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Therapeutic modalities designed specifically to inhibit COVID-19 infection and replication would limit progressive COVID-19-associated pulmonary disease in infected patients and prevent or limit systemic disease. If effective, antivirals could reduce viral transmission rates by reducing viral burden and allow time for immune clearance. For individuals infected with acute-stage disease, antivirals in support of the existing vaccines could reduce COVID-19 hospitalizations and deaths. Here, we evaluate MRCV-19, a phosphorodiamidate morpholino oligo with delivery dendrimer (Vivo-Morpholino), to prevent coronavirus infection in a cell culture model. This is a novel antiviral that effectively inhibits SARS-CoV-2 replication in vitro. By design, MRCV-19 targets the SARS-CoV-2 5’UTR and overlaps the pp1a start site of translation in order to block access of the translation initiation complex to the start. MRCV-19 testing is conducted in a high-throughput, 384-well plate format with a 10-point dose-response curve (common ratio of 2) assayed in duplicate with parallel cytotoxicity evaluations. MRCV-19 was shown to be more effective than hydroxychloroquine and remdesivir in our CPE reduction assay with low toxicity. The clinical translational impact of this study is providing the basis for evaluating MRCV-19 on a large scale in an appropriate infection model for toxicity and systemic high-level inhibition of SARS-CoV-2, which could lead in time to phase I testing in humans.

In recent years CRISPR-Cas9 knockouts (KO) have become increasingly ultilised to study gene function. MicroRNAs (miRNAs) are short non-coding RNAs, 20-22 nucleotides long, which affect gene expression through post-transcriptional repression. We previously identified miRNAs-196a and −219 as implicated in the development of Xenopus neural crest (NC). The NC is a multipotent stem-cell population, specified during early neurulation. Following EMT NC cells migrate to various points in the developing embryo where they give rise to a number of tissues including parts of the peripheral nervous system and craniofacial skeleton. Dysregulation of NC development results in many diseases grouped under the term neurocristopathies. As miRNAs are so small it is difficult to design CRISPR sgRNAs that reproducibly lead to a KO. We have therefore designed a novel approach using two guide RNAs to effectively ‘drop out’ a miRNA. We have knocked out miR-196a and miR-219 and compared the results to morpholino knockdowns (KD) of the same miRNAs. Validation of efficient CRISPR miRNA KO and phenotype analysis included use of whole-mount in situ hybridization of key NC and neural plate border markers such as Pax3, Xhe2, Sox10 and Snail2, q-RT-PCR and Sanger sequencing. miRNA-219 and miR-196a KO’s both show loss of NC, altered neural plate and hatching gland phenotypes. Tadpoles show gross craniofacial and pigment phenotypes.

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish CYP20A1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1−/− mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Duchenne muscular dystrophy (DMD) afflicts 1 in 5000 newborn males, leading to progressive muscle weakening and the loss of ambulation between the ages of 8 and 12. Typically, DMD patients pass away from heart failure or respiratory failure. Currently, there is no cure, though exon-skipping therapy including eteplirsen (brand name Exondys 51), a synthetic antisense oligonucleotide designed to skip exon 51 of the dystrophin gene, is considered especially promising. Applicable to approximately 14% of DMD patients, a phosphorodiamidate morpholino oligomer (PMO) antisense oligonucleotide eteplirsen received accelerated approval by the US Food and Drug Administration (FDA) in 2016. Throughout clinical trials, eteplirsen has been well tolerated by patients with no serious drug-related adverse events. The most common events observed are balance disorder, vomiting, and skin rash. Despite its safety and promise of functional benefits, eteplirsen remains controversial due to its low production of dystrophin. In addition, unmodified PMOs have limited efficacy in the heart. To address these concerns of efficacy, eteplirsen has been conjugated to a proprietary cell-penetrating peptide; the conjugate is called SRP-5051. Compared to eteplirsen, SRP-5051 aims to better prompt exon-skipping and dystrophin production but may have greater toxicity concerns. This paper reviews and discusses the available information on the efficacy, safety, and tolerability data of eteplirsen and SRP-5051 from preclinical and clinical trials. Issues faced by eteplirsen and SRP-5051, including efficacy and safety, are identified. Lastly, the current state of eteplirsen and exon-skipping therapy in general as a strategy for the treatment of DMD are discussed.

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.

The mechanisms by which tissues and organs achieve their final size and shape during development are largely unknown. Although we have learned much about the mechanisms that control growth, little is known about how those play out to achieve a structure's specific final size and shape. The wings of insects are attractive systems for the study of the control of morphogenesis, because they are perfectly flat and two-dimensional, composed of two closely appressed cellular monolayers in which morphogenetic processes can be easily visualized. The wings of Lepidoptera arise from imaginal disks whose structure is always perfectly congruent with that of the adult wing, so that it is possible to fate-map corresponding positions on the larval disk to those of the adult wing. Here we show that the forewing imaginal disks of Junonia coenia are subdivided into four domains, with characteristic patterns of expression of known patterning genes Spalt (Sal), Engrailed (En), and Cubitus interruptus (Ci). We show that DNA and protein synthesis, as well as mitoses, are spatially patterned in a domain-specific way. Knockdown of Sal and En using produced domain-specific reductions in the shape of the forewing. Knockdown of signaling pathways involved in the regulation of growth likewise altered the shape of the forewing in a domain-specific way. Our results reveal a multi-level regulation of forewing shape involving hormones and growth-regulating genes.

