Morpholino Oligonucleotides Research Articles

Vivo-Morpholino-Based Antiviral for SARS-CoV-2: Implications for Novel Therapies in the Treatment of Acute COVID-19 Disease.

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.

Crosslinking of CD38 Receptors Triggers Apoptosis of Malignant B Cells.

Recently, we designed an inventive paradigm in nanomedicine—drug-free macromolecular therapeutics (DFMT). The ability of DFMT to induce apoptosis is based on biorecognition at cell surface, and crosslinking of receptors without the participation of low molecular weight drugs. The system is composed of two nanoconjugates: a bispecific engager, antibody or Fab’ fragment—morpholino oligonucleotide (MORF1) conjugate; the second nanoconjugate is a multivalent effector, human serum albumin (HSA) decorated with multiple copies of complementary MORF2. Here, we intend to demonstrate that DFMT is a platform that will be effective on other receptors than previously validated CD20. We appraised the impact of daratumumab (DARA)- and isatuximab (ISA)-based DFMT to crosslink CD38 receptors on CD38+ lymphoma (Raji, Daudi) and multiple myeloma cells (RPMI 8226, ANBL-6). The biological properties of DFMTs were determined by flow cytometry, confocal fluorescence microscopy, reactive oxygen species determination, lysosomal enlargement, homotypic cell adhesion, and the hybridization of nanoconjugates. The data revealed that the level of apoptosis induction correlated with CD38 expression, the nanoconjugates meet at the cell surface, mitochondrial signaling pathway is strongly involved, insertion of a flexible spacer in the structure of the macromolecular effector enhances apoptosis, and simultaneous crosslinking of CD38 and CD20 receptors increases apoptosis.

Microinjection of Xenopus tropicalis Embryos.

Microinjection is an important technique used to study development in the oocyte and early embryo. In Xenopus, substances such as DNA, mRNA, and morpholino oligonucleotides have traditionally been injected into Xenopus laevis, because of their large embryo size and the relatively long time from their fertilization to first division. In the past few decades, Xenopus tropicalis has become an important model in developmental biology; it is particularly useful in genetic studies. The advent and rapid development of CRISPR-Cas9 technology has provided an array of targeted gene manipulations for which X. tropicalis is particularly suited. The equipment and protocol for X. tropicalis microinjection is broadly transferable from X. laevis There are important differences between the species to consider, however, including the smaller embryo size and faster embryo development time in X. tropicalis There are a number of solutions and reagents that differ in concentration and composition as well. Here we describe a microinjection protocol specifically for studies in X. tropicalis.

ETV6/TEL1 Is Required for HSC Emergence during Embryonic Development.

ETV6/TEL1 (hereinafter referred to as TEL1) is an ets family transcription factor that is frequently involved in chromosomal translocations associated with leukaemia, including t(12:21) which generates the TEL-AML1 fusion gene, the most common genetic abnormality in childhood cancer. The role of TEL1 in normal haemopoiesis has been extensively studied in the mouse where knock-out studies have indicated that TEL1 is not required for development of primitive (ie yolk sac-derived) haemopoiesis. TEL1-knockout mice die at day E10.5 due to a defect in angiogenesis precluding direct analysis of the definitive (ie intraembryonically-derived) haematopoietic system. Analysis of chimaeric mice generated using TEL1−/− ES cells suggest that TEL1 is not required for generation of the definitive haemopoietic system during ontogeny but is required for HSC survival perinatally at bone marrow stages. Conditional knockout studies have subsequently identified a role for TEL1 in the survival of adult bone marrow-derived HSC. We now report the analysis of TEL1 function in amphibian (Xenopus) embryos where haemopoietic development can be studied even in embryos with defective angiogenesis. We characterised the TEL1 expression pattern and knocked down its function using antisense morpholino oligonucleotide injection. Consistent with the mouse knockout, specification of the primitive haemopoietic system was unaffected, however a defect in maturation of primitive erythroid cells, not previously reported in the mouse, was also apparent. We next analysed the development of the definitive haemopoietic system, thought to arise as clusters of HSCs in the floor of the dorsal aorta. But, no clusters of HSCs were found. Formation of the dorsal aorta however, proceeded normally, indicating that TEL1 controls the decision between the arterial and HSC programmes. Given, the critical role for TEL1 in the development of HSC indicated by these studies, a temporal analysis of emergence of intraembryonic HSC in TEL-deficient mice may be warranted.

