Search

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.

Purpose Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) have shown promise in treating Duchenne muscular dystrophy (DMD). We evaluated a semi-mechanistic pharmacokinetic (PK) and pharmacodynamic (PD) model to capture the relationship between plasma and muscle tissue exposure/response in mdx mice treated by mouse surrogate PPMO. Methods A single or repeated (every 4 weeks for 20 weeks) intravenous PPMO dose was administered to mdx mice (n = 6/timepoint). A PK/PD model was built to characterize data via sequential modeling. A 2-compartment model was used to describe plasma PK. A simultaneous tissue PK/PD model was subsequently developed: 2-compartment model to describe muscle PK; linked to an indirect response model describing stimulation of synthesis of skipped transcript, which was in turn linked to stimulation of synthesis of dystrophin protein expression. Results Model performance assessment via goodness-of-fit plots, visual predictive checks, and accurate parameter estimation indicated robust fits of plasma PK and muscle PK/PD data. The model estimated a PPMO tissue half-life of 5 days—a useful parameter in determining the longevity of PPMOs in tissue and their limited accumulation after multiple doses. Additionally, the model successfully described dystrophin expression after single dosing and associated protein accumulation after multiple dosing (increasing ~ twofold accumulation from the first to last dose). Conclusions This first PK/PD model of a PPMO in a DMD disease model will help characterize and predict the time course of PK/PD biomarkers in mdx mice. Furthermore, the model framework can be used to develop clinical PK/PD models and can be extended to other exon-skipping therapies and species.

Zinc biology, featuring intertwining signaling networks and of critical importance to human health, witnesses exciting opportunities in the big data era of physiology. Here, we report a class of red- and far-red-emitting Zn 2+ probes with K d values ranging from 190 nM to 74 μM, which are particularly suitable for real-time monitoring the high concentration of Zn 2+ /insulin co-released during vesicular secretory events. Compared to the prototypical rhodamine-based Zn 2+ probes, the new class exploits morpholino auxochromes which eliminates phototoxicity during long-term live recording from isolated islets. A Si-rhodamine-based Zn 2+ probe with high turn-on ratio (> 100), whose synthesis was enabled by a new route featuring late-stage N-alkylation, allowed simultaneous recording of Ca 2+ influx, mitochondrial signal, and insulin secretion in isolated mouse islets. The time-lapse multicolor fluorescence movies and their analysis, enabled by red-shifted Zn 2+ and other orthogonal physiological probes, highlight the potential impact of biocompatible fluorophores on the fields of islet endocrinology and system biology.

Freshwater fish live in environments where pH levels fluctuate more than those in seawater. During acidic stress, the acid–base balance in these fish is regulated by ionocytes in the gills, which directly contact water and function as an external kidney. In ionocytes, apical acid secretion is largely mediated by H+-ATPase and the sodium/hydrogen exchanger (NHE). Control of this system was previously proposed to depend on the hormone, cortisol, mostly based on studies of zebrafish, a stenohaline fish, which utilize H+-ATPase as the main route for apical acid secretion. However, the role of cortisol is poorly understood in euryhaline fish species that preferentially use NHE as the main transporter. In the present study, we explored the role of cortisol in NHE-mediated acid secretion in medaka larvae. mRNA expression levels of transporters related to acid secretion and cortisol-synthesis enzyme were enhanced by acidic FW treatment (pH 4.5, 2 days) in medaka larvae. Moreover, exogenous cortisol treatment (25 mg/L, 2 days) resulted in upregulation of nhe3 and rhcg1 expression, as well as acid secretion in 7 dpf medaka larvae. In loss-of-function experiments, microinjection of glucocorticoid receptor (GR)2 morpholino (MO) caused reductions in nhe3 and rhcg1 expression and diminished acid secretion, but microinjection of mineralocorticoid receptor (MR) and GR1 MOs did not. Together, these results suggest a conserved action of cortisol and GR2 on fish body fluid acid–base regulation.

A morpholine-based nucleotide analog was developed as a building block for hepatic siRNA targeting and stabilization. Attachment of an asialoglycoprotein-binding GalNAc ligand at the morpholine nitrogen was realized with different linkers. The obtained morpholino GalNAc scaffolds were coupled to the sense strand of a transthyretin-targeting siRNA and tested for their knockdown potency in vitro and in vivo. A clear structure-activity relationship was developed with regard to the linker type and length as well as the attachment site of the morpholino GalNAc moieties at the siRNA sense strand. Further, simple alkylation of the morpholine nitrogen led to a nucleotide analog, which increased siRNA stability, when used as a double 3'-overhang at the sense strand sequence. Combination of the best morpholino GalNAc building blocks as targeting nucleotides with an optimized stabilizing alkyl-substituted morpholine as 3'-overhangs resulted in siRNAs without any phosphorothioate stabilization in the sense strand and clearly improved the duration of action in vivo.

