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Antimicrobial Agents and Chemotherapy Nov 2014Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate... (Randomized Controlled Trial)
Randomized Controlled Trial
Safety and pharmacokinetic profiles of phosphorodiamidate morpholino oligomers with activity against ebola virus and marburg virus: results of two single-ascending-dose studies.
Two identical single-ascending-dose studies evaluated the safety and pharmacokinetics (PK) of AVI-6002 and AVI-6003, two experimental combinations of phosphorodiamidate morpholino oligomers with positive charges (PMOplus) that target viral mRNA encoding Ebola virus and Marburg virus proteins, respectively. Both AVI-6002 and AVI-6003 were found to suppress disease in virus-infected nonhuman primates in previous studies. AVI-6002 (a combination of AVI-7537 and AVI-7539) or AVI-6003 (a combination of AVI-7287 and AVI-7288) were administered as sequential intravenous (i.v.) infusions of a 1:1 fixed dose ratio of the two subcomponents. In each study, 30 healthy male and female subjects between 18 and 50 years of age were enrolled in six-dose escalation cohorts of five subjects each and received a single i.v. infusion of active study drug (0.005, 0.05, 0.5, 1.5, 3, and 4.5 mg/kg per component) or placebo in a 4:1 ratio. Both AVI-6002 and AVI-6003 were safe and well tolerated at the doses studied. A maximum tolerated dose was not observed in either study. The four chemically similar PMOplus components exhibited generally similar PK profiles. The mean peak plasma concentration and area under the concentration-time curve values of the four components exhibited dose-proportional PK. The estimated plasma half-life of all four components was 2 to 5 h. The safety of the two combinations and the PK of the four components were similar, regardless of the target RNA sequence.
Topics: Adult; Animals; Area Under Curve; Double-Blind Method; Ebolavirus; Female; Hemorrhagic Fever, Ebola; Humans; Infusions, Intravenous; Male; Marburg Virus Disease; Marburgvirus; Middle Aged; Morpholinos; Placebos; Young Adult
PubMed: 25155593
DOI: 10.1128/AAC.03442-14 -
Methods in Molecular Biology (Clifton,... 2015Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role(s) of a gene product. Because of its genetic tractability and...
Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role(s) 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), which comprise 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; Base Sequence; Embryo, Nonmammalian; Female; Galectins; Gene Expression Regulation; Gene Knockdown Techniques; Gene Knockout Techniques; Injections; Male; Morpholinos; Phenotype; RNA; Zebrafish
PubMed: 25253151
DOI: 10.1007/978-1-4939-1396-1_22 -
BMC Developmental Biology Mar 2018Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are...
BACKGROUND
Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in embryogenesis remain to be fully understood.
RESULTS
Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the 1-cell stage for several hours before dying by 10 h post fertilization. We demonstrate that dusp6 is expressed in gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults.
CONCLUSIONS
The fact that offspring of homozygous dusp6 mutants stall prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis and/or parentally-directed early development. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for proper embryogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other regulators of the ERK pathway compensate for loss of dusp6 at later stages.
Topics: Alleles; Animals; Base Sequence; CRISPR-Cas Systems; Cell Division; Dual Specificity Phosphatase 6; Embryo, Nonmammalian; Embryonic Development; Female; Gastrulation; Gene Editing; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; Germ Cells; Homozygote; Male; Morpholinos; Mutation; Ovary; Phenotype; Rhombencephalon; Testis; Zebrafish; Zebrafish Proteins
PubMed: 29544468
DOI: 10.1186/s12861-018-0164-6 -
PloS One 2014Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways....
Secreted molecules of the Wnt family regulate key decisions in embryogenesis and adult tissue homeostasis by activating a complex network of Wnt signaling pathways. Although the different branches of Wnt signaling have been studied for more than 25 years, fluorophore tagged constructs for live cell imaging of Wnt molecules activating the Wnt/β-catenin pathway have become available only recently. We have generated a fluorophore tagged Wnt construct of the Xenopus Wnt5a protein (Xwnt5A) with the enhanced green fluorescent protein (EGFP), Xwnt5A-EGFP. This construct activates non-canonical Wnt pathways in an endocytosis dependent manner and is capable of compensating for the loss of endogenous Xwnt5A in Xenopus embryos. Strikingly, non-canonical Wnt pathway activation was restricted to short-range signaling while an inhibitory effect was observed in transwell cell cultures taken as long-range signaling model sytem. We used our Xwnt5A-EGFP construct to analyze in vivo binding of Wnt5A to its co-receptor ROR2 on the microscopic and on the molecular level. On the microscopic level, Xwnt5A-EGFP clusters in the membrane and recruits ROR2-mCherry to these clusters. Applying dual-colour dual-focus line-scanning fluorescence correlation spectroscopy on dorsal marginal zone explants, we identified membrane tethered Xwnt5A-EGFP molecules binding to ROR2-mCherry molecules. Our data favour a model, in which membrane-tethered Wnt-5A recruits ROR2 to form large ligand/receptor clusters and signals in an endocytosis-dependent manner.