Donepezil Reduces Amyloid Precursor Protein Endocytosis by Resulting from Increase in the Expression of Sorting Nexin Protein 33

Donepezil, the most widely used drug for the treatment of Alzheimer's disease (AD), is an acetylcholinesterase (AChE) inhibitor and is thought to improve cognition by stimulating cholinergic neurotransmission. However, no correlation has yet been established between the inhibitory role of AChE inhibitors and their therapeutic effects when used in AD patients. The cleavage pathway of amyloid precursor protein (APP) includes amyloidgenic (β, γ-cleavage) and non-amyloidgenic (α-cleavage) pathways. The intracellular transportation of APP is important in determining these cleavage pathways. It has been suggested that sorting nexin (SNX) family proteins regulates the intracellular transport of APP, thereby enhancing α-cleavage. In this study, we examined the effects of donepezil on SNX33 expression changes and APP processing in primary cultures of fetal rat cortical neurons. While donepezil treatment increased the levels of SNX33 expression and soluble APPα (sAPPα) in culture media, no changes were observed regarding full-length APP expression in the cell lysate. Donepezil also reduced the release of amyloid β (Aβ) into culture media in a concentration- and time-dependent manner. This reduction was not affected by acetylcholine receptor antagonists. The membrane surface expression of APP was elevated by donepezil. Furthermore, SNX knockdown by antisense morpholino oligos prevented the effects of donepezil. These results indicated that donepezil increased APP expression at the surface of the plasma membrane by decreasing APP endocytosis through upregulation of SNX33, suggesting donepezil might stimulate the non-amyloidogenic pathway. This new mechanism of action for the currently used anti-AD drug may provide a valuable basis for future drug discovery.

The cytokine FAM3B/PANDER is an FGFR ligand that promotes posterior development in Xenopus

Fibroblast growth factor (FGF)/extracellular signal-regulated kinase (ERK) signaling plays a crucial role in anterior-posterior (A-P) axial patterning of vertebrate embryos by promoting posterior development. In our screens for novel developmental regulators in Xenopus embryos, we identified Fam3b as a secreted factor regulated in ectodermal explants. Family with sequence similarity 3 member B (FAM3B)/PANDER (pancreatic-derived factor) is a cytokine involved in glucose metabolism, type 2 diabetes, and cancer in mammals. However, the molecular mechanism of FAM3B action in these processes remains poorly understood, largely because its receptor is still unidentified. Here we uncover an unexpected role of FAM3B acting as a FGF receptor (FGFR) ligand in Xenopus embryos. fam3b messenger RNA (mRNA) is initially expressed maternally and uniformly in the early Xenopus embryo and then in the epidermis at neurula stages. Overexpression of Xenopus fam3b mRNA inhibited cephalic structures and induced ectopic tail-like structures. Recombinant human FAM3B protein was purified readily from transfected tissue culture cells and, when injected into the blastocoele cavity, also caused outgrowth of tail-like structures at the expense of anterior structures, indicating FGF-like activity. Depletion of fam3b by specific antisense morpholino oligonucleotides in Xenopus resulted in macrocephaly in tailbud tadpoles, rescuable by FAM3B protein. Mechanistically, FAM3B protein bound to FGFR and activated the downstream ERK signaling in an FGFR-dependent manner. In Xenopus embryos, FGFR activity was required epistatically downstream of Fam3b to mediate its promotion of posterior cell fates. Our findings define a FAM3B/FGFR/ERK-signaling pathway that is required for axial patterning in Xenopus embryos and may provide molecular insights into FAM3B-associated human diseases.

Proapoptotic Bad Involved in Brain Development, When Severely Defected, Induces Dramatic Malformation in Zebrafish.