The Hedgehog (Hh)/Gli signaling pathway controls cell proliferation and differentiation, is critical for the development of nearly every tissue and organ in vertebrates and is also involved in tumorigenesis. In this study, we characterize the oncoprotein SET/I2PP2A as a novel regulator of Hh signaling. Our previous work has shown that the zebrafish homologs of SET are expressed during early development and localized in the ciliated organs. In the present work, we show that CRISPR/Cas9-mediated knockdown of setb gene in zebrafish embryos resulted in cyclopia, a characteristic patterning defect previously reported in Hh mutants. Consistent with these findings, targeting setb gene using CRISPR/Cas9 or a setb morpholino, reduced Gli1-dependent mCherry expression in the Hedgehog reporter zebrafish line Tg(12xGliBS:mCherry-NLS). Likewise, SET loss of function by means of pharmacological inhibition and gene knockdown prevented the increase of Gli1 expression in mammalian cells in vitro. Conversely, overexpression of SET resulted in an increase of the expression of a Gli-dependent luciferase reporter, an effect likely attributable to the relief of the Sufu-mediated inhibition of Gli1. Collectively, our data support the involvement of SET in Gli1-mediated transcription and suggest the oncoprotein SET/I2PP2A as a new modulator of Hedgehog signaling.

Von Willebrand A domain-containing protein 8 (VWA8), also named KIAA0564, is a poorly characterized, mitochondrial matrix-targeted protein having a putative ATPase activity. VWA8 is comprising of ATPase-associated domains and a VWFA domain associated with ATPase activity inside the cell. In the present study, we describe a large consanguineous family of Saudi origin segregating a complex developmental syndrome in an autosomal recessive fashion. All the affected individuals exhibited severe developmental disorders. DNA from three patients was subjected to whole-exome sequencing followed by Sanger sequencing. VWA8 knock-down zebrafish morpholinos were used to study the phenotypic effect of this gene on zebrafish development. A homozygous missense variant [c.947A > G; p.(Asp316Gly)] was identified in exon 8 of the VWA8 gene, which perfectly segregated with the disease phenotype. Using zebrafish morpholino, we observed delayed development at an early stage, lack of movement, light sensitivity, severe skeletal deformity such as scoliosis, and facial dysmorphism. This is the first homozygous variant identified in the VWA8 gene underlying global developmental delay, microcephaly, scoliosis, limbs, and cardiovascular malformations in humans. We provide genetic and molecular evidence using zebrafish morpholino for a homozygous variant in the VWA8 gene, associated with such a complex developmental syndrome in humans.

Background Kdm6b, a specific histone 3 lysine 27 (H3K27) demethylase, has been reported to be implicated in a variety of developmental processes including cell differentiation and cell fate determination and multiple organogenesis. Here, we regulated the transcript level of kdm6bb to study the potential role in controlling the hearing organ development of zebrafish. Methods A morpholino antisense oligonucleotide (MO) strategy was used to induce Kdm6b deficiency; immunohistochemical staining and in situ hybridization analysis were conducted to figure out the morphologic alterations and embryonic mechanisms. Results Kdm6bb is expressed in the primordium and neuromasts at the early stage of zebrafish embryogenesis, suggesting a potential function of Kdm6b in the development of mechanosensory organs. Knockdown of kdm6bb severely influences the cell migration and proliferation in posterior lateral line primordium, abates the number of neuromasts along the trunk, and mRNA-mediated rescue test can partially renew the neuromasts. Loss of kdm6bb might be related to aberrant expressions of chemokine genes encompassing cxcl12a and cxcr4b/cxcr7b in the migrating primordium. Moreover, inhibition of kdm6bb reduces the expression of genes in Fgf signaling pathway, while it increases the axin2 and lef1 expression level of Wnt/β-catenin signaling during the migrating stage. Conclusions Collectively, our results revealed that Kdm6b plays an essential role in guiding the migration of primordium and in regulating the deposition of zebrafish neuromasts by mediating the gene expression of chemokines and Wnt and Fgf signaling pathway. Since histone methylation and demethylation are reversible, targeting Kdm6b may present as a novel therapeutic regimen for hearing disorders.

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.

In contrast to humans and other mammals, zebrafish can successfully regenerate and remyelinate central nervous system (CNS) axons following injury. In addition to common myelin proteins found in mammalian myelin, 36K protein is a major component of teleost fish CNS myelin. Although 36K is one of the most abundant proteins in zebrafish brain, its function remains unknown. Here we investigate the function of 36K using translation-blocking Morpholinos. Morphant larvae showed fewer dorsally migrated oligodendrocyte precursor cells as well as upregulation of Notch ligand. A gamma secretase inhibitor, which prevents activation of Notch, could rescue oligodendrocyte precursor cell numbers in 36K morphants, suggesting that 36K regulates initial myelination through inhibition of Notch signaling. Since 36K like other short chain dehydrogenases might act on lipids, we performed thin layer chromatography and mass spectrometry of lipids and found changes in lipid composition in 36K morphant larvae. Altogether, we suggest that during early development 36K regulates membrane lipid composition, thereby altering the amount of transmembrane Notch ligands and the efficiency of intramembrane gamma secretase processing of Notch and thereby influencing oligodendrocyte precursor cell differentiation and further myelination. Further studies on the role of 36K short chain dehydrogenase in oligodendrocyte precursor cell differentiation during remyelination might open up new strategies for remyelination therapies in human patients.