Topics: Activating Transcription Factor 2; Animals; Embryo, Nonmammalian; Embryonic Development; Genes, Reporter; Green Fluorescent Proteins; Luminescent Proteins; Microscopy, Confocal; Morpholinos; RNA, Messenger; Receptor Tyrosine Kinase-like Orphan Receptors; Signal Transduction; Wnt Proteins; Wnt-5a Protein; Xenopus; Xenopus Proteins; Red Fluorescent Protein
PubMed: 25313906
DOI: 10.1371/journal.pone.0109428 -
Nature Communications Jul 2021Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense...
Rapid development of antisense therapies can enable on-demand responses to new viral pathogens and make personalized medicine for genetic diseases practical. Antisense phosphorodiamidate morpholino oligomers (PMOs) are promising candidates to fill such a role, but their challenging synthesis limits their widespread application. To rapidly prototype potential PMO drug candidates, we report a fully automated flow-based oligonucleotide synthesizer. Our optimized synthesis platform reduces coupling times by up to 22-fold compared to previously reported methods. We demonstrate the power of our automated technology with the synthesis of milligram quantities of three candidate therapeutic PMO sequences for an unserved class of Duchenne muscular dystrophy (DMD). To further test our platform, we synthesize a PMO that targets the genomic mRNA of SARS-CoV-2 and demonstrate its antiviral effects. This platform could find broad application not only in designing new SARS-CoV-2 and DMD antisense therapeutics, but also for rapid development of PMO candidates to treat new and emerging diseases.
Topics: Animals; COVID-19; Chemistry Techniques, Synthetic; Chemistry, Pharmaceutical; Chlorocebus aethiops; Communicable Diseases, Emerging; Disease Models, Animal; High-Throughput Screening Assays; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides, Antisense; Precision Medicine; RNA, Messenger; RNA, Viral; SARS-CoV-2; Time Factors; Vero Cells; COVID-19 Drug Treatment
PubMed: 34285203
DOI: 10.1038/s41467-021-24598-4 -
Scientific Reports Jan 2021LIM homeobox 4 (LHX4) is expressed in the photoreceptors (PRs) of the outer nuclear layer (ONL) and bipolar cells (BCs) of the inner nuclear layer (INL) in mouse and...
LIM homeobox 4 (LHX4) is expressed in the photoreceptors (PRs) of the outer nuclear layer (ONL) and bipolar cells (BCs) of the inner nuclear layer (INL) in mouse and chicken retina. It regulates the subtype-specific development of rod BCs and cone BCs in the mouse retina. However, no report has been published on its expression and function in the zebrafish retina. In this study, we assessed the expression of Lhx4 using in situ hybridization (ISH) technique and explored its role in zebrafish (Danio rerio) retinal development via morpholino (MO) technology. We found that the expression of lhx4 in the zebrafish retina begins 48 h post-fertilization (hpf) and is continuously expressed in the ONL and INL. A zebrafish model constructed with lhx4 knockdown in the eyes through vivo-MO revealed that: lhx4 knockdown inhibits the differentiation of Parvalbumin amacrine cells (ACs) and Rhodopsin rod photoreceptors (RPs), enhances the expression of visual system homeobox 2 (vsx2); and damages the responses of zebrafish to light stimulus, without affecting the differentiation of OFF-BCs and rod BCs, and apoptosis in the retina. These findings reveal that lhx4 regulates neural differentiation in the retina and visual function during zebrafish embryonic development.
Topics: Amacrine Cells; Animals; Cell Differentiation; Embryonic Development; Gene Knockdown Techniques; Humans; In Situ Hybridization; LIM-Homeodomain Proteins; Mice; Morpholinos; Neurons; Retina; Retinal Bipolar Cells; Retinal Cone Photoreceptor Cells; Retinal Rod Photoreceptor Cells; Rhodopsin; Transcription Factors; Vision, Ocular; Zebrafish; Zebrafish Proteins
PubMed: 33479361
DOI: 10.1038/s41598-021-81211-w -
Scientific Reports Nov 2020We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense...