Introduction RNA-binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (muscleblind-like) is sequestered by toxic CUG repeats, leading to missplicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Herein we sought to evaluate the functional role of the splicing factor hnRNP L in normal and DM1 muscle cells. Methods Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and splicing profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. In human myoblasts downregulation of hnRNP L was achieved with shRNAi. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated. Results Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued missplicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells. Discussion Together, these studies revealed that hnRNP L can modulate DM1 pathologies and is a potential therapeutic target.

The synthesis of novel 6- chloro/morpholino/amino/-9-sulfonylpurine derivatives was accomplished in two ways, either (i) involving the condensation reaction of 6- chloropurine with commercially available arylsulfonyl chlorides in acetone and the presence of aqueous KOH at 0 °C, followed by the substitution of C6-chlorine with morpholine, or (ii) employing a reversed synthetic approach where 6-morpholinopurine and commercially available adenine bases were reacted with the corresponding alkyl, 2-arylethene and arylsulfonyl chlorides giving the N9 sulfonylated products, the latter particularly used where prior nonselective sulfonylation was observed. In both approaches, the sulfonylation reaction occurred regioselectively at the purine N9 position lacking any concurrent N7 derivatives, except in the case of a smaller methyl substituent on SO2 and the free amino group at C6 of the purine ring. The tautomeric features of initial N9 unsubstituted purines, as well as stability trends among the prepared N-9-sulfonylpurine derivates, were investigated using DFT calculations with an important conclusion that electron-donating C6 substituents are beneficial for the synthesis as they both promote the predominance of the desired N9 tautomers and help to assure the stability of the final products. The newly synthesized 6- morpholino and 6-amino-9-sulfonylpurine derivatives showed antiproliferative activity on human carcinoma, lymphoma, and leukemia cells. Among the tested compounds, 6-morpholino 17 and 6- amino 22 derivatives, with trans-β-styrenesulfonyl group attached at the N9 position of purine, proved to be the most effective antiproliferative agents, causing accumulation of leukemia cells in subG0 cell cycle phase.

Oxygen uptake (ṀO2) in larval zebrafish prior to maturation of the gill relies on cutaneous O2 transfer. Under normoxic conditions, rates of cutaneous O2 transfer are unaffected by haemoglobin availability but are diminished in fish lacking a functional circulatory system, suggesting that internal convection is critically involved in setting the resting ṀO2 in zebrafish larvae, even when relying on cutaneous O2 transfer. The reliance of ṀO2 on blood circulation led to the first objective of the current study, to determine whether loss of internal convection would reduce acute hypoxia performance (as determined by measuring critical PO2; Pcrit) in larval zebrafish under conditions of moderate hypoxia (PO2 = 55 mmHg) at 28.5 and 34 °C. Internal convection was eliminated by preventing development of blood vessels using morpholino knockdown of vascular endothelial growth factor (VEGF); these fish are termed VEGF morphants. Breathing frequency (fV) and heart rate (fH) also were measured (at 28.5 °C) to determine whether any detriment in performance might be linked to cardiorespiratory dysfunction. Although ṀO2 was reduced in the VEGF morphants, there was no significant effect on Pcrit at 28.5 °C. Raising temperature to 34 °C resulted in the VEGF morphants exhibiting a higher Pcrit than the shams, suggesting an impairment of hypoxia tolerance in the morphants at the higher temperature. The usual robust increase in fV during hypoxia was absent or attenuated in VEGF morphants at 4 and 5 days post fertilization (dpf), respectively. Resting fH was reduced in the VEGF morphants and unlike the sham fish, the morphants did not exhibit hypoxic tachycardia at 4 or 5 dpf. The number of cutaneous neuroepithelial cells (presumptive O2 chemoreceptors) was significantly higher in the VEGF morphants and thus the cardiorespiratory impairment in the morphants during hypoxia was unlikely related to inadequate peripheral O2 sensing.

A trifluoroacetic-acid-mediated desulfurilative sulfonylthiolation of arenes using SS-morpholino dithiosulfonate is described. This system is based on selective activation of the morpholino group over the tosyl group of the doubly transformable sulfur surrogate. Mechanistic studies suggested that the reaction proceeds through electrophilic aromatic substitution followed by sulfur extrusion. The wide substrate scope of this reaction and the transformability of the resulting thiosulfonates enable expeditious access to divergent multifunctionalized sulfides.

Synthetic antisense oligonucleotides (ASOs) have emerged as one of the most promising therapeutic approaches. So far, nine ASO drugs have received approval for clinical use, and four of them are based on splice-switching principles demonstrating the impact of ASO-mediated splice modulation. Notably, three among them (Exondys 51, Vyondys 53 and Viltepso) are based on phosphorodiamidate morpholino (PMO) chemistry whereas Spinraza is based on 2′-O-methoxyethyl phosphorothioate (2′-MOE PS) chemistry. Although systemic delivery of PMOs has displayed a good safety profile even at high doses, the 2′-O-methyl phosphorothioate modified (2′-OMe PS) ASO drug candidate (drisapersen) failed due to safety issues. The potency of 2′-modified RNA for splice-switching needs to be further improved by novel design strategies for broad applicability. Towards this goal, in this study, we evaluated the potential of incorporating DNA segments at appropriate sites in 2′-OMe PS and 2′-MOE PS ASOs to induce exon skipping. For this purpose, a four-nucleotide DNA segment was systematically incorporated into a 20-mer 2′-OMe PS and 2′-MOE PS ASO designed to skip exon 23 in mdx mouse myotubes in vitro. Our results demonstrated that 2′-modified RNA PS ASOs containing four or less PS DNA nucleotides at the 3′-end yielded improved exon 23 skipping efficacy in line with fully modified ASO controls. Based on these results, we firmly believe that the present study opens new avenues towards designing splice modulating ASOs with limited chemical modifications for enhanced safety and therapeutic efficacy.