The BH3-only molecule Bad regulates cell death via its differential protein phosphorylation, but very few studies address its effect on early embryonic development in vertebrate systems. In this work, we examined the novel role of zebrafish Bad in the initial programmed cell death (PCD) for brain morphogenesis through reducing environmental stress and cell death signaling. Bad was considered to be a material factor that because of the knockdown of Bad by morpholino oligonucleotides, PCD was increased and the reactive oxygen species (ROS) level was enhanced, which correlated to trigger a p53/caspase-8 involving cell death signaling. This Bad knockdown-mediated environmental stress and enhanced cell dying can delay normal cell migration in the formation of the three germ layers, especially the ectoderm, for further brain development. Furthermore, Bad defects involved in three-germ-layers development at 8 hpf were identified by in situ hybridization approach on cyp26, rtla, and Sox17 pattern expression markers. Finally, the Bad knockdown-induced severely defected brain was examined by tissue section from 24 to 48 h postfertilization (hpf), which correlated to induce dramatic malformation in the hindbrain. Our data suggest that the BH3-only molecule Bad regulates brain development via controlling programmed cell death on overcoming environmental stress for reducing secondary cell death signaling, which suggests that correlates to brain developmental and neurological disorders in this model system.

RNA‐binding protein Elavl1/HuR is required for maintenance of cranial neural crest specification

Neural crest (NC) cells are a multipotent cell population, unique to the vertebrate species, which gather at the dorsal neural tube and undergo an epithelial-to-mesenchymal transition (EMT). NC then migrate throughout the developing embryo contributing to various cell types such as craniofacial cartilage, neurons, and elements of the peripheral nervous system. While a gene regulatory network (GRN) underlies establishment of NC formation, recent studies suggest a role for post-transcriptional regulation in modulating the output of these regulatory circuits. In this study, we examined RNA-sequencing data from avian embryos at different stages of NC development and found enrichment of Elav1, a transcript encoding for the RNA-binding protein HuR. Using immunohistochemical analyses we characterized the spatiotemporal expression of HuR. We observed HuR expression following the establishment of the neural plate border and upon closure of the neural tube, with an enrichment in expression coinciding with NC formation. Using antisense morpholino oligo to perturb HuR expression, we observed loss of early neural crest specifier FoxD3 and its activator Axud1, as well as the EMT regulator Draxin. In addition, we identified several putative HuR binding sites within the predicted secondary structure of Draxin 3’UTR. RNA pulldown further showed Draxin is a specific target of HuR. Importantly, overexpression of exogenous Draxin was able to rescue NC specification defects observed in HuR knockdowns. Our results demonstrate a critical role for HuR in maintenance of cranial NC specification, at least partially via Draxin mRNA stabilization. Together, these data highlight an important intersection of post-transcriptional regulation with modulation of the neural crest specification GRN.

Radiation enhances the delivery of antisense oligonucleotides and improves chemo-radiation efficacy in brain tumor xenografts

Overexpression of O6-methylguanine DNA methyltransferase (MGMT) contributes to resistance to chemo-radiation therapy (CRT) in brain tumors. We previously demonstrated that non-ablative radiation improved delivery of anti-MGMT morpholino oligonucleotides (AMONs) to reduce MGMT levels in subcutaneous tumor xenografts. We evaluate this approach to enhance CRT efficacy in rat brain tumor xenograft models. The impact of radiation on targeted delivery was evaluated using fluorescent oligonucleotides (f-ON). In vitro, f-ON was localized to clathrin-coated vesicles, endosomes, and lysosomes using confocal microscopy in T98G glioma cells. In vivo, fluorescence was detected in pre-radiated, but not non-radiated Long Evans (non-tumor bearing) rat brains. Cranial radiation (2 Gy) followed by AMONs (intravenous, 10.5 mg/kg) reduced MGMT expression by 50% in both orthotopic cerebellar D283 medulloblastoma and intracerebral H460 non-small cell lung carcinoma (NSCLC) xenograft models. To evaluate the efficacy, AMONs concurrent with CRT (2 Gy radiation plus oral 20 mg/kg temozolomide ×4 days) reduced tumor volumes in the medulloblastoma model (p = 0.012), and a similar trend was found in the NSCLC brain metastasis model. We provide proof of concept for the use of non-ablative radiation to guide and enhance the delivery of morpholino oligonucleotides into brain tumor xenograft models to reduce MGMT levels and improve CRT efficacy.

