Mice, Inbred mdx; Morpholinos; Muscle Fibers, Skeletal; Oligonucleotides, Antisense; Primary Cell Culture; RNA Interference; SMN Complex Proteins; Transfection
PubMed: 31408997
DOI: 10.3390/molecules24162922 -
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 -
The International Journal of... 2018The neural crest is a multipotent and highly migratory cell type that contributes to many of the defining features of vertebrates, including the skeleton of the head and... (Review)
Review
The neural crest is a multipotent and highly migratory cell type that contributes to many of the defining features of vertebrates, including the skeleton of the head and most of the peripheral nervous system. 150 years after the discovery of the neural crest, avian embryos remain one of the most important model organisms for studying neural crest development. In this review, we describe aspects of neural crest induction, migration and axial level differences, highlighting what is known about the underlying gene regulatory mechanisms. Past and emerging technologies continue to improve the resolution with which we can examine important questions of neural crest development, with modern avian molecular embryology continuing to make important contributions.
Topics: Animals; Body Patterning; CRISPR-Cas Systems; Cell Lineage; Cell Movement; Chick Embryo; Embryology; Gene Expression Regulation, Developmental; History, 19th Century; History, 20th Century; History, 21st Century; Humans; Morpholinos; Neural Crest; Oligonucleotides, Antisense; Stem Cells
PubMed: 29616727
DOI: 10.1387/ijdb.180038sg -
Journal of Controlled Release :... Oct 2022Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without...
Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2) to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'-MORF1 and Fab'-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'-MORF1 and Fab'-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI < 1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.
Topics: Animals; Antigens, CD20; Apoptosis; Caspases; Humans; Immunoglobulin Fab Fragments; Macromolecular Substances; Mice; Morpholinos; Rituximab; Serum Albumin, Human
PubMed: 36037975
DOI: 10.1016/j.jconrel.2022.08.045