Background: Congenital anomalies are increasingly becoming a global pediatric health concern, which requires immediate attention to its early diagnosis, preventive strategies, and efficient treatments. Guanine nucleotide binding protein, alpha inhibiting activity polypeptide 3 (Gnai3) gene mutation has been demonstrated to cause congenital small jaw deformity, but the functions of Gnai3 in the disease-specific microRNA (miRNA) upregulations and their downstream signaling pathways during osteogenesis have not yet been reported. Our previous studies found that the expression of Mir24-2-5p was significantly downregulated in the serum of young people with overgrowing mandibular, and bioinformatics analysis suggested possible binding sites of Mir24-2-5p in the Gnai3 3'UTR region. Therefore, this study was designed to investigate the mechanism of Mir24-2-5p-mediated regulation of Gnai3 gene expression and explore the possibility of potential treatment strategies for bone defects. Methods: Synthetic miRNA mimics and inhibitors were transduced into osteoblast precursor cells to regulate Mir24-2-5p expression. Dual-luciferase reporter assay was utilized to identify the direct binding of Gnai3 and its regulator Mir24-2-5p. Gnai3 levels in osteoblast precursor cells were downregulated by shRNA (shGnai3). Agomir, Morpholino Oligo (MO), and mRNA were microinjected into zebrafish embryos to control mir24-2-5p and gnai3 expression. Relevant expression levels were determined by the qRT-PCR and Western blotting. CCK-8 assay, flow cytometry, and transwell migration assays were performed to assess cell proliferation, apoptosis, and migration. ALP, ARS and Von Kossa staining were performed to observe osteogenic differentiation. Alcian blue staining and calcein immersions were performed to evaluate the embryonic development and calcification of zebrafish. Results: The expression of Mir24-2-5p was reduced throughout the mineralization process of osteoblast precursor cells. miRNA inhibitors and mimics were transfected into osteoblast precursor cells. Cell proliferation, migration, osteogenic differentiation, and mineralization processes were measured, which showed a reverse correlation with the expression of Mir24-2-5p. Dual-luciferase reporter gene detection assay confirmed the direct interaction between Mir24-2-5p and Gnai3 mRNA. Moreover, in osteoblast precursor cells treated with Mir24-2-5p inhibitor, the expression of Gnai3 gene was increased, suggesting that Mir24-2-5p negatively targeted Gnai3. Silencing of Gnai3 inhibited osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization. Promoting effects of osteoblast precursor cells proliferation, migration, osteogenic differentiation, and mineralization by low expression of Mir24-2-5p was partially rescued upon silencing of Gnai3. In vivo, mir24-2-5p Agomir microinjection into zebrafish embryo resulted in shorter body length, smaller and retruded mandible, decreased cartilage development, and vertebral calcification, which was partially rescued by microinjecting gnai3 mRNA. Notably, quite similar phenotypic outcomes were observed in gnai3 MO embryos, which were also partially rescued by mir24-2-5p MO. Besides, the expression of phospho-JNK (p-JNK) and p-p38 were increased upon Mir24-2-5p inhibitor treatment and decreased upon shGnai3-mediated Gnai3 downregulation in osteoblast precursor cells. Osteogenic differentiation and mineralization abilities of shGnai3-treated osteoblast precursor cells were promoted by p-JNK and p-p38 pathway activators, suggesting that Gnai3 might regulate the differentiation and mineralization processes in osteoblast precursor cells through the MAPK signaling pathway. Conclusions: In this study, we investigated the regulatory mechanism of Mir24-2-5p on Gnai3 expression regulation in osteoblast precursor cells and provided a new idea of improving the prevention and treatment strategies for congenital mandibular defects and mandibular protrusion.