English

Long non-coding RNAs (lncRNA) are increasingly being regard as potential key regulators of biological process, however, little is known about the function of most of them. The involvement of a novel lncRNA (ENSDART000000150571), previously named TCONS_00028652, in intersegmental vessel development was investigated in this study. TCONS_00028652, having a single exon (568 bp), is located in chromosome 16 at position from 15786804 to 15786237, which was previously identified as an embryonic and adult heart-enrichedlncRNA. Bioinformatics analysis and annotation of TCONS_00028652 was performed using online databases. Its protein-coding potential was assessed using online softwares: the Coding Potential Assessing Tool (CPAT) and the Coding Potential Calculator (CPC). To verify its real existence, a cloned fragment was proliferated by designing primers against 5’ and 3’ exon-flanking sequence. Subsequently, spatiotemporal expression during zebrafish embryonic development was determined using real-time quantitative PCR (qPCR) and whole mount in situ hybridization. We found that lncRNA TCONS_00028652 was generally expressed throughout early stages of zebrafish embryonic development and predominantly in embryonic brain, tail, and heart. Knockdown of TCONS_00028652 using morpholino oligonucleotides (MO) resulted in intersegmental vessel defects, suggesting that TCONS_00028652 is indispensable for embryonic vascular development in zebrafish. Using Tg (flil:EGFP) transgenic fish expressing a cardiovascular marker gene, loss of function experiments confirmed that TCONS_00028652 was involved in embryonic intersegmental vessel development. Our results may lead to valuable understanding of lncRNAs functions in zebrafish embryonic development and molecular mechanisms of embryonic cardiovascular development. © 2021 Friends Science Publishers

Rapid and unamplified identification of COVID-19 with morpholino-modified graphene field-effect transistor nanosensor

SARS-CoV-2 RNA is identified as a pivotal player to bolster energizing zones of COVID-19 detection. Herein, we develop a rapid and unamplified nanosensing platform for detection of SARS-CoV-2 RNA in human throat swab specimens. A gold nanoparticle (AuNP)-decorated graphene field-effect transistor (G-FET) sensor was fabricated, after which complementary phosphorodiamidate morpholino oligos (PMO) probe was immobilized on the AuNP surface. This sensor allowed for highly sensitive testing of SARS-CoV-2 RdRp as PMO does not have charges, leading to low background signal. Not only did the method present a low limit of detection in PBS (0.37 fM), throat swab (2.29 fM), and serum (3.99 fM), but also it achieved a rapid response to COVID-19 patients’ samples within 2 min. The developed nanosensor was capable of analyzing RNA extracts from 30 real clinical samples. The results show that the sensor could differentiate the healthy people from infected people, which are in high agreement with RT-PCR results (Kappa index = 0.92). Furthermore, a well-defined distinction between SARS-CoV-2 RdRp and SARS-CoV RdRp was also made. Therefore, we believe that this work provides a satisfactory, attractive option for COVID-19 diagnosis.

Functional Validation of a Pathogenic Missense Variant in Human Filamin C Cardiomyopathy through Disruption of a Zebrafish Homologue Recapitulates Cardiac Disease

Purpose We recently performed whole-genome sequencing (WGS) of an East Asian proband and family members with diverse cardiac diseases, including arrhythmias, cardiomyopathies, premature heart failure requiring transplant, and identified a pathogenic missense variant (p.G2151S) in exon 39 of the filamin C gene, FLNC. In this study, we aim to demonstrate recapitulation of the pathogenic effects of the variant using a zebrafish (Danio rerio) model. Methods The proband, 3 other affected and 3 unaffected family members of a four-generation nonconsanguineous Hong Kong Chinese pedigree of 25 individuals were sequenced by WGS with 30X coverage. Single nucleotide variants and insertion-deletions that met our inclusion criteria were shortlisted, and bioinformatic analysis and variant prioritization were performed using Kaviar, gnomAD and dbNSFP databases to identify damaging variants in known cardiac-specific genes. The study was approved by the university/hospital's institutional review board. Sequence alignment of the pathogenic gene region with the zebrafish homologue was performed using the GENESTREAM network server, Institut de Genetique Humaine, Montpellier, France. Knockdown of the region was carried out using microinjection of morpholino oligonucleotides (MO), or control oligomers. Embryonic lethality, survival after birth, animal and cardiac morphology, heart rate, and histopathology were analyzed. Results Using a zebrafish model, we performed sequence alignment and identified the corresponding point mutation in flnca and flncb. Knockdown of the homologous sequences using antisense morpholino oligonucleotides resulted in stunted growth and development of the animal, cardiomegaly, pericardial edema, dose-dependent increase in embryonic lethality (survival rates of 95.8%, 46.6% and 9.7% at day 3 post-fertilization for control oligomer (n=24), 0.1 ng MO (n=88) and 0.5 ng MO (n=154), respectively), and reduced heart rate (134, 108 and 103 beats per minute, respectively; P Conclusion Damaging effects of the missense variant in human filamin C cardiomyopathy have been confirmed in the homologous region in this zebrafish model. The variable phenotypic manifestations within this East Asian family remain to be elucidated.