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.

Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45–55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45–55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45–55 skipping with removing 3, 8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45–55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.

OBJECTIVE: The Popeye domain containing 3 (POPDC3) gene encodes a membrane protein involved in cyclic adenosine monophosphate (cAMP) signaling. Besides gastric cancer, no disease association has been described. We describe a new muscular dystrophy associated with this gene.METHODS: We screened 1,500 patients with unclassified limb girdle weakness or hyperCKemia for pathogenic POPDC3 variants. Five patients carrying POPDC3 variants were examined by muscle magnetic resonance imaging (MRI), muscle biopsy, and cardiac examination. We performed functional analyses in a zebrafish popdc3 knockdown model and heterologous expression of the mutant proteins in Xenopus laevis oocytes to measure TREK-1 current.RESULTS: We identified homozygous POPDC3 missense variants (p.Leu155His, p.Leu217Phe, and p.Arg261Gln) in 5 patients from 3 ethnically distinct families. Variants affected highly conserved residues in the Popeye (p.Leu155 and p.Leu217) and carboxy-terminal (p.Arg261) domains. The variants were almost absent from control populations. Probands' muscle biopsies were dystrophic, and serum creatine kinase levels were 1,050 to 9,200U/l. Muscle weakness was proximal with adulthood onset in most patients and affected lower earlier than upper limbs. Muscle MRI revealed fat replacement of paraspinal and proximal leg muscles; cardiac investigations were unremarkable. Knockdown of popdc3 in zebrafish, using 2 different splice-site blocking morpholinos, resulted in larvae with tail curling and dystrophic muscle features. All 3 mutants cloned in Xenopus oocytes caused an aberrant modulation of the mechano-gated potassium channel, TREK-1.INTERPRETATION: Our findings point to an important role of POPDC3 for skeletal muscle function and suggest that pathogenic variants in POPDC3 are responsible for a novel type of autosomal recessive limb girdle muscular dystrophy. ANN NEUROL 2019;86:832-843.

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.

Background Matrix metalloproteinases 13 (MMP13) is a potent endopeptidase that regulate cell growth, migration, and extracellular matrix remodeling. However, its role in fin regeneration remains unclear. Results mmp13a expression is strongly upregulated during blastema formation and persists in the distal blastema. mmp13a knockdown via morpholino electroporation impairs regenerative outgrowth by decreasing cell proliferation, which correlates with a downregulation of fgf10a and sall4 expression in the blastema. Laminin distribution in the basement membrane is also affected in mmp13a MO-injected rays. Another impact of mmp13a knockdown is observed in the skeletal elements of the fin rays. Expression of two main components of actinotrichia, Collagen II and Actinodin 1 is highly reduced in mmp13a MO-injected rays leading to highly disorganized actinotrichia pattern. Inhibition of mmp13a strongly affects bone formation as shown by a reduction of Zns5 and sp7 expression and of bone matrix mineralization in rays. These defects are accompanied by a significant increase in apoptosis in mmp13a MO-injected fin regenerates. Conclusion Defects of expression of this multifunctional proteinase drastically affects osteoblast differentiation, bone and actinotrichia formation as well as Laminin distribution in the basement membrane of the fin regenerate, suggesting the important role of Mmp13 during the regenerative process.

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.

Myotubularin related protein 7 (MTMR7), a key member of the MTMR family, depicts phosphatase activity and is involved in myogenesis and tumor growth. We have previously identified MTMR7 in the proteomic profile of mouse spermatogonial stem cell (SSC) maturation and differentiation, implying that MTMR7 is associated with neonatal testicular development. In this study, to further explore the distribution and function of MTMR7 in mouse testis, we studied the effect of Mtmr7 knockdown on neonatal testicular development by testicular and SSC culture methods. Our results revealed that MTMR7 is exclusively located in early germ cells. Deficiency of MTMR7 by morpholino in neonatal testis caused excessive SSC proliferation, which was attributable to the aberrant PI3K/AKT signaling activation. Altogether, our study demonstrates that MTMR7 maintains SSC homeostasis by inhibiting PI3K/AKT signaling activation.

Background/Aims: Neural crest cells play a vital role in craniofacial development, microRNA-1 (miR-1) is essential in development and disease of the cardiac and skeletal muscle, the objective of our study is to investigate effects of miR-1 on neural crest cell in the craniofacial development and its molecular mechanism. Methods: We knocked down miR-1 in zebrafish by miR-1 morpholino (MO) microinjection and observed phenotype of neural crest derivatives. We detected neural crest cell migration by time-lapse. Whole-mount in situ hybridization was used to monitor the expressions of genes involved in neural crest cell induction, specification, migration and differentiation. We performed a quantitative proteomics study (iTRAQ) and bioinformatics prediction to identify the targets of miR-1 and validate the relationship between miR-1 and its target gene sec63. Results: We found defects in the tissues derived from neural crest cells: a severely reduced lower jaw and delayed appearance of pigment cells. miR-1 MO injection also disrupted neural crest cell migration. At 24 hours post fertilization (hpf), reduced expression of tfap2a, dlx2, dlx3b, ngn1 and crestin indicated that miR-1 deficiency affected neural crest cell differentiation. iTRAQ and luciferase reporter assay identified SEC63 as a direct target gene of miR-1. The defects of miR-1 deficiency could be reversed, at least in part, by specific suppression of sec63 expression. Conclusion: miR-1 is involved in the regulation of neural crest cell development, and that it acts, at least partially, by targeting sec63 expression.