EVALUATION OF THE CEREBROPROTECTIVE ACTION OF 1-ADAMANTY- LOXY-3-MORPHOLINO-2-PROPANOL HYDROCHLORIDE IN RATS WITH EXPERIMENTAL HEMORRHAGIC STROKE ACCORDING TO THE DYNAMICS OF NEUROCYTOLYSIS, NEUROAPOPTOSIS AND NEUROPROLIFERATION MARKERS

A nontoxic delivery vehicle is essential for the therapeutic applications of antisense phosphorodiamidate morpholino oligonucleotides (PMOs). Though guanidinium-rich or arginine-rich cellular transporter conjugated Vivo-PMO or PPMO has been developed for in vivo application, however, either their toxicity or stability has become an issue. Previously, we reported nonpeptidic internal guanidinium transporter (IGT) mediated delivery of PMO for gene silencing and got encouraging results. In this paper, we report the synthesis of IGT using a Hg-free method for scale up and N-terminal modification of IGT with a suitable hydrophobic or lipophilic group to improve the cell permeability, endosomal escape, and mitochondrial localization and to reduce toxicity in the MTT assay. For the delivery of PMO, IGT-PMO conjugate was synthesized to target NANOG in cells, a transcription factor required for cancer stem cell proliferation and embryonic development and is involved in many cancers. Our data shows IGT-PMO-facilitated NANOG inhibition, and thereby the prevention of EpCAM-N-Cadherin-Vimentin axis mediated epithelial to mesenchymal transition (EMT) in MCF-7 cells. Moreover, unlike taxol, NANOG inhibition influences the expression of stemness factor c-Myc, Hh-Gli signaling proteins, other cancer related factors, and their respective phenotypes in cancer cells. To the best of our knowledge, this is the first report to illustrate that the IGT-PMO-mediated NANOG inhibition increases the therapeutic potential of taxol and induces G0-G1 arrest in cancer cells to prevent cancer progression. However, it warrants further investigation in other cancer cells and preclinical platforms.

This Article outlines the optimized chemical synthesis and preliminary biochemical characterization of a new oligonucleotide analogue called thiophosphoramidate morpholinos (TMOs). Their rational design hinges upon integrating two well-studied pharmacophores, namely, phosphorothioates (pS) and morpholinos, to create morpholino-pS hybrid oligonucleotides. Our simple synthesis strategy enables the easy incorporation of morpholino-pS moieties and therapeutically relevant sugar modifications in tandem to create novel oligonucleotide (ON) analogues that are hitherto unexplored in the oligotherapeutics arena. Exclusively TMO-modified ONs demonstrate high stability toward 3'-exonuclease. Hybridization studies show that TMO chimeras consisting of alternating TMO and DNA-pS subunits exhibit higher binding affinity toward complementary RNA relative to the canonical DNA/RNA duplex (∼10 °C). Oligonucleotides that consist entirely of TMO linkages also show higher RNA binding affinity but do not recruit ribonuclease H1 (RNase H1). Chimeric TMO analogues demonstrate high gene silencing efficacy, comparable to that of a chimeric 2'-OMe-pS/pO control, during in vitro bioassay screens designed to evaluate their potential as microRNA inhibitors of hsa-miR-15b-5p in HeLa cells.

On continuing the investigation with the reaction between aminophosphines and Cu(I) halides, the reaction of aminophosphine, viz (Diisopropylamino)(morpholino)(phenyl)phosphine with CuX (X = Br, I)...

Receptor tyrosine kinase EphA7 is specifically expressed in otic region in Xenopus early development. However, its role in otocyst development remains unknown. Knockdown of EphA7 by a specific morpholino oligonucleotide (MO) reduced the size of the otocyst and triggered otic epithelial cell extrusion. Interestingly, EphA7 depletion attenuated the membrane level of the tight junction protein Claudin6 (CLDN6). Utilizing the Cldn6 MO, we further confirmed that CLDN6 attenuation also led to otic epithelial cell extrusion. Our work suggested that EphA7 modulates the otic epithelial homeostasis through stabilizing the CLDN6 membrane level.

N6-methyladenosine (m6A) is the most prevalent epigenetic modification of messenger RNA (mRNA) in higher eukaryotes; this modification is mainly catalyzed by a methyltransferase complex including methyltransferase-like 3 (METTL3) as a key factor. Although m6A modification has been proven to play an essential role in diverse biological processes, our knowledge of Mettl3 is still limited because Mettl3 mutations are lethal to embryos in both mammals and plants. In this study, we knocked down Mettl3 by microinjection of its specific short interfering RNAs (siRNAs) or morpholino into fully grown germinal vesicle (GV) oocytes. As a result, we demonstrated that knocking down Mettl3 in female germ cells severely inhibited oocyte maturation by decreasing mRNA translation efficiency and led to defects in the maternal-to-zygotic transition, probably due to its interference in disrupting mRNA degradation. The discovery from this study suggests that the reversible m6A modification has vital functions in mammalian oocyte maturation and pre-implantation embryonic development processes.

There is an ongoing effort to increase the efficiency of gene delivery for the regulation of diseases-related genes. In this report, we demonstrate the efficiency of a DNA-based nanostructure to deliver morpholino antisense oligonucleotides for the upregulated genes in breast cancer as an alternative to the currently used delivery systems. A DNA-tile structure is constructed by embedding antisense oligonucleotides targeting the HER2 and ER alpha genes. Then, the sticky ends of the DNA-tile nanostructures are hybridized to gold nanoparticles (AuNPs) coated with the complementary oligonucleotides to enhance their cellular uptake. It is found that the morpholino antisense oligonucleotide embedded DNA-tile-AuNPs structure is 30% more effective than the liposome-based system to deliver morpholinos and induce gene silencing in breast cancer cells. The results of the study suggest that the prepared novel nanostructure is an effective and biocompatible carrier that can be used in in vitro gene silencing studies and can be further pursued in in vivo studies.