In Vivo Modeling of Zebrafish Zinc Finger, MIZ-Type Containing 1 Expression and Its Effect on Pigmentation

Objective: The zinc finger, MIZ-type containing 1 (ZMIZ1) gene has been identified as a possible susceptibility gene associated with vitiligo, therefore we conducted this study to investigate the role of ZMIZ1 in pigmentation. Methods: We generate a zebrafish loss-of-function model using morpholino oligonucleotides (MOs), and two orthologs of human ZMIZ1 have been annotated (ZMIZ1a and ZMIZ1b). The expression profiles of ZMIZ1a and ZMIZ1b and their effects on the pigmentation in zebrafish were evaluated by using whole-mount in situ hybridization and melanin quantification. Statistical analysis was performed using the unpaired Student t-test or one-way analysis. Results: Investigation of the temporal and spatial expressions of these two transcripts suggested that the expressions of ZMIZ1a and ZMIZ1b in the brain start to emerge in a ubiquitous fashion from 2 days post-fertilization onwards. After the successful design and validation of MOs, we observed that ZMIZ1a and ZMIZ1b MOs caused embryonic developmental delays and malformations in zebrafish. Further analysis of the melanin content in the morphants revealed that ZMIZ1a significantly (49.1% for 0.667 mmol/L in ZMIZI1a group, P = 0.03) reduced the melanin content in a dose-dependent manner, but only the highest concentration of injected ZMIZ1b MOs significantly (50% for 0.667 mmol/L in ZMIZ1b group, P = 0.02) reduced the melanin content. A tyrosinase inhibition assay indicated no significant difference between the morphants and wild-type zebrafish. Conclusion: This study successfully modeled a susceptibility gene identified by genome-wide association studies in a zebrafish loss-of-function model and provides insights into the biological mechanism of pigmentation.

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133–150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133–150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.

The Atypical Kinase RIOK3 Limits RVFV Propagation and Is Regulated by Alternative Splicing.

In recent years, transcriptome profiling studies have identified changes in host splicing patterns caused by viral invasion, yet the functional consequences of the vast majority of these splicing events remain uncharacterized. We recently showed that the host splicing landscape changes during Rift Valley fever virus MP-12 strain (RVFV MP-12) infection of mammalian cells. Of particular interest, we observed that the host mRNA for Rio Kinase 3 (RIOK3) was alternatively spliced during infection. This kinase has been shown to be involved in pattern recognition receptor (PRR) signaling mediated by RIG-I like receptors to produce type-I interferon. Here, we characterize RIOK3 as an important component of the interferon signaling pathway during RVFV infection and demonstrate that RIOK3 mRNA expression is skewed shortly after infection to produce alternatively spliced variants that encode premature termination codons. This splicing event plays a critical role in regulation of the antiviral response. Interestingly, infection with other RNA viruses and transfection with nucleic acid-based RIG-I agonists also stimulated RIOK3 alternative splicing. Finally, we show that specifically stimulating alternative splicing of the RIOK3 transcript using a morpholino oligonucleotide reduced interferon expression. Collectively, these results indicate that RIOK3 is an important component of the mammalian interferon signaling cascade and its splicing is a potent regulatory mechanism capable of fine-tuning the host interferon response.

CRISPR/Cas9-mediated grna gene knockout leads to neurodevelopmental defects and motor behavior changes in zebrafish.

