-
Methods in Molecular Biology (Clifton,... 2017Noncoding RNAs have essential biochemical functions in different areas of cellular metabolism, including protein synthesis, RNA splicing, protein secretion, and DNA...
Noncoding RNAs have essential biochemical functions in different areas of cellular metabolism, including protein synthesis, RNA splicing, protein secretion, and DNA replication. We have successfully used Morpholino antisense oligonucleotides for the functional inactivation of small noncoding RNAs required for DNA replication (Y RNAs in vertebrates and stem-bulge RNAs in nematodes). Here we discuss specific issues of targeting functional noncoding RNAs for inactivation by Morpholino antisense oligonucleotides. We present protocols for the design, preparation, and efficacy controls of Morpholino antisense oligonucleotides, as well as brief descriptions for their delivery into vertebrate and nematode embryos.
Topics: Animals; Gene Expression Regulation; Gene Knockdown Techniques; Gene Targeting; Gene Transfer Techniques; Morpholinos; Oligonucleotides, Antisense; Phenotype; RNA, Untranslated; Statistics as Topic
PubMed: 28364241
DOI: 10.1007/978-1-4939-6817-6_13 -
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 -
Annual Review of Pharmacology and... Jan 2019Oligonucleotides (ONs) can interfere with biomolecules representing the entire extended central dogma. Antisense gapmer, steric block, splice-switching ONs, and short... (Review)
Review
Oligonucleotides (ONs) can interfere with biomolecules representing the entire extended central dogma. Antisense gapmer, steric block, splice-switching ONs, and short interfering RNA drugs have been successfully developed. Moreover, antagomirs (antimicroRNAs), microRNA mimics, aptamers, DNA decoys, DNAzymes, synthetic guide strands for CRISPR/Cas, and innate immunity-stimulating ONs are all in clinical trials. DNA-targeting, triplex-forming ONs and strand-invading ONs have made their mark on drug development research, but not yet as medicines. Both design and synthetic nucleic acid chemistry are crucial for achieving biologically active ONs. The dominating modifications are phosphorothioate linkages, base methylation, and numerous 2'-substitutions in the furanose ring, such as 2'-fluoro, O-methyl, or methoxyethyl. Locked nucleic acid and constrained ethyl, a related variant, are bridged forms where the 2'-oxygen connects to the 4'-carbon in the sugar. Phosphorodiamidate morpholino oligomers, carrying a modified heterocyclic backbone ring, have also been commercialized. Delivery remains a major obstacle, but systemic administration and intrathecal infusion are used for treatment of the liver and brain, respectively.
Topics: Clinical Trials as Topic; Humans; Morpholinos; Nucleic Acids; Oligonucleotides; Oligonucleotides, Antisense
PubMed: 30285540
DOI: 10.1146/annurev-pharmtox-010818-021050 -
Methods in Molecular Biology (Clifton,... 2022Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and... (Review)
Review
Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are β-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.
Topics: Animals; Galectins; Gene Knockdown Techniques; Mammals; Morpholinos; RNA; Zebrafish
PubMed: 35320539
DOI: 10.1007/978-1-0716-2055-7_23 -
The Lancet. Child & Adolescent Health Aug 2018Inherited neuromuscular disorders encompass a broad group of genetic conditions, and the discovery of these underlying genes has expanded greatly in the past three... (Review)
Review
Inherited neuromuscular disorders encompass a broad group of genetic conditions, and the discovery of these underlying genes has expanded greatly in the past three decades. The discovery of such genes has enabled more precise diagnosis of these disorders and the development of specific therapeutic approaches that target the genetic basis and pathophysiological pathways. Such translational research has led to the approval of two genetic therapies by the US Food and Drug Administration: eteplirsen for Duchenne muscular dystrophy and nusinersen for spinal muscular atrophy, which are both antisense oligonucleotides that modify pre-mRNA splicing. In this Review we aim to discuss new genetic therapies and ongoing clinical trials for Duchenne muscular dystrophy, spinal muscular atrophy, and other less common childhood neuromuscular disorders.
Topics: Child; Genetic Therapy; Humans; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Neuromuscular Diseases; Oligonucleotides
PubMed: 30119719
DOI: 10.1016/S2352-4642(18)30140-8 -
Nanomedicine (London, England) May 2016Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering... (Review)
Review
Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics.