Conditionally activated, caged morpholino antisense agents (cMOs) are tools that enable the temporal and spatial investigation of gene expression, regulation, and function during embryonic development. Cyclic MOs are conformationally gated oligonucleotide analogs that do not block gene expression until they are linearized through the application of an external trigger, such as light or enzyme activity. Here, we describe the first examples of small molecule-responsive cMOs, which undergo rapid and efficient decaging via a Staudinger reduction. This is enabled by a highly flexible linker design that offers opportunities for the installation of chemically activated, self-immolative motifs. We synthesized cyclic cMOs against two distinct, developmentally relevant genes and demonstrated phosphine-triggered knockdown of gene expression in zebrafish embryos. This represents the first report of a small molecule-triggered antisense agent for gene knockdown, adding another bioorthogonal entry to the growing arsenal of gene knockdown tools.

Coagulation factor V (FV) is a liver-derived protein encoded by the F5 gene. Alternative splicing of F5 exon 13 produces a low-abundance splicing isoform, known as FV-short, which binds the anticoagulant protein tissue factor pathway inhibitor (TFPI alpha) with high affinity, stabilising it in the circulation and potently enhancing its anticoagulant activity. Accordingly, rare F5 gene mutations that up-regulate FV-short splicing are associated with bleeding. In this study we have explored the possibility of decreasing FV-short splicing by antisense-based splicing modulation. To this end, we have designed morpholino antisense oligonucleotides (MAOs) targeting the FV-short-specific donor and acceptor splice sites and tested their efficacy in a liver cell line (HepG2) that naturally expresses full-length FV and FV-short. Cells were treated with 0-20 mu M MAO, and full-length FV and FV-short mRNA expression was analysed by RT-(q)PCR. Both MAOs, alone or in combination, decreased the FV-short/full-length FV mRNA ratio down to ~50% of its original value in a specific and dose-dependent manner. This pilot study provides proof-of-principle for the possibility to decrease FV-short expression by antisense-mediated splicing modulation. In turn, this may form the basis for novel therapeutic approaches to bleeding disorders caused by FV-short over-expression and/or elevated TFPI alpha (activity) levels.

Numerous studies have demonstrated that the aberrant enzymatic activity of SIRTs has been linked to various diseases like diabetes, cancer, inflammation, cardiovascular diseases, neurodegenerative disorders and therefore, the modulation of SIRTs is of therapeutic importance. The pharmacological action of substituted pyridazinones has been broadly studied and has received much attention on account of especially their cardiovascular, anticancer, anti-inflammatory, and analgesic activity. From this point of view, we evaluated our in-house compounds bearing substituted pyridazinone skeleton for their in vitro inhibitory potency against SIRT2 and the results were supported both by the compatibility with the pharmacophore model previously developed for selective SIRT2 inhibitors and by the interaction fingerprints in hSIRT2 active site. The results revealed that 4-aryl-6-morpholino-3(2H)-pyridazinone functionalized amide scaffold showing 48% inhibition against SIRT2 may be considered as a starting point in the development of new type SIRT2 inhibitors.

Animal models of human neurodegenerative disease have been investigated for several decades. In recent years, zebrafish (Danio rerio) and medaka (Oryzias latipes) have become popular in pathogenic and therapeutic studies about human neurodegenerative diseases due to their small size, the optical clarity of embryos, their fast development, and their suitability to large-scale therapeutic screening. Following the emergence of a new generation of molecular biological technologies such as reverse and forward genetics, morpholino, transgenesis, and gene knockout, many human neurodegenerative disease models, such as Parkinson’s, Huntington’s, and Alzheimer’s, were constructed in zebrafish and medaka. These studies proved that zebrafish and medaka genes are functionally conserved in relation to their human homologues, so they exhibit similar neurodegenerative phenotypes to human beings. Therefore, fish are a suitable model for the investigation of pathologic mechanisms of neurodegenerative diseases and for the large-scale screening of drugs for potential therapy. In this review, we summarize the studies in modelling human neurodegenerative diseases in zebrafish and medaka in recent years.

The polyanionic phosphodiester backbone of nucleic acids contributes to high nuclease sensitivity and low cellular uptake and is therefore a major obstacle to the biological application of native oligonucleotides. Backbone modifications, particularly charge alterations is a proven strategy to provide artificial oligonucleotides with improved properties. Here, we describe the synthesis of a new type of oligonucleotide analogues consisting of a morpholino and a ribo- or deoxyribonucleoside in which the 5′-amino group of the nucleoside unit provides the nitrogen of the morpholine ring. The synthetic protocol is compatible with trityl and dimethoxytrityl protecting groups and azido functionality, and was extended to the synthesis of higher oligomers. The chimeras are positively charged in aqueous medium, due to the N-alkylated tertiary amine structure of the morpholino unit.

