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Nucleic Acids Research Apr 2023Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides... (Review)
Review
Eighteen nucleic acid therapeutics have been approved for treatment of various diseases in the last 25 years. Their modes of action include antisense oligonucleotides (ASOs), splice-switching oligonucleotides (SSOs), RNA interference (RNAi) and an RNA aptamer against a protein. Among the diseases targeted by this new class of drugs are homozygous familial hypercholesterolemia, spinal muscular atrophy, Duchenne muscular dystrophy, hereditary transthyretin-mediated amyloidosis, familial chylomicronemia syndrome, acute hepatic porphyria, and primary hyperoxaluria. Chemical modification of DNA and RNA was central to making drugs out of oligonucleotides. Oligonucleotide therapeutics brought to market thus far contain just a handful of first- and second-generation modifications, among them 2'-fluoro-RNA, 2'-O-methyl RNA and the phosphorothioates that were introduced over 50 years ago. Two other privileged chemistries are 2'-O-(2-methoxyethyl)-RNA (MOE) and the phosphorodiamidate morpholinos (PMO). Given their importance in imparting oligonucleotides with high target affinity, metabolic stability and favorable pharmacokinetic and -dynamic properties, this article provides a review of these chemistries and their use in nucleic acid therapeutics. Breakthroughs in lipid formulation and GalNAc conjugation of modified oligonucleotides have paved the way to efficient delivery and robust, long-lasting silencing of genes. This review provides an account of the state-of-the-art of targeted oligo delivery to hepatocytes.
Topics: Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; RNA; RNA Interference
PubMed: 36881759
DOI: 10.1093/nar/gkad067 -
American Journal of Human Genetics Oct 2020Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney...
Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.
Topics: Amphibian Proteins; Animals; Case-Control Studies; Child; Child, Preschool; DNA-Binding Proteins; Epigenesis, Genetic; Family; Female; Forkhead Transcription Factors; Heterozygote; Humans; Infant; Larva; Male; Mice; Mice, Knockout; Morpholinos; Mutation; Pedigree; Protein Binding; Repressor Proteins; Transcription Factors; Urinary Tract; Urogenital Abnormalities; Exome Sequencing; Xenopus
PubMed: 32891193
DOI: 10.1016/j.ajhg.2020.08.013 -
Journal of Neuromuscular Diseases 2021BackgroundEteplirsen received accelerated FDA approval for treatment of Duchenne muscular dystrophy (DMD) with mutations amenable to exon 51 skipping, based on...
BackgroundEteplirsen received accelerated FDA approval for treatment of Duchenne muscular dystrophy (DMD) with mutations amenable to exon 51 skipping, based on demonstrated dystrophin production.ObjectiveTo report results from PROMOVI, a phase 3, multicenter, open-label study evaluating efficacy and safety of eteplirsen in a larger cohort.MethodsAmbulatory patients aged 7-16 years, with confirmed mutations amenable to exon 51 skipping, received eteplirsen 30 mg/kg/week intravenously for 96 weeks. An untreated cohort with DMD not amenable to exon 51 skipping was also enrolled.Results78/79 eteplirsen-treated patients completed 96 weeks of treatment. 15/30 untreated patients completed the study; this cohort was considered an inappropriate control group because of genotype-driven differences in clinical trajectory. At Week 96, eteplirsen-treated patients showed increased exon skipping (18.7-fold) and dystrophin protein (7-fold) versus baseline. Post-hoc comparisons with patients from eteplirsen phase 2 studies (4658-201/202) and mutation-matched external natural history controls confirmed previous results, suggesting clinically notable attenuation of decline on the 6-minute walk test over 96 weeks (PROMOVI: -68.9 m; phase 2 studies: -67.3 m; external controls: -133.8 m) and significant attenuation of percent predicted forced vital capacity annual decline (PROMOVI: -3.3%, phase 2 studies: -2.2%, external controls: -6.0%; p < 0.001). Adverse events were generally mild to moderate and unrelated to eteplirsen. Most frequent treatment-related adverse events were headache and vomiting; none led to treatment discontinuation.ConclusionsThis large, multicenter study contributes to the growing body of evidence for eteplirsen, confirming a positive treatment effect, favorable safety profile, and slowing of disease progression versus natural history.
Topics: Adolescent; Child; Disease Progression; Dystrophin; Exons; Humans; Male; Morpholinos; Muscular Dystrophy, Duchenne; Mutation; Vital Capacity
PubMed: 34120909
DOI: 10.3233/JND-210643 -
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 -
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 -
Developmental Biology Oct 2022The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of...
The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.