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

Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified epoa being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for epoa have not been described so far. In order to compare a transient knockdown vs. permanent knockout for epoa in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate epoa knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to epoa morphants, epoa-/- zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named epob, was identified and upregulated in epoa-/- mutants. epob knockdown altered pronephros development, which was further aggravated in epoa-/- mutants. Likewise, epoa and epob morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of epoa during embryonic development can be compensated by epob leading to phenotypical discrepancies in epoa knockdown and knockout zebrafish embryos.

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.

Sea urchins are used as a model organism for research on developmental biology and gene regulatory networks during early development. Gene knockdown by microinjection of morpholino antisense oligonucleotide (MASO) has been used to analyze gene function in early sea urchin embryos. However, as the effect of MASO is not long lasting, it is impossible to perturb genes expressed during late development by MASO. Recent advances in genome editing technologies have enabled gene modification in various organisms. We previously reported genome editing in the sea urchin Hemicentrotus pulcherrimus using zinc-finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN); however, the efficiencies of these technologies were not satisfactory. Here, we applied clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology to knock out the Pks1 gene in H. pulcherrimus. When sgRNAs targeting Pks1, which is required for the biosynthesis of larval pigment, were microinjected into fertilized eggs with SpCas9 mRNA, high-efficiency mutagenesis was achieved within 24 hr post fertilization and SpCas9/sgRNA-injected pluteus larvae had an albino phenotype. One of the sgRNAs yielded 100% mutagenesis efficiency, and no off-target effect was detected. In addition, the albino phenotype was maintained in juvenile sea urchins after metamorphosis, and the knockout sea urchins survived for at least one year and grew to albino adult sea urchins. These findings suggest that knockout adult sea urchins were successfully established and the CRISPR-Cas9 system is a feasible method for analyzing gene functions from late developmental to adult stage.

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.

Antisense oligonucleotide (AO) therapy has been the specific treatment for Duchenne muscular dystrophy, with ongoing clinical trials. However, therapeutic applications of AOs remain limited, particularly because of the lack of efficient cellular delivery methods imperative for achieving efficacy. In this study, we investigated a few aminoglycosides (AGs) for their potential to improve the delivery of antisense phosphorodiamidate morpholino oligomer (PMO) both in vitro and in vivo. AGs had lower cytotoxicity compared with Endoporter, the currently most effective delivery reagent for PMO in vitro, and improved efficiency in PMO delivery 9- to 15-fold over PMO alone. Significant enhancement in systemic PMO-targeted dystrophin exon 23 skipping was observed in mdx mice, up to a 6-fold increase with AG3 (kanamycin) and AG7 (sisomicin) compared with PMO only. No muscle damage could be detected clearly with the test dosages. These results establish AGs as PMO delivery-enhancing agents for treating muscular dystrophy or other diseases., Graphical Abstract

mTOR has become a promising target for many types of cancer like breast, lung and renal cell carcinoma. CoMFA, CoMSIA, Topomer CoMFA and HQSAR were performed on the series of 39 triazine morpholino derivatives. CoMFA analysis showed q2 value of 0.735, r2cv value of 0.722 and r2pred value of 0.769. CoMSIA analysis (SEHD) showed q2 value of 0.761, r2cv value of 0.775 and r2pred value of 0.651. Topomer CoMFA analysis showed q2 value of 0.693, r2 (conventional correlation coefficient) value of 0.940 and r2pred value of 0.720. HQSAR analysis showed q2,r2and r2pred values of 0.694, 0.920 and 0.750, respectively. HQSAR analysis with the combination of atomic number (A), bond type (B) and atomic connections showed q2 and r2 values of 0.655 and 0.891, respectively. Contour maps from all studies provided significant insights. Molecular docking studies with molecular dynamics simulations were carried out on the highly potent compound 36. Furthermore, four acridine derivatives were designed and docking results of these designed compounds showed the same interactions as that of the standard PI-103 which proved the efficiency of 3D-QSAR and MD/MS study. In future, this study might be useful prior to synthesis for the designing of novel mTOR inhibitors.

Signal transducer and activator of transcription 3 (STAT3), a transcription factor responsive to the activation of cytokine receptors, is known for its oncogenic actions. Whilst STAT3α is the predominant spliceform in most tissues, alternative splicing of the STAT3 gene can generate a shorter STAT3β spliceform. Redirecting splicing to enhance STAT3β levels can result in tumor suppression in vivo, and so we evaluated the cellular basis underlying the anti-tumorigenic properties of STAT3β. To investigate the impact of increased STAT3β levels in cancer cells, we implemented a Morpholino-based antisense oligonucleotide strategy to modulate STAT3 spliceform expression in the MCF10CA1h cancer cells of the MCF10 series of human breast cancer cells. We employed nonsense-mediated decay (NMD) oligonucleotides and STAT3α-to-β expression switching (SWI) oligonucleotides to successfully induce STAT3 knockdown and redirect alternative splicing to increase STAT3β levels in MCF10CA1h cells, respectively. Importantly, assessment of the impacts of STAT3 splicing modulation on tumor cell biology showed that the SWI treatment significantly reduced MCF10CA1h cell growth, viability, and migration, whereas NMD treatment was without significant impact, although neither NMD nor SWI oligonucleotides significantly inhibited MCF10CA1h cell invasion through a semi-solid matrix. In conclusion, our data demonstrate that reduced breast cancer cell growth, viability and migration, but not invasion, follow the redirection of STAT3α-to-β expression switching to favour STAT3β expression.

