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Methods in Molecular Biology (Clifton,... 2021The zebrafish has emerged as a valuable and important model organism for studying vascular development and vascular biology. Here, we discuss some of the approaches used...
The zebrafish has emerged as a valuable and important model organism for studying vascular development and vascular biology. Here, we discuss some of the approaches used to study vessels in fish, including loss-of-function tools such as morpholinos and genetic mutants, along with methods and considerations for assessing vascular phenotypes. We also provide detailed protocols for methods used for vital imaging of the zebrafish vasculature, including microangiography and long-term time-lapse imaging. The methods we describe, and the considerations we suggest using for assessing phenotypes observed using these methods, will help ensure reliable, valid conclusions when assessing vascular phenotypes following genetic or experimental manipulation of zebrafish.
Topics: Angiography; Animals; Blood Vessels; Morpholinos; Neovascularization, Physiologic; Phenotype; Zebrafish; Zebrafish Proteins
PubMed: 32754820
DOI: 10.1007/978-1-0716-0916-3_15 -
Nucleic Acid Therapeutics Jun 2018RNA interference (RNAi) is a fundamental cellular process for the posttranscriptional regulation of gene expression. RNAi can exogenously be modulated by small RNA... (Review)
Review
RNA interference (RNAi) is a fundamental cellular process for the posttranscriptional regulation of gene expression. RNAi can exogenously be modulated by small RNA oligonucleotides, such as microRNAs (miRNAs) and small interfering RNAs (siRNAs), or by antisense oligonucleotides. These small oligonucleotides provided the scientific community with powerful and versatile tools to turn off the expression of genes of interest, and hold out the promise of new therapeutic solutions against a wide range of gene-associated pathologies. However, unmodified nucleic acids are highly instable in biological systems, and their weak interaction with plasma proteins confers an unfavorable pharmacokinetics. In this review, we first provide an overview of the most efficient chemical strategies that, over the past 30 years, have been used to significantly improve the therapeutic potential of oligonucleotides. Oligonucleotides targeting and delivery technologies are then presented, including covalent conjugates between oligonucleotides and targeting ligand, and noncovalent association with lipid or polymer nanoparticles. Finally, we specifically focus on the endosomal escape step, which represents a major stumbling block for the effective use of oligonucleotides as therapeutic agents. The need for approaches to quantitatively measure endosomal escape and cytosolic arrival of biomolecules is discussed in the context of the development of efficient oligonucleotide targeting and delivery vectors.
Topics: Amyloidosis; Animals; Biological Transport; Cell-Penetrating Peptides; Cytosol; Endosomes; Gene Transfer Techniques; Genetic Therapy; Humans; Lipids; MicroRNAs; Morpholinos; Nanoparticles; Neoplasms; Oligonucleotides; Oligonucleotides, Antisense; Peptide Nucleic Acids; Phosphorothioate Oligonucleotides; RNA, Small Interfering
PubMed: 29883296
DOI: 10.1089/nat.2017.0716 -
Journal of Visualized Experiments : JoVE Aug 2022The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have...
The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.
Topics: Animals; Embryo, Nonmammalian; Gene Knockdown Techniques; Morpholinos; Oligonucleotides, Antisense; Phenotype; RNA, Messenger; Zebrafish; Zebrafish Proteins
PubMed: 36036621
DOI: 10.3791/63324 -
Journal of the American Chemical Society Sep 2022MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate...
MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradiation, precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.
Topics: Animals; Embryo, Nonmammalian; MicroRNAs; Morpholinos; Oligonucleotides, Antisense; Zebrafish; Zebrafish Proteins
PubMed: 36073798
DOI: 10.1021/jacs.2c04479 -
Nature Biotechnology Nov 2016
Topics: Clinical Trials as Topic; Decision Making; Drug Approval; Humans; Morpholinos; Muscular Dystrophy, Duchenne; United States; United States Food and Drug Administration
PubMed: 27824847
DOI: 10.1038/nbt.3733 -
Nature Medicine Nov 2016
Topics: Data Accuracy; Drug Approval; Evidence-Based Medicine; Humans; Morpholinos; Muscular Dystrophy, Duchenne; United States; United States Food and Drug Administration
PubMed: 27824816
DOI: 10.1038/nm.4234 -
Methods in Molecular Biology (Clifton,... 2023Viltolarsen is a phosphorodiamidate morpholino antisense oligonucleotide (PMO) designed to skip exon 53 of the DMD gene for the treatment of Duchenne muscular dystrophy...