We recently developed a piggyback knockdown method that was used to knockdown genes in adult zebrafish. In this method, a vivo morpholino (VMO) piggybacks an antisense deoxyoligonucleotide (dO) into the somatic cells and reduces the cognate mRNA levels. In this paper, we tested whether we can piggyback more than one dO with one VMO. We designed various hybrids that had more than one dO that could be piggybacked with one VMO. We chose f7, f8, and αIIb genes and tested their knockdown by the appropriate assays. The knockdown with piggybacking either two or three dOs by one VMO yielded > 85% knockdown efficiency. We also performed knockdown of argonautes and rnaseh separately along with f7. We found the knockdown of f7 occurs when knockdown of argonautes happens and not when rnaseh knockdown was performed, suggesting that RNaseH is involved in mRNA degradation. In conclusion, we developed a method where we could knockdown three genes at one time, and by increasing the concentration of VMO by twofold, we could knockdown six genes simultaneously. These multiple gene knockdowns will not only increase the efficiency of the method in whole genome-wide knockdowns but will also be useful to study multifactorial disorders.
Topics: Animals; Embryo, Nonmammalian; Gene Knockdown Techniques; Morpholinos; Oligonucleotides, Antisense; Zebrafish; Zebrafish Proteins
PubMed: 33214638
DOI: 10.1038/s41598-020-76655-5 -
Nucleic Acid Therapeutics Jun 2021Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases....
Chitosan-Shelled Nanobubbles Irreversibly Encapsulate Morpholino Conjugate Antisense Oligonucleotides and Are Ineffective for Phosphorodiamidate Morpholino-Mediated Gene Silencing of .
Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model. cell-free analysis demonstrated a good loading capacity of PMO into NBs, while experiments in cell cultures showed lack of therapeutic effect since expression of DUX4 and its targets remained unmodified. We conclude that these types of chitosan-shelled NBs do not release PMO-AON and are therefore not ideal for PMO AON-related therapies.
Topics: Chitosan; Gene Silencing; Morpholinos; Oligonucleotides, Antisense
PubMed: 32679000
DOI: 10.1089/nat.2020.0862 -
Scientific Reports Feb 2021CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders....
CYP3A5 is the primary CYP3A subfamily enzyme expressed in the human kidney and its aberrant expression may contribute to a broad spectrum of renal disorders. Pharmacogenetic studies have reported inconsistent linkages between CYP3A5 expression and hypertension, however, most investigators have considered CYP3A5*1 as active and CYP3A5*3 as an inactive allele. Observations of gender specific differences in CYP3A5*3/*3 protein expression suggest additional complexity in gene regulation that may underpin an environmentally responsive role for CYP3A5 in renal function. Reconciliation of the molecular mechanism driving conditional restoration of functional CYP3A5*3 expression from alternatively spliced transcripts, and validation of a morpholino-based approach for selectively suppressing renal CYP3A5 expression, is the focus of this work. Morpholinos targeting a cryptic splice acceptor created by the CYP3A5*3 mutation in intron 3 rescued functional CYP3A5 expression in vitro, and salt-sensitive cellular mechanisms regulating splicing and conditional expression of CYP3A5*3 transcripts are reported. The potential for a G-quadruplex (G4) in intron 3 to mediate restored splicing to exon 4 in CYP3A5*3 transcripts was also investigated. Finally, a proximal tubule microphysiological system (PT-MPS) was used to evaluate the safety profile of morpholinos in proximal tubule epithelial cells, highlighting their potential as a therapeutic platform for the treatment of renal disease.
Topics: Cell Line; Cytochrome P-450 CYP3A; Drug Discovery; G-Quadruplexes; HEK293 Cells; Humans; Kidney Diseases; Morpholinos; Mutation; Oligonucleotides, Antisense
PubMed: 33633318
DOI: 10.1038/s41598-021-84194-w -
Methods in Molecular Biology (Clifton,... 2022Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and...
Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.
Topics: Anthrax; Antigens, Bacterial; Bacterial Toxins; Escherichia coli; Humans; Morpholinos; Oligonucleotides, Antisense
PubMed: 35213014
DOI: 10.1007/978-1-0716-2010-6_8