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Journal of Biosciences 2023Duchenne muscular dystrophy (DMD) is an X-linked genetic disease primarily affecting boys causing loss of the dystrophin protein, ultimately leading to muscle wastage... (Review)
Review
Duchenne muscular dystrophy (DMD) is an X-linked genetic disease primarily affecting boys causing loss of the dystrophin protein, ultimately leading to muscle wastage and death by cardiac or respiratory failure. The genetic mutation involved can be overcome with antisense oligonucleotides which bind to a pre-mRNA and results in reading frame restoration by exon skipping. Phosphorodiamidate morpholino oligonucleotides (PMOs) are a class of antisense agents with a neutral backbone derived from RNA which can induce effective exon skipping. In this review, the evolution of PMOs in exon skipping therapy for the last two decades has been detailed with the gradual structural and functional advancements. Even though the success rate of PMObased therapy has been high with four FDA approved drugs, several key challenges are yet to overcome, one being the dystrophin restoration in cardiac muscle. The current scenario in further improvement of PMOs has been discussed along with the future perspectives that have the potential to revolutionize the therapeutic benefits in DMD.
Topics: Male; Humans; Morpholinos; Dystrophin; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; Exons
PubMed: 37846020
DOI: No ID Found -
Methods in Molecular Biology (Clifton,... 2017Modifications at either end, both ends, or in-between the ends of a Morpholino oligo provide functional groups for further conjugation. Amino groups are the most useful... (Review)
Review
Modifications at either end, both ends, or in-between the ends of a Morpholino oligo provide functional groups for further conjugation. Amino groups are the most useful and efficient reactive entities for chemical bonding with other molecules. The combination of modifications at both ends, especially with double functionalization at the 3'-end, yields myriad opportunities for diverse applications. An orthogonally protected diamine for advanced 3'-end double modification on the solid phase synthesis support allows the convenient assembly of a vast variety of custom-designed molecules. A particular application is the assembly of a class of Vis-Vivo-Morpholino where at the 3'-end an optically visible fluorophore is installed at one side for fluorescent detection and an in vivo delivery moiety is attached at the other side for intracellular activity studies.
Topics: Amines; Animals; Humans; Morpholinos; Oligonucleotides, Antisense; Solid-Phase Synthesis Techniques
PubMed: 28364232
DOI: 10.1007/978-1-4939-6817-6_4 -
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 -
Clinical Pharmacology and Therapeutics Jan 2016The development of genetic and molecular biology tools permitting the connection of specific genes to their functions has accelerated our understanding of molecular... (Review)
Review
The development of genetic and molecular biology tools permitting the connection of specific genes to their functions has accelerated our understanding of molecular pathways underlying health and disease. The resulting gains in knowledge have propelled gene targeting to the forefront of promising therapeutic strategies. Here we discuss the uniquely powerful and adaptable approach of morpholino-driven modification of normal and mutant gene expression as a pathway to health.
Topics: Animals; Disease; Drug Carriers; Gene Expression; Gene Targeting; Genetic Engineering; Humans; Models, Genetic; Morpholinos; Preventive Medicine
PubMed: 26474085
DOI: 10.1002/cpt.276 -
Methods in Molecular Biology (Clifton,... 2020Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function....
Antisense morpholino oligonucleotides (MOs) have become a valuable method to knockdown protein levels, to block with mRNA splicing and to interfere with miRNA function. MOs are widely used to alter gene expression in development of Xenopus and Zebrafish, where they are typically injected into the fertilized egg or blastomeres. Here we present methods to use electroporation to target delivery of MOs to the central nervous system of Xenopus laevis or Xenopus tropicalis tadpoles. Briefly, MO electroporation is accomplished by injecting MO solution into the brain ventricle and driving the MOs into cells of the brain with current passing between 2 platinum plate electrodes, positioned on either side of the target brain area. The method is relatively straightforward and uses standard equipment found in many neuroscience labs. A major advantage of electroporation is that it allows spatial and temporal control of MO delivery and therefore knockdown. Co-electroporation of MOs with cell type-specific fluorescent protein expression plasmids allows morphological analysis of cellular phenotypes. Furthermore, co-electroporation of MOs with rescuing plasmids allows assessment of specificity of the knockdown and phenotypic outcome. By combining MO-mediated manipulations with sophisticated assays of neuronal function, such as electrophysiological recording, behavioral assays, or in vivo time-lapse imaging of neuronal development, the functions of specific proteins and miRNAs within the developing nervous system can be elucidated. These methods can be adapted to apply antisense morpholinos to study protein and RNA function in a variety of complex tissues.