This paper is not just about the benefits of the smart plastics, but also about the potential remedies of the environmental concerns of their use. It is also a discussion about a potential pathway to an efficient use of fossil (combustion) fuels and the reduction of the emissions caused due to their use, in general. Morpholino-poly (piperazinyl-morpholinyl-triazin) imparts sustainable fire-safety and subdued incineration behaviour to plastics combined with weight reduction, and facilitates their melt processing. Additionally, it enables their ultimate environmentally friendly disposal as waste, after the ultimate use, coupled with simultaneous generation of usable energy. The knowledge & experience thus gained from energy extraction of waste plastics provide guidelines for future research to increase the efficiency of energy extraction and the reduction of emissions from combustion fuels in general. The technology is also intended to safeguard against open burning of plastics that leads to air-pollution flashover and forest fires. The various aspects of the experimental data generated are presented and plausible mechanisms of the technology are discussed.

Different forms of sudden cardiac death have been described, including a recently identified form of genetic arrhythmogenic disorder, named “Triadin KnockOut Syndrome” (TKOS). TKOS is associated with recessive mutations in the TRDN gene, encoding for TRIADIN, but the pathogenic mechanism underlying the malignant phenotype has yet to be completely defined. Moreover, patients with TKOS are often refractory to conventional treatment, substantiating the need to identify new therapeutic strategies in order to prevent or treat cardiac events. The zebrafish (Danio rerio) heart is highly comparable to the human heart in terms of functions, signal pathways and ion channels, representing a good model to study cardiac disorders. In this work, we generated the first zebrafish model for trdn loss-of-function, by means of trdn morpholino injections, and characterized its phenotype. Although we did not observe any gross cardiac morphological defect between trdn loss-of-function embryos and controls, we found altered cardiac rhythm that was recovered by the administration of arrhythmic drugs. Our model will provide a suitable platform to study the effect of TRDN mutations and to perform drug screening to identify new pharmacological strategies for patients carrying TRDN mutations.

Osteosarcoma (OS) is the most common primary bone malignancy. Chemotherapy plays an essential role in OS treatment, potentially doubling 5-year event-free survival if tumor necrosis can be stimulated, but long-term treatment results in detriment to health and quality of life. The canonical Wnt inhibitor Dickkopf-1 (Dkk-1) enhances OS survival in part through upregulation of aldehyde-dehydrogenase-1A1 (ALDH1A1) which neutralizes reactive oxygen species from nutritional stress and chemotherapeutic challenge. Dkk-1 also inhibits bone repair, exacerbating formation of osteolytic lesions caused by tumor infiltration. Therefore, targeting the expression of Dkk-1 in OS could reduce tumor burden and increase susceptibility to chemotherapeutics while restoring bone repair. Herein, we report that inhibiting Dkk-1 transcription by means of a vivo morpholino (DkkMo) reduced the expansion of experimental OS tumors, preserved bone volume and architecture, and stimulated tumor necrosis. This was observed in the presence or absence of doxorubicin (DRB), and as a single agent, inhibition of tumor expansion by DkkMo was equivalent to that achieved by DRB. DkkMo stimulated apoptotic and necrotic mechanisms in tumors and appeared to deplete the tumor stroma. These results indicate that administration of DkkMo with or without chemotherapeutics can substantially improve OS outcome with respect to tumor expansion and osteolytic corruption of bone.

Deltamethrin (DM), a type II pyrethroid insecticide, is widely used to control agricultural pests. However, its excessive use exerts a detrimental effect on the ecological environment and human health, indicating the need to study its potential risks in detail. In the present study, zebrafish embryos were exposed to varying concentrations of DM (0.1, 1, 10, and 25 μg/L) for 96 h to assess the alterations in the transcript levels of proteins of the estrogenic and dopaminergic pathways. In addition, its effect on zebrafish locomotor activity was studied. The mRNA expression of cyp19a1b, erα, erβ2, fshr, gnrh2, gnrhr3, vtg3, dat, and dr1 significantly changed after exposing the embryos to DM. Deltamethrin at 10 and 25 μg/L significantly reduced the average swimming speed of zebrafish larvae. In addition, embryos injected with zebrafish estrogen receptor α (erα) and β (erβ) morpholinos and co-exposed to 25 μg/L DM for 96 h showed reduced expression of vtg3 mRNA compared to embryos exposed to 25 μg/L DM only. The locomotor activity of erα and erβ knockdown zebrafish following DM exposure was increased significantly when compared with that of larvae exposed to 25 μg/L DM only. Our results demonstrated that DM altered the locomotor activity of zebrafish larvae and the transcript levels of the components of estrogenic and dopaminergic pathways; erα and erβ knockdown weakened these effects.

