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Organic Letters Feb 2023We report the synthesis of piperidino nucleoside phosphoramidates functionalized with uracil, cytosine, guanine, and adenine and their incorporation into oligomers....
We report the synthesis of piperidino nucleoside phosphoramidates functionalized with uracil, cytosine, guanine, and adenine and their incorporation into oligomers. High-performance liquid chromatography analyses demonstrated that a phosphorodiamidate piperidino oligomer (PPO) is more lipophilic than a phosphorodiamidate morpholino oligomer (PMO) of the same tetrameric sequence. A PMO containing piperidino residues formed duplexes with both DNA and RNA, and the PPO had higher stability at endosomolytic pH and higher hydrophobicity than the PMO.
Topics: Oligonucleotides, Antisense; Morpholinos
PubMed: 36734846
DOI: 10.1021/acs.orglett.2c04067 -
Drug Metabolism and Disposition: the... Oct 2023Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike...
Several modified antisense oligonucleotides (ASOs) have recently been approved for clinical use. Some are phosphorodiamidate morpholino oligomers (PMOs), which, unlike other nucleic acids, are not negatively charged. Thus, PMOs differ from other ASOs in their pharmacokinetic (PK) properties. Drugs with a PMO backbone have been administered to Duchenne muscular dystrophy pediatric patients; however, appropriate methodologies are not currently available to predict their human PK from nonclinical data. In this study, we used viltolarsen as a representative PMO to investigate the applicability of the allometric scaling approach to human PK prediction. We first summarized the nonclinical and clinical PK data for viltolarsen as showing high total clearance, low serum protein binding, metabolic resistance, and urinary excretion as the unchanged drug in both animals and humans. We then investigate the PK of viltolarsen in mice, rats, cynomolgus monkeys, and dogs and used the results, with body weight, to extrapolate to humans by several methods. The estimate of human total clearance obtained from cynomolgus monkeys was the best, and body weight may be the key factor in accurately predicting human total clearance. In contrast, all of the well-known prediction methods for the volume of distribution at steady state gave underestimates. However, the human PK profiles predicted from the PK parameters in cynomolgus monkeys fit the observed human plasma concentrations well. These results are expected to contribute to the further development of PMOs. SIGNIFICANCE STATEMENT: We investigated how to predict the human PK of phosphorodiamidate morpholino oligomers from nonclinical data. The estimates of human PK parameters and profiles determined from cynomolgus monkeys by an allometric scaling approach were the most suitable, and the cynomolgus monkey body weight may be the key factor in accurately predicting human total clearance.
Topics: Humans; Rats; Mice; Animals; Dogs; Child; Morpholinos; Muscular Dystrophy, Duchenne; Macaca fascicularis; Body Weight
PubMed: 37468285
DOI: 10.1124/dmd.123.001425 -
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 -
Bioorganic & Medicinal Chemistry Letters Jul 2016Many cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) inhibitors, such as resveratrol, have planar, hydrophobic, aromatic rings in their structure and exhibit anti-cancer...
Many cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) inhibitors, such as resveratrol, have planar, hydrophobic, aromatic rings in their structure and exhibit anti-cancer activity. Aryl morpholino triazenes have similar structural features and in addition contain a triazene unit consisting of three consecutive, conjugated nitrogen atoms. Several aryl morpholino triazenes, including 4-[(E)-2-(3,4,5-trimethoxyphenyl)diazenyl]-morpholine (2), were prepared from a reaction involving morpholine and a diazonium ion produced from different aniline derivatives, such as 3,4,5-trimethoxyaniline. The aryl morpholino triazenes were then screened at 100μM for their ability to inhibit CYP1A1 and CYP1B1 using ethoxyresorufin as the substrate. Triazenes that inhibited the enzymes to less than 80% of the uninhibited enzyme activity were assayed to determine their IC50 value. Compound 2 was the only triazene to inhibit both CYP1A1 and CYP1B1 to the same degree as resveratrol with IC50 values of 10μM and 18μM, respectively. Compounds 3 and 6 selectively inhibited CYP1B1 over CYP1A1 with IC values of 2μM and 7μM, respectively. Thus, aryl morpholino triazenes are a new class of compounds that can inhibit CYP1A1 and CYP1B1 and potentially prevent cancer.
Topics: Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Humans; Molecular Structure; Morpholinos; Structure-Activity Relationship; Triazenes
PubMed: 27265259
DOI: 10.1016/j.bmcl.2016.05.064 -
The New England Journal of Medicine Jan 2019
Review
Topics: Gene Expression; Humans; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Oligonucleotides; RNA, Messenger; RNA, Small Interfering
PubMed: 30601736
DOI: 10.1056/NEJMra1705346 -
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