Exon skipping is a promising genetic therapeutic strategy for restoring dystrophin expression in the treatment of Duchenne muscular dystrophy (DMD). The potential for newly synthesized dystrophin to trigger an immune response in DMD patients, however, is not well established. We have evaluated the effect of chronic phosphorodiamidate morpholino oligomer (PMO) treatment on skeletal muscle pathology and asked whether sustained dystrophin expression elicits a dystrophin-specific autoimmune response. Here, two independent cohorts of dystrophic mdx mice were treated chronically with either 800 mg/kg/month PMO for 6 months (n = 8) or 100 mg/kg/week PMO for 12 weeks (n = 11). We found that significant muscle inflammation persisted after exon skipping in skeletal muscle. Evaluation of humoral responses showed serum-circulating antibodies directed against de novo dystrophin in a subset of mice, as assessed both by Western blotting and immunofluorescent staining; however, no dystrophin-specific antibodies were observed in the control saline-treated mdx cohorts (n = 8) or in aged (12-month-old) mdx mice with expanded 'revertant' dystrophin-expressing fibers. Reactive antibodies recognized both full-length and truncated exon-skipped dystrophin isoforms in mouse skeletal muscle. We found more antigen-specific T-cell cytokine responses (e.g. IFN-g, IL-2) in dystrophin antibody-positive mice than in dystrophin antibody-negative mice. We also found expression of major histocompatibility complex class I on some of the dystrophin-expressing fibers along with CD8+ and perforin-positive T cells in the vicinity, suggesting an activation of cell-mediated damage had occurred in the muscle. Evaluation of complement membrane attack complex (MAC) deposition on the muscle fibers further revealed lower MAC deposition on muscle fibers of dystrophin antibody-negative mice than on those of dystrophin antibody-positive mice. Our results indicate that de novo dystrophin expression after exon skipping can trigger both cell-mediated and humoral immune responses in mdx mice. Our data highlights the need to further investigate the autoimmune response and its long-term consequences after exon-skipping therapy. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antisense oligonucleotide (AO)-mediated splice modulation has been established as a therapeutic approach for tackling genetic diseases. Recently, Exondys51, a drug that aims to correct splicing defects in the dystrophin gene was approved by the US Food and Drug Administration (FDA) for the treatment of Duchenne muscular dystrophy (DMD). However, Exondys51 has relied on phosphorodiamidate morpholino oligomer (PMO) chemistry which poses challenges in the cost of production and compatibility with conventional oligonucleotide synthesis procedures. One approach to overcome this problem is to construct the AO with alternative nucleic acid chemistries using solid-phase oligonucleotide synthesis via standard phosphoramidite chemistry. 2′-Fluoro (2′-F) is a potent RNA analogue that possesses high RNA binding affinity and resistance to nuclease degradation with good safety profile, and an approved drug Macugen containing 2′-F-modified pyrimidines was approved for the treatment of age-related macular degeneration (AMD). In the present study, we investigated the scope of 2′-F nucleotides to construct mixmer and gapmer exon skipping AOs with either 2′-O-methyl (2′-OMe) or locked nucleic acid (LNA) nucleotides on a phosphorothioate (PS) backbone, and evaluated their efficacy in inducing exon-skipping in mdx mouse myotubes in vitro. Our results showed that all AOs containing 2′-F nucleotides induced efficient exon-23 skipping, with LNA/2′-F chimeras achieving better efficiency than the AOs without LNA modification. In addition, LNA/2′-F chimeric AOs demonstrated higher exonuclease stability and lower cytotoxicity than the 2′-OMe/2′-F chimeras. Overall, our findings certainly expand the scope of constructing 2′-F modified AOs in splice modulation by incorporating 2′-OMe and LNA modifications.

Duchenne muscular dystrophy is a severe, progressive muscle-wasting disease that is caused by mutations that abolish the production of functional dystrophin protein. The exon skipping approach aims to restore the disrupted dystrophin reading frame, to allow the production of partially functional dystrophins, such as found in the less severe Becker muscular dystrophy. Exon skipping is achieved by antisense oligonucleotides (AONs). Several chemical modifications have been tested in nonclinical and clinical trials. The morpholino phosphorodiamidate oligomer eteplirsen has been approved by the Food and Drug Administration, whereas clinical development with the 2 '-O-methyl phosphorothioate (2OMePS) AON drisapersen was recently stopped. In this study, we aimed to study various aspects of 2OMePS AONs in nonclinical animal studies. We show that while efficiency of exon skipping restoration is comparable in young and older C57BL/10ScSn-Dmd(mdx)/J (mdx/BL10) mice, functional improvement was only observed for younger treated mice. Muscle quality did not affect exon skipping efficiency as exon skip and dystrophin levels were similar between mdx/BL10 and more severely affected, age-matched D2-mdx mice. We further report that treadmill running increases AON uptake and dystrophin levels in mdx/BL10 mice. Finally, we show that even low levels of exon skipping and dystrophin restoration are sufficient to significantly increase the survival of mdx-utrn-/- mice from 70 to 97 days.