Progranulin (PGRN) is a secreted glycoprotein with multiple biological functions in early embryogenesis, anti-inflammation, and neurodegeneration. A good model for the functional study of PGRN is the zebrafish with knockdown or knockout of grn, the gene encoding PGRN. Morpholino oligonucleotides (MOs) and zinc finger nucleases have been used to generate zebrafish grn models, yet they have shown inconsistent phenotypes due to either the neurotoxicity of the MOs or possible genetic compensation responses during gene editing. In this study, we generated stable grna (one of the major grn homologues of zebrafish) knockout zebrafish by using CRISPR/Cas9-mediated genome editing. A grna sgRNA was designed to target the similar repeated sequence shared by exon 13, exon 15, and exon 19 in zebrafish. The F1 generation with the frameshift mutation of+4bp (the addition of 4bp to exon15), which causes a premature termination, was obtained and subjected to morphological and behavioral evaluation. The grna knockout zebrafish showed neurodevelopmental defects, including spinal motor neurons with shorter axons, decreased sensory hair cells, thinning of the outer nuclear layer and thickening of the inner nuclear layer of the retina, decreased expression of rhodopsin in the cone cells, and motor behavior changes. Moreover, the phenotypes of grna knockout zebrafish could be rescued with the Tol2 system carrying the grna gene. The grna knockout zebrafish model generated in this study provides a useful tool to study PGRN function and has potential for high-throughput drug screening for disease therapy.

SNRNP200 Mutations Cause Autosomal Dominant Retinitis Pigmentosa.

The small nuclear ribonucleoprotein 200 kDa (SNRNP200) gene plays a key role in the maturation of pre-message RNA (pre-mRNA) splicing with the indication for the etiology of retinitis pigmentosa (RP). Gene recognition can facilitate the diagnosis of these patients for better clinical management, treatment and counseling. This study aimed to outline the causative mutation in a Chinese family and the pathogenic mechanism of this SNRNP200 mutation in RP. Eighteen individuals from the affected family underwent a complete ophthalmic examination. Whole exome sequencing (WES) was conducted to identify the pathogenic variant in the proband, which was then confirmed by Sanger sequencing. Expression of the SNRNP200 transcript in zebrafish was identified via whole mount in situ hybridization. Morpholino oligonucleotide (MO) and SNRNP200 wild and mutant mRNA were injected into zebrafish embryos followed by analyses of the systemic changes and retinal phenotypes using immunofluorescence. Heterozygous SNRNP200c.C6088T (p.Arg2030Cys) mutation was ascertained in two members of this family: the proband and his father (II-2). Overexpression of SNRNP200Arg2030Cys, but not SNRNP200WT caused systemic deformities in the wild-type zebrafish embryos with the retina primarily injured, and significantly increased death rates in the morphant embryos, in which the orthologous zebrafish SNRNP200 gene was blocked. In conclusion, this study reports a novel heterozygous SNRNP200c.C6088T mutation, which is evidenced to cause RP via a dominant-negative effect.

Microtubule Severing Protein Fignl2 Contributes to Endothelial and Neuronal Branching in Zebrafish Development.

Previously, fidgetin (fign) and its family members fidgetin-like 1 (fignl1) and fidgetin-like 2 (fignl2) were found to be highly expressed during zebrafish brain development, suggesting their functions in the nervous system. In this study, we report the effects of loss-of-function of these genes on development. We designed and identified single-guide RNAs targeted to generate fign, fignl1, and fignl2 mutants and then observed the overall morphological and behavioral changes. Our findings showed that while fign and fignl1 null mutants displayed no significant defects, fignl2 null zebrafish mutants displayed pericardial edema, reduced heart rate, and smaller eyes; fignl2 null mutants responded to the light-darkness shift with a lower swimming velocity. fignl2 mRNAs were identified in vascular endothelial cells by in situ hybridization and re-analysis of an online dataset of single-cell RNAseq results. Finally, we used morpholino oligonucleotides to confirm that fignl2 knockdown resulted in severe heart edema, which was caused by abnormal vascular branching. The zebrafish fignl2 morphants also showed longer axonal length and more branches of caudal primary neurons. Taken together, we summarize that Fignl2 functions on cellular branches in endothelial cells and neurons. This study reported for the first time that the microtubule-severing protein Fignl2 contributes to cell branching during development.

A globin-family protein, Cytoglobin 1, is involved in the development of neural crest-derived tissues and organs in zebrafish

The zebrafish is an excellent model