Background: Mitochondrial aminoacyl-tRNA synthetases (mtARSs) catalyze the binding of specific amino acids to their cognate tRNAs and play an essential role in the synthesis of proteins encoded by mitochondrial DNA. Defects in mtARSs have been linked to human diseases, but their tissue-specific pathophysiology remains elusive. Here we examined the role of mitochondrial phenylalanyl-tRNA synthetase (FARS2) in developmental angiogenesis and its potential contribution to the pathogenesis of cardiovascular disease.Methods: Morpholinos were injected into fertilized zebrafish ova to establish an in vivo fars2 knock-down model. A visualization of the vasculature was achieved by using Tg (fli1: EGFP)y1 transgenic zebrafish. In addition, small interference RNAs (siRNAs) were transferred into human umbilical vein endothelial cells (HUVECs) to establish an in vitro FARS2 knock-down model. Cell motility, proliferation, and tubulogenesis were determined using scratch-wound CCK8, transwell-based migration, and tube formation assays. In addition, mitochondria- and non-mitochondria-related respiration were evaluated using a Seahorse XF24 analyzer and flow cytometry assays. Analyses of the expression levels of transcripts and proteins were performed using qRT-PCR and western blotting, respectively.Results: The knock-down of fars2 hampered the embryonic development in zebrafish and delayed the formation of the vasculature in Tg (fli1: EGFP)y1 transgenic zebrafish. In addition, the siRNA-mediated knock-down of FARS2 impaired angiogenesis in HUVECs as indicated by decreased cell motility and tube formation capacity. The knock-down of FARS2 also produced variable decreases in mitochondrial- and non-mitochondrial respiration in HUVECs and disrupted the regulatory pathways of angiogenesis in both HUVECs and zebrafish.Conclusion: Our current work offers novel insights into angiogenesis defects and cardiovascular diseases induced by FARS2 deficiency.

The paper reports the synthesis of a series of antisense oligonucleotides (aONs) directed against different segments of the influenza A virus genome (H1N1) and screening of their antiviral activity in the influenza A virus (H1N1)/MDCK cells and influenza A virus (H1N1)/A549 cells systems. The results of screening have shown that PB1-2AUG-R aON targeted towards the AUG codon region of the segment 2 of virus genome possessed the highest antiviral activity. The synthesized morpholino analog (PMO) and the new phosphoryl guanidine oligodeoxyribonucleotide (PGO) with the sequence of oligonucleotide PB1-2AUG-R provided a comparable biological effects: the influenza virus titer in MDCK cell culture was reduced by 15 times compared to the control when PGO was used in the concentration of 10 µM and by 40 times when PMO was used in the concentration of 20 µM. For the delivery of electrically neutral analogs of oligonucleotides (PGO and PMO), the perforation method proposed for PMO was used.

Thioredoxin is an evolutionarily conserved antioxidant protein that plays a crucial role for fundamental cellular processes and embryonic development. Growing evidence support that Thioredoxin influences cellular response to chemicals insults, particularly those accompanying oxidative stress. The mechanisms underlying the functions of Thioredoxin1 in the embryonic development under the environmental toxicant exposure remain, however, largely unexplored. We report here that thioredoxin1 becomes differentially expressed in zebrafish embryos after exposure to 9 out of 11 environmental chemicals. In situ gene expression analysis show that thioredoxin1 is expressed in neurons, olfactory epithelia, liver and swim bladder under normal conditions. After MeHg exposure, however, thioredoxin1 is ectopically induced in the hair cells of the lateral line and in epithelia cells of the pharynx. Knockdown of Thioredoxin1 induces hydrocephalus and increases cell apoptosis in the brain ventricular epithelia cells. In comparison with 5% malformation in embryos injected with control morpholino, MeHg induces more than 77% defects in Thioredoxin1 knockdown embryos. Our data suggest that there is an association between hydrocephalus and Thioredoxin1 malfunction in embryonic development, and provide valuable information to elucidate the protective role of Thioredoxin1 against chemicals disruption.

Previous studies have shown that ATP synthase α subunit (ATP5A1) plays multiple roles, but our understanding of its biologic functions remains poor and incomprehensive. Here, we clearly demonstrated that zebrafish ATP5A1 was a newly characterized lipoteichoic acid (LTA)- and LPS-binding protein abundantly stored in the eggs and embryos of zebrafish. Zebrafish ATP5A1 acted not only as a pattern recognition receptor, capable of identifying LTA and LPS, but also as an effector molecule, capable of inhibiting the growth of both gram-positive and -negative bacteria. ATP5A1 could disrupt the bacterial membranes by a combined action of membrane depolarization and permeabilization. We also found that the N-terminal 65 residues were critical for the antibacterial activity of zebrafish ATP5A1. In particular, we showed that microinjection of exogenous recombinant (r)ATP5A1 into early embryos could promote their resistance against pathogenic Aeromonas hydrophila challenge, and this pathogen-resistant activity was markedly reduced by the coinjection of anti-ATP5A1 antibody or by the knockdown with morpholino for atp5a1 but not by the coinjection of anti-actin antibody. Moreover, each egg/embryo contains a sufficient amount of ATP5A1 in vivo to kill A. hydrophila. Furthermore, the N-terminal 65 residues 1–65 of ATP5A1 α subunit (rA(1–65)) with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the N-terminal 69 residues 66–134 (rA(66–134)) or C-terminal residues 135–551 (rA(135–551)) of ATP5A1 α subunit without in vitro antibacterial activity did not. Finally, we showed that the antibacterial activity of the N-terminal 65 residues of ATP5A1 α subunit was conserved throughout animal evolution. Collectively, these results indicate that ATP5A1 is a novel maternal immunocompetent factor that can protect the early embryos of zebrafish from bacterial infection. This work also provides a new viewpoint for understanding the biologic roles of ATP5A1, which is ubiquitously present in animals.—Ni, S., Zhou, Y., Chen, Y., Du, X., Zhang, S. Identification of ATP synthase α subunit as a new maternal factor capable of protecting zebrafish embryos from bacterial infection.