The molecular chaperon MRJ (DNAJB6) exhibits two splice isoforms that have different roles in human viral infection, but the regulatory mechanism of MRJ isoform expression is yet unclear. In this study, we show that reduction of the polyadenylation factor CstF64 was correlated with the increase of the MRJ large isoform (MRJ-L) in human macrophages and elucidate the mechanism underlying CstF64-modulated MRJ isoform expression. Moreover, we exploited an antisense strategy targeting MRJ-L for virus replication. A morpholino oligonucleotide complementary to the 5′ splice site of MRJ intron 8 downregulated MRJ-L expression and suppressed the replication of not only HIV-1 but also respiratory syncytial virus (RSV). We demonstrated that downregulation of the MRJ-L level reduced HIV-1 replication as well as the subgenomic mRNA and viral production of RSV. The present findings that two human health-threatening viruses take advantage of MRJ-L for infection suggest MRJ-L as a potential target for broad-spectrum antiviral strategy., Graphical Abstract

Mutation of FOXC1 causes Axenfeld-Rieger Syndrome (ARS) with early onset or congenital glaucoma. We assessed retinal ganglion cell (RGC) number in zebrafish due to CRISPR-mediated mutation and antisense inhibition of two-forkhead box transcription factors, foxc1a and foxc1b. These genes represent duplicated homologues of human FOXC1. Using a CRISPR induced null mutation in foxc1b, in combination with antisense inhibition of foxc1a, we demonstrate reduced cell number in the retinal ganglion cell layer of developing zebrafish eyes. As early as 5 days post fertilization (dpf), fewer RGCs are found in foxc1b homozygous mutants injected with foxc1a morpholinos, and a thinner optic nerve results. Our data illustrates that foxc1 is required for the expression of atonal homolog 7 (atoh7), a gene that is necessary for RGC differentiation. As markers of differentiated RGCs (pou4f2) are downregulated in foxc1b−/− mutants injected with foxc1a morpholinos and no cell death is observed, our results are consistent with defects in the differentiation of RGCs leading to reduced cell number, as opposed to increased cell death of RGCs or off targets effects of morpholino injection. Our zebrafish model demonstrates that aberrant regulation of RGC number could act in concert with other known glaucoma risk factors to influence the development of congenital and early onset glaucoma due to FOXC1 mutation.

Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) is a promising treatment strategy for Duchenne muscular dystrophy (DMD). The most significant limitation of these clinically used compounds is their lack of delivery systems that target muscles; thus, cell-penetrating peptides are being developed to enhance uptake into muscles. Recently, we reported that uptake of peptide-conjugated PMOs into myofibers was mediated by scavenger receptor class A (SR-A), which binds negatively charged ligands. However, the mechanism by which the naked PMOs are taken up into fibers is poorly understood. In this study, we found that PMO uptake and exon-skipping efficiency were promoted in dystrophin-deficient myotubes via endocytosis through a caveolin-dependent pathway. Interestingly, SR-A1 was upregulated and localized in juxtaposition with caveolin-3 in these myotubes and promoted PMO-induced exon skipping. SR-A1 was also upregulated in the skeletal muscle of mdx52 mice and mediated PMO uptake. In addition, PMOs with neutral backbones had negative zeta potentials owing to their nucleobase compositions and interacted with SR-A1. In conclusion, PMOs with negative zeta potential were taken up into dystrophin-deficient skeletal muscle by upregulated SR-A1. Therefore, the development of a drug delivery system targeting SR-A1 could lead to highly efficient exon-skipping therapies for DMD. Keywords: phosphorodiamidate morpholino oligomer, exon skipping, oligonucleotide uptake, scavenger receptor, zeta potential, Duchenne muscular dystrophy, endocytosis, splice switching, mdx52, delivery

International audience; Primary ciliary dyskinesia (PCD) is a genetic disorder in which impaired ciliary function leads to chronic airway disease. Exome sequencing of a PCD subject identified an apparent homozygous frameshift variant, c.887_890delTAAG (p.Val296Glyfs∗13), in exon 5; this frameshift introduces a stop codon in amino acid 308 of the growth arrest-specific protein 2-like 2 (GAS2L2). Further genetic screening of unrelated PCD subjects identified a second proband with a compound heterozygous variant carrying the identical frameshift variant and a large deletion (c.867_∗343+1207del; p.?) starting in exon 5. Both individuals had clinical features of PCD but normal ciliary axoneme structure. In this research, using human nasal cells, mouse models, and X.laevis embryos, we show that GAS2L2 is abundant at the apical surface of ciliated cells, where it localizes with basal bodies, basal feet, rootlets, and actin filaments. Cultured GAS2L2-deficient nasal epithelial cells from one of the affected individuals showed defects in ciliary orientation and had an asynchronous and hyperkinetic (GAS2L2-deficient = 19.8 Hz versus control = 15.8 Hz) ciliary-beat pattern. These results were recapitulated in Gas2l2-/- mouse tracheal epithelial cell (mTEC) cultures and in X. laevis embryos treated with Gas2l2 morpholinos. In mice, the absence of Gas2l2 caused neonatal death, and the conditional deletion of Gas2l2 impaired mucociliary clearance (MCC) and led to mucus accumulation. These results show that a pathogenic variant in GAS2L2 causes a genetic defect in ciliary orientation and impairs MCC and results in PCD