Topics: Animals; Brain; Electrophysiological Phenomena; Electroporation; Gene Knockdown Techniques; Morpholinos; Oligonucleotides, Antisense; Phenotype; Time-Lapse Imaging; Xenopus
PubMed: 31552666
DOI: 10.1007/978-1-4939-9732-9_21 -
Methods in Molecular Biology (Clifton,... 2017A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and... (Review)
Review
A good Morpholino experiment starts with oligos that have been carefully designed to minimize off-target RNA binding. Performing a successful, reproducible, and well-controlled Morpholino experiment requires oligos that are single stranded and in solution at a known concentration. The outcome of treatment with the oligo needs to be checked for specificity, that is, that the observed outcome is due to interaction with the intended RNA and not an interaction with an unexpected RNA. In this chapter, I will discuss Morpholino use mostly in the context of embryonic microinjection experiments, though many techniques and warnings will be applicable to cell culture or adult animal experiments as well. Controls are critical to a good experiment, but good techniques in designing, preparing, storing, and using the oligos can improve the strength and specificity of the knockdown. Finally, it is important to know the solution concentration of the oligo to ensure that the results are reproducible.
Topics: Animals; Base Pairing; Gene Knockdown Techniques; Gene Targeting; Humans; Microinjections; Morpholinos; Oligonucleotides, Antisense; Preservation, Biological; Quality Control; RNA Stability
PubMed: 28364230
DOI: 10.1007/978-1-4939-6817-6_2 -
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 -
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 -
Methods in Molecular Biology (Clifton,... 2019Morpholino antisense oligonucleotides are used as routine tools in developmental biology to investigate gene function during early embryogenesis. These chemically...
Morpholino antisense oligonucleotides are used as routine tools in developmental biology to investigate gene function during early embryogenesis. These chemically modified oligos contain morpholine ring connected with phosphorodiamidate linkages as backbone but carry unmodified nucleobases. In this chapter, we describe the methods to further modify the nucleobases using palladium-catalyzed cross-coupling reactions. The key reactions used are halogenations of the nucleobases in suitable position and subsequent Pd-catalyzed Sonogashira and Suzuki reactions. The sequential synthetic steps are described in detail in this chapter, and the examples are shown in tables.
Topics: Catalysis; Cross-Linking Reagents; Molecular Structure; Morpholinos; Oligonucleotides, Antisense; Palladium
PubMed: 31016698
DOI: 10.1007/978-1-4939-9216-4_7 -
Expert Opinion on Drug Metabolism &... Nov 2021Antisense oligonucleotides (ASOs) have emerged as a promising novel drug modality that aims to address unmet medical needs. A record of six ASO drugs have been approved... (Review)
Review
INTRODUCTION
Antisense oligonucleotides (ASOs) have emerged as a promising novel drug modality that aims to address unmet medical needs. A record of six ASO drugs have been approved since 2016, and more candidates are in clinical development. ASOs are the most advanced class within the RNA-based therapeutics field.
AREAS COVERED
This review highlights the two major backbones that are currently used to build the most advanced ASO platforms - the phosphorodiamidate morpholino oligomers (PMOs) and the phosphorothioates (PSs). The absorption, distribution, metabolism, and excretion (ADME) properties of the PMO and PS platforms are discussed in detail.
EXPERT OPINION
Understanding the ADME properties of existing ASOs can foster further improvement of this cutting-edge therapy, thereby enabling researchers to safely develop ASO drugs and enhancing their ability to innovate.
ABBREVIATIONS
2'-MOE, 2'-O-methoxyethyl; 2'PS, 2 modified PS; ADME, absorption, distribution, metabolism, and excretion; ASO, antisense oligonucleotide; AUC, area under the curve; BNA, bridged nucleic acid; CPP, cell-penetrating peptide; CMV, cytomegalovirus; CNS, central nervous system; CYP, cytochrome P; DDI, drug-drug interaction; DMD, Duchenne muscular dystrophy; FDA, Food and Drug Administration; GalNAc3, triantennary N-acetyl galactosamine; IT, intrathecal; IV, intravenous; LNA, locked nucleic acid; mRNA, messenger RNA; NA, not applicable; PBPK, physiologically based pharmacokinetics; PD, pharmacodynamic; PK, pharmacokinetic; PMO, phosphorodiamidate morpholino oligomer; PMOplus, PMOs with positionally specific positive molecular charges; PPMO, peptide-conjugated PMO; PS, phosphorothioate; SC, subcutaneous; siRNA, small-interfering RNA; SMA, spinal muscular atrophy.
Topics: Humans; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; Pharmaceutical Preparations; RNA
PubMed: 34643122
DOI: 10.1080/17425255.2021.1992382