Viltolarsen is a phosphorodiamidate morpholino antisense oligonucleotide (PMO) designed to skip exon 53 of the DMD gene for the treatment of Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders characterized by progressive degeneration of skeletal muscles and cardiomyopathy. It was developed by Nippon Shinyaku in collaboration with the National Center of Neurology and Psychiatry (NCNP) in Japan based on the preclinical studies conducted in the DMD dog model at the NCNP. After showing hopeful results in pre-clinical trials and several clinical trials across North America and Japan, it received US Food and Drug Administration (FDA) approval for DMD in 2020. Viltolarsen restores the reading frame of the DMD gene by skipping exon 53 and produces a truncated but functional form of dystrophin. It can treat approximately 8-10% of the DMD patient population. This paper aims to summarize the development of viltolarsen from preclinical trials to clinical trials to, finally, FDA approval, and discusses the challenges that come with fighting DMD using antisense therapy.
Topics: United States; Humans; Dogs; Animals; United States Food and Drug Administration; Oligonucleotides; Morpholinos; Muscular Dystrophy, Duchenne
PubMed: 36401022
DOI: 10.1007/978-1-0716-2772-3_2 -
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 -
Methods in Molecular Biology (Clifton,... 2017Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular...
Morpholino oligomers are effective antisense molecules to regulate gene expression and the US FDA has approved a Morpholino drug for the treatment of Duchenne muscular dystrophy. However, it has been observed that the antisense activities of aqueous solutions of some Morpholinos decrease over time. We hypothesize that the decreased activity is caused by the formation of soluble aggregates of the Morpholinos. Here, we analyzed three Morpholino sequences by size exclusion chromatography and found two of them have over time formed soluble aggregates in water. The degree of aggregation is sequence-, temperature-, and time-dependent. We describe a simple procedure for detecting and breaking down the aggregates to return the Morpholinos to their monomeric forms.
Topics: Base Pairing; Base Sequence; Chromatography, High Pressure Liquid; Morpholinos; Oligonucleotides, Antisense; Solubility; Solutions
PubMed: 28364231
DOI: 10.1007/978-1-4939-6817-6_3 -
Expert Opinion on Therapeutic Targets Dec 2018Gene regulation is the term used to describe the mechanisms by which a cell increases or decreases the amount of a gene product (RNA or protein). In complex organs such... (Review)
Review
Gene regulation is the term used to describe the mechanisms by which a cell increases or decreases the amount of a gene product (RNA or protein). In complex organs such as the brain, gene regulation is of the utmost importance; aberrations in the regulation of specific genes can lead to neurological disorders. Understanding these mechanisms can create new strategies for targeting these disorders and progress is being made. Two drugs that function at the RNA level (nusinersen and eteplirsen) have now been approved by the FDA for the treatment of Spinomuscular atrophy and Duchenne muscular dystrophy, respectively; several other compounds for neurological disease are currently being investigated in preclinical studies and clinical trials. Areas covered: We highlight how gene regulation at the level of RNA molecules can be used as a therapeutic strategy to treat neurological disorders. We provide examples of how such an approach is being studied or used and discuss the current hurdles. Expert opinion: Targeting gene expression at the RNA level is a promising strategy to treat genetic neurological disorders. Safe administration, long-term efficacy, and potential side effects, however, still need careful evaluation before RNA therapeutics can be applied on a larger scale.
Topics: Animals; Drug Development; Gene Expression Regulation; Humans; Morpholinos; Nervous System Diseases; Oligonucleotides; RNA
PubMed: 30372655
DOI: 10.1080/14728222.2018.1542429