Insufficient delivery of oligonucleotides to muscle and heart remains a barrier for clinical implementation of antisense oligonucleotide (AO)-mediated exon-skipping therapeutics in Duchenne muscular dystrophy (DMD), a lethal monogenic disorder caused by frame-disrupting mutations in the DMD gene. We previously demonstrated that hexose, particularly an equal mix of glucose:fructose (GF), significantly enhanced oligonucleotide delivery and exon-skipping activity in peripheral muscles of mdx mice; however, its efficacy in the heart remains limited. Here we show that co-administration of GF with peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO, namely, BMSP-PMO) induced an approximately 2-fold higher level of dystrophin expression in cardiac muscles of adult mdx mice compared to BMSP-PMO in saline at a single injection of 20 mg/kg, resulting in evident phenotypic improvement in dystrophic mdx hearts without any detectable toxicity. Dystrophin expression in peripheral muscles also increased. However, GF failed to potentiate BMSP-PMO efficiency in aged mdx mice. These findings demonstrate that GF is applicable to both PMO and PPMO. Furthermore, GF potentiates oligonucleotide activity in mdx mice in an age-dependent manner, and, thus, it has important implications for its clinical deployment for the treatment of DMD and other muscular disorders. Keywords: hexose, peptide-conjugated morpholino oligomer, Duchenne muscular dystrophy, exon skipping, cardiac functions

Morpholino-containing aza-BODIPYs at 3,5-positions were synthesized. The maxima absorption and emission of these dyes locate at the near-infrared region. Aza-BODIPY 1 with the morpholino group as a pH-sensitive functionality could be used to be a pH probe, and the dramatic increase in fluorescence intensity at 675 nm by about 1500 folds. Moreover, the singlet oxygen generation of PS 2 with the dibromo groups at 2,6-positions was more effective than that of the parent dye 1.

Podocytes are important to glomerular filtration barrier integrity and maintenance of size selectivity in protein filtration in the kidney. Although there is evidence to suggest that triptolide has direct protective effects on podocyte injuries, the mechanism mediating this process remains largely unexplored. In this study, we found triptolide suppresses podocyte p53 and GADD45B expression in vivo and in vitro. We used our previously developed in vivo zebrafish model of inducible podocyte-targeted injury and found that triptolide or the inhibition of p53 and gadd45ba with morpholino (MO) alleviated metronidazole (MTZ) induced edema in zebrafish, while the overexpression of gadd45ba in podocytes blocked the protective effect of triptolide and p53 MO on podocyte injury in zebrafish. Further study showed that p53 directly transactivated GADD45B. Triptolide inhibited p53 binding to the GADD45B promoter and subsequent GADD45B transcription. We further demonstrated that p53 may indirectly regulate GADD45B expression via NF-κB signaling. Taken together, our findings demonstrated that triptolide maintained glomerular barrier function via the inhibition of p53-NF-κB-GADD45B signaling, which provides a new understanding of the antiproteinuric effects of triptolide in glomerular diseases.

ABCD4, a member of the ATP-binding cassette transporter superfamily, is associated with the transport of vitamin B12 which is crucial for the development of red blood cells (RBCs) and may also be involved in its metabolism. However, the molecular function of ABCD4 during RBC development in zebrafish is mostly unknown. Using a morpholino-based knockdown approach, we found that abcd4-knockdown resulted in abnormal RBCs of irregular shapes and various sizes. o-Dianisidine staining, as an indicator of hemoglobin in RBCs, further confirmed that abcd4 morphants possessed fewer hemoglobinized cells and impaired blood circulation. Multiple protein sequence alignment revealed that the amino acid sequence for residues 13-292, which is the domain of vitamin B12 transport, of the zebrafish Abcd4 was highly conserved compared to that of other species. Accordingly, the abcd4 morphants can be rescued with human ABCD4, demonstrating a conserved role of ABCD4 in vertebrates. Notably, the vitamin B12-deficient phenotype in abcd4 morphants, which causes anemia, was recapitulated in the newly-established abcd4 mutant, indicating the possibility that the abcd4 mutant could be used as a disease model of vitamin B12-deficiency anemia. Our study provides an insight that the analysis of the newly-established abcd4 mutant may contribute to understanding its roles in ABCD4-related vitamin B12-deficiency anemia and the associated pathogeneses in humans.