Topics: Animals; Diagnostic Imaging; Drug Carriers; Drug Delivery Systems; Humans; MicroRNAs; Morpholinos; Nanoparticles; Neoplasms; Oligonucleotides; RNA, Small Interfering; Theranostic Nanomedicine
PubMed: 27102380
DOI: 10.2217/nnm-2016-0035 -
Journal of Microbiological Methods Jul 2017This review covers a brief history of antisense RNAs and its applications, and summarizes the current stage of antisense technologies used in Toxoplasma gondii, a... (Review)
Review
This review covers a brief history of antisense RNAs and its applications, and summarizes the current stage of antisense technologies used in Toxoplasma gondii, a fascinating model organism with a unique characteristic blend of genetic regulatory systems normally found in plants or animals. Based on the current knowledge of regulatory RNAs and non-coding RNA (ncRNA), the antisense technologies are reviewed according to the classification of ncRNAs, which are roughly categorized into small, ranging from ~20-200 nucleotides in length, and long >200 nucleotides. Techniques utilizing small regulatory RNAs such as siRNA, miRNA, antagomirs, ribozymes and morpholino oligomers are discussed along with long non-coding RNA (lncRNA) including antisense and double stranded. These antisense technologies can be used in forward and reverse genetics studies. The future of technologies is limitless, particularly by combining these technologies with conventional methods, and should allow for ever greater understanding of gene regulation of the organism and related pathogenic microorganisms.
Topics: Antagomirs; MicroRNAs; Morpholinos; Oligoribonucleotides, Antisense; RNA, Catalytic; RNA, Small Interfering; Toxoplasma; Toxoplasmosis
PubMed: 26724749
DOI: 10.1016/j.mimet.2015.12.013 -
The Journal of Biological Chemistry Jul 2023Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature...
Functional depletion of the U1 small nuclear ribonucleoprotein (snRNP) with a 25 nt U1 AMO (antisense morpholino oligonucleotide) may lead to intronic premature cleavage and polyadenylation of thousands of genes, a phenomenon known as U1 snRNP telescripting; however, the underlying mechanism remains elusive. In this study, we demonstrated that U1 AMO could disrupt U1 snRNP structure both in vitro and in vivo, thereby affecting the U1 snRNP-RNAP polymerase II interaction. By performing chromatin immunoprecipitation sequencing for phosphorylation of Ser2 and Ser5 of the C-terminal domain of RPB1, the largest subunit of RNAP polymerase II, we showed that transcription elongation was disturbed upon U1 AMO treatment, with a particular high phosphorylation of Ser2 signal at intronic cryptic polyadenylation sites (PASs). In addition, we showed that core 3'processing factors CPSF/CstF are involved in the processing of intronic cryptic PAS. Their recruitment accumulated toward cryptic PASs upon U1 AMO treatment, as indicated by chromatin immunoprecipitation sequencing and individual-nucleotide resolution CrossLinking and ImmunoPrecipitation sequencing analysis. Conclusively, our data suggest that disruption of U1 snRNP structure mediated by U1 AMO provides a key for understanding the U1 telescripting mechanism.
Topics: Morpholinos; Oligonucleotides, Antisense; Polyadenylation; Ribonucleoprotein, U1 Small Nuclear; RNA Polymerase II; RNA Precursors; Humans; HeLa Cells; Gene Knockdown Techniques; Cleavage And Polyadenylation Specificity Factor; Cleavage Stimulation Factor; Transcription, Genetic
PubMed: 37224962
DOI: 10.1016/j.jbc.2023.104854 -
Human Molecular Genetics Aug 2018Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by progressive muscle wasting that has currently no cure. Exon-skipping strategy represents... (Review)
Review
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder characterized by progressive muscle wasting that has currently no cure. Exon-skipping strategy represents one of the most promising therapeutic approaches that aim to restore expression of a shorter but functional dystrophin protein. The antisense field has remarkably progress over the last years with recent accelerated approval of the first antisense oligonucleotide-based therapy for DMD, Exondys 51, though the therapeutic benefit remains to be proved in patients. Despite clinical advances, the poor effective delivery to target all muscle remains the main hurdle for antisense drug therapy. This review describes the antisense-based exon-skipping approach for DMD, from proof-of-concept to first marketed drug. We discuss the main obstacles to achieve a successful exon-skipping therapy and the latest advances of the international community to develop more powerful chemistries and more sophisticated delivery systems in order to increase potency, bioavailability and safety. Finally, we highlight the importance of collaborative efforts and early dialogue between drug developers and regulatory agencies in order to overcome difficulties, find appropriate outcome markers and collect useful data.
Topics: Alternative Splicing; Animals; Dystrophin; Exons; Genetic Therapy; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; RNA Splicing; RNA, Antisense
PubMed: 29771317
DOI: 10.1093/hmg/ddy171 -
Medecine Sciences : M/S Nov 2016
Topics: Drug Approval; Exons; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Paris; United States; United States Food and Drug Administration
PubMed: 27869079
DOI: 10.1051/medsci/201632s217