Topics: Animals; CRISPR-Cas Systems; Gene Editing; Morpholinos; RNA, Guide, CRISPR-Cas Systems; Sea Urchins
PubMed: 35917936
DOI: 10.1016/j.ydbio.2022.07.008 -
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 -
European Journal of Cell Biology Jun 2023Spinal muscular atrophy (SMA), the most common genetic cause of infantile death, is caused by a mutation in the survival of motor neuron 1 gene (SMN1), leading to the... (Review)
Review
Spinal muscular atrophy (SMA), the most common genetic cause of infantile death, is caused by a mutation in the survival of motor neuron 1 gene (SMN1), leading to the death of motor neurons and progressive muscle weakness. SMN1 normally produces an essential protein called SMN. Although humans possess a paralogous gene called SMN2, ∼90% of the SMN it produces is non-functional. This is due to a mutation in SMN2 that causes the skipping of a required exon during splicing of the pre-mRNA. The first treatment for SMA, nusinersen (brand name Spinraza), was approved by the FDA in 2016 and by the EMU in 2017. Nusinersen is an antisense oligonucleotide-based therapy that alters the splicing of SMN2 to make functional full-length SMN protein. Despite the recent advancements in antisense oligonucleotide therapy and SMA treatment development, nusinersen is faced with a multitude of challenges, such as intracellular and systemic delivery. In recent years, the use of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) in antisense therapy has gained interest. These are antisense oligonucleotides conjugated to cell-penetrating peptides such as Pips and DG9, and they have the potential to address the challenges associated with delivery. This review focuses on the historic milestones, development, current challenges, and future perspectives of antisense therapy for SMA.
Topics: Humans; Oligonucleotides, Antisense; Muscular Atrophy, Spinal; Morpholinos; Motor Neurons; RNA Splicing
PubMed: 37295266
DOI: 10.1016/j.ejcb.2023.151326 -
Indian Pediatrics Feb 2020Synthetic Antisense oligonucleotides (ASOs) are novel and efficient laboratory tools to regulate the expression of specific genes, and have only recently come into... (Review)
Review
Synthetic Antisense oligonucleotides (ASOs) are novel and efficient laboratory tools to regulate the expression of specific genes, and have only recently come into clinical use. These are synthetic single-stranded DNA analogs, whose sequence is complementary to a target nucleotide and alter protein synthesis by several mechanisms. We herein provide a primer on the topic for pediatricians, as this group of drugs is likely to see many more drugs for previously incurable diseases.
Topics: Adolescent; Child; Child, Preschool; Humans; Infant; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Oligonucleotides; Oligonucleotides, Antisense; Randomized Controlled Trials as Topic
PubMed: 32060244
DOI: No ID Found -
Journal of the American Society of... Jun 2018Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex...
Podocyte loss and effacement of interdigitating podocyte foot processes are the major cause of a leaky filtration barrier and ESRD. Because the complex three-dimensional morphology of podocytes depends on the actin cytoskeleton, we studied the role in podocytes of the actin bundling protein palladin, which is highly expressed therein. We knocked down palladin in cultured podocytes by siRNA transfection or in zebrafish embryos by morpholino injection and studied the effects by immunofluorescence and live imaging. We also investigated kidneys of mice with podocyte-specific knockout of palladin (PodoPalld-/- mice) by immunofluorescence and ultrastructural analysis and kidney biopsy specimens from patients by immunostaining for palladin. Compared with control-treated podocytes, palladin-knockdown podocytes had reduced actin filament staining, smaller focal adhesions, and downregulation of the podocyte-specific proteins synaptopodin and -actinin-4. Furthermore, palladin-knockdown podocytes were more susceptible to disruption of the actin cytoskeleton with cytochalasin D, latrunculin A, or jasplakinolide and showed altered migration dynamics. In zebrafish embryos, palladin knockdown compromised the morphology and dynamics of epithelial cells at an early developmental stage. Compared with PodoPalld+/+ controls, PodoPalld-/- mice developed glomeruli with a disturbed morphology, an enlarged subpodocyte space, mild effacement, and significantly reduced expression of nephrin and vinculin. Furthermore, nephrotoxic serum injection led to significantly higher levels of proteinuria in PodoPalld-/- mice than in controls. Kidney biopsy specimens from patients with diabetic nephropathy and FSGS showed downregulation of palladin in podocytes as well. Palladin has an important role in podocyte function and .
Topics: Actins; Animals; Cytoskeletal Proteins; Cytoskeleton; Female; Focal Adhesions; Gene Expression; Gene Silencing; Humans; Kidney Glomerulus; Male; Mice, Knockout; Microfilament Proteins; Morpholinos; Phosphoproteins; Podocytes; RNA, Messenger; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 29720549
DOI: 10.1681/ASN.2017091039