Drug-free macromolecular therapeutics (DFMT) is a new paradigm for the treatment of B cell malignancies. Apoptosis is initiated by the biorecognition of complementary oligonucleotide motifs at the cell surface resulting in crosslinking of CD20 receptors. DMFT is composed from two nanoconjugates: 1) bispecific engager, Fab’-MORF1 (anti-CD20 Fab’ fragment conjugated with morpholino oligonucleotide), and 2) a crosslinking (effector) component P-(MORF2)(X) (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer grafted with multiple copies of complementary morpholino oligonucleotide). We evaluated this concept in 44 samples isolated from patients diagnosed with various subtypes of B cell malignancies. Apoptosis was observed in 65.9% of the samples tested. Pretreatment of cells with gemcitabine (GEM) or polymer-gemcitabine conjugate (2P-GEM) enhanced CD20 expression levels thus increasing apoptosis induced by DFMT. These positive results demonstrated that DFMT has remarkable therapeutic potential in various subtypes of B cell malignancies.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Trim-Away is a recent technique to rapidly deplete a protein from any cell type. Guided by antibodies, TRIM21 selects proteins for destruction. However, the applicability of this method in model organisms has not been investigated. Here, we show that Trim-Away can degrade proteins in zebrafish embryos. Trim-Away depletes proteins faster than morpholinos, which enables analysis of protein function during early embryogenesis. Furthermore, Trim-Away can be applied to evaluate the role of maternally contributed proteins in zebrafish embryos. Our findings indicate that Trim-Away is a powerful tool to perform functional analysis of proteins during zebrafish development. Electronic supplementary material The online version of this article (10.1186/s13059-019-1624-4) contains supplementary material, which is available to authorized users.

Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response.

A new cyclic enantiopure β-amino acid, named β-Morph, containing the morpholino ring, was obtained in gram scale starting from an α-D-glucopyranose enantiopure material, focusing on the “environmental sustainability” concept. A series of ultrashort model peptides containing β-Morph were prepared. β-Morph was found to be able to induce an inverse γ-turn in Leu-Val containing sequences. The key element for the γ-turn formation might be the oxygen atom of the morpholino ring that could drive the spatial orientation of the NH of amino acid i + 1, through an unusual hydrogen bond. This datum was confirmed by QTAIM calculations. Interestingly, when two β-Morph-Leu-Val repeats are present in the peptide, two γ-turns can be formed, as supported by NMR experiments and aMD and H-REMD calculations.

The centrosomal protein γ-tubulin is part of the cytoplasmic γ-tubulin small (γ-TuSCs) and large complexes (γ-TuRCs). Both, molecular and cellular evidence indicate that γ-tubulin plays a central role in microtubule nucleation and mitotic spindle formation. However, the molecular mechanisms of complex formation and subsequent biological roles in animal development remain unclear. Here, we used γ-tubulin gene knockdown in the zebrafish early embryo model to gain insights into its activity and cellular contribution during vertebrate embryogenesis. γ-Tubulin loss-of-function impaired γ-TuSC formation, impacting the microtubule nucleation rate in vitro. Moreover, decreased γ-tubulin synthesis caused dramatic defects in nuclear dynamics and cell cycle progression, leading to developmental arrest at the mid-gastrula stage. At the subcellular level, microtubule organization and function were altered, affecting chromosome segregation and triggering cell proliferation arrest and apoptosis. Our results suggest that de novo translated γ-tubulin participates in γ-TuSC formation required for early animal development. Importantly, formation of this complex is essential for both centrosome assembly and function, and cell proliferation. Thus, γ-TuSC integrity appears to be critical for cell cycle progression, and concomitantly, for coordinating the many distinct activities carried out by the early embryo. Our findings identify a novel role for γ-TuSC in the regulation of early vertebrate embryogenesis, providing molecular and biochemical starting points for future in depth studies of γ-tubulin functionality and its specific role in development.

The potency of antisense oligonucleotide (ASO) drugs has significantly improved in the clinic after exploiting asialoglycoprotein receptor (ASGR) mediated delivery to hepatocytes. To further this technology, we evaluated the structure-activity relationships of oligonucleotide chemistry on in vivo potency of GalNAc-conjugated Gapmer ASOs. GalNAc conjugation improved potency of ASOs containing 2'-O-methyl (2'-O-Me), 3'-fluoro hexitol nucleic acid (FHNA), locked nucleic acid (LNA), and constrained ethyl bicyclo nucleic acid (cEt BNA) 10-20-fold compared to unconjugated ASOs. We further demonstrate that GalNAc conjugation improves activity of 2'-O-(2-methoxyethyl) (2'-O-MOE) and Morpholino ASOs designed to correct splicing of survival motor neuron (SMN2) pre-mRNA in liver after subcutaneous administration. GalNAc modification thus represents a viable strategy for enhancing potency of ASO with diverse nucleic acid modifications and mechanisms of action for targets expressed in hepatocytes.

Dysferlinopathy is a progressive myopathy caused by mutations in the dysferlin (DYSF) gene. Dysferlin protein plays a major role in plasma-membrane resealing. Some patients with DYSF deletion mutations exhibit mild symptoms, suggesting some regions of DYSF can be removed without significantly impacting protein function. Antisense-mediated exon-skipping therapy uses synthetic molecules called antisense oligonucleotides to modulate splicing, allowing exons harboring or near genetic mutations to be removed and the open reading frame corrected. Previous studies have focused on DYSF exon 32 skipping as a potential therapeutic approach, based on the association of a mild phenotype with the in-frame deletion of exon 32. To date, no other DYSF exon-skipping targets have been identified, and the relationship between DYSF exon deletion pattern and protein function remains largely uncharacterized. In this study, we utilized a membrane-wounding assay to evaluate the ability of plasmid constructs carrying mutant DYSF, as well as antisense oligonucleotides, to rescue membrane resealing in patient cells. We report that multi-exon skipping of DYSF exons 26–27 and 28–29 rescues plasma-membrane resealing. Successful translation of these findings into the development of clinical antisense drugs would establish new therapeutic approaches that would be applicable to ∼5%–7% (exons 26–27 skipping) and ∼8% (exons 28–29 skipping) of dysferlinopathy patients worldwide. Keywords: exon skipping, antisense, morpholino, dysferlin, dysferlinopathy, limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy, plasma membrane, membrane wounding

Glioblastoma (GBM) is a deadly disease due to its ability to quickly invade and destroy brain tissue. Slowing or stopping GBM cell progression is crucial to help those inflicted with the disease. Our lab created an embryo-larval zebrafish xenograft model as a tool to study human GBM progression in an observable brain environment. The zebrafish brain is a dynamic and complex environment providing an optimal setting for studying GBM cell progression. Here we demonstrate the ability of our model to quantitate GBM proliferation, dispersal, blood vessel association, microtumor formation, and individual cell invasion by evaluating the importance of an extracellular matrix protein, laminin alpha 5 (lama5), on U251MG cell progression. Lama5 has been implicated in cancer cell survival, proliferation and invasion and is a known adhesion site for GBM cells. While lama5 is highly expressed in endothelial cells in the brain, it is unknown how lama5 affects GBM behavior. Using a lama5 morpholino, we discovered that lama5 decreased U251MG dispersal by 23% and doubles the formation of blood vessel dependent microtumors. Despite lama5 being a known attachment site for GBM, lama5 expression had no effect on U251MG association with blood vessels. Analysis of individual U251MG cells revealed lama5 significantly lowered invasion as mobile U251MG cells traveled 32.5 μm less, invaded 5.0 μm/hr slower and initiated invasion 60% few times per cell.

Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole-exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells, and it restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping, and it is potentially applicable to 10.1% of patients with DMD. Keywords: Duchenne muscular dystrophy, dystrophin, exon 53, exon skipping, antisense therapeutics, morpholino

This review discusses why the embryo requires vitamin E (VitE) and shows that its lack causes metabolic dysregulation and impacts morphological changes at very early stages in development, which occur prior to when a woman knows she is pregnant. VitE halts the chain reactions of lipid peroxidation (LPO). Metabolomic analyses indicate that thiols become depleted in E− embryos because LPO generates products that require compensation using limited amino acids and methyl donors that are also developmentally relevant. Thus, VitE protects metabolic networks and the integrated gene expression networks that control development. VitE is critical especially for neurodevelopment, which is dependent on trafficking by the α-tocopherol transfer protein (TTPa). VitE-deficient (E−) zebrafish embryos initially appear normal, but by 12 and 24 h post-fertilization (hpf) E− embryos are developmentally abnormal with expression of pax2a and sox10 mis-localized in the midbrain-hindbrain boundary, neural crest cells and throughout the spinal neurons. These patterning defects indicate cells that are especially in need of VitE-protection. They precede obvious morphological abnormalities (cranial-facial malformation, pericardial edema, yolksac edema, skewed body-axis) and impaired behavioral responses to locomotor activity tests. The TTPA gene (ttpa) is expressed at the leading edges of the brain ventricle border. Ttpa knockdown using morpholinos is 100% lethal by 24 hpf, while E− embryo brains are often over- or under-inflated at 24 hpf. Further, E− embryos prior to 24 hpf have increased expression of genes involved in glycolysis and the pentose phosphate pathway, and decreased expression of genes involved in anabolic pathways and transcription. Combined data from both gene expression and the metabolome in E− embryos at 24 hpf suggest that the activity of the mechanistic Target of Rapamycin (mTOR) signaling pathway is decreased, which may impact both metabolism and neurodevelopment. Further evaluation of VitE deficiency in neurogenesis and its subsequent impact on learning and behavior is needed.

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

The focal adhesion protein Kindlin2 is essential for integrin activation, a process that is fundamental to cell-extracellular matrix adhesion. Kindlin 2 (Fermt2) is widely expressed in mouse embryos, and its absence causes lethality at the peri-implantation stage due to the failure to trigger integrin activation. The function of kindlin2 during embryogenesis has not yet been fully elucidated as a result of this early embryonic lethality. Here, we showed that kindlin2 is essential for neural crest (NC) formation in Xenopus embryos. Loss-of-function assays performed with kindlin2-specific morpholino antisense oligos (MOs) or with CRISPR/Cas9 techniques in Xenopus embryos severely inhibit the specification of the NC. Moreover, integrin-binding-deficient mutants of Kindlin2 rescued the phenotype caused by loss of kindlin2, suggesting that the function of kindlin2 during NC specification is independent of integrins. Mechanistically, we found that Kindlin2 regulates the fibroblast growth factor (FGF) pathway, and promotes the stability of FGF receptor 1. Our study reveals a novel function of Kindlin2 in regulating the FGF signaling pathway and provides mechanistic insights into the function of Kindlin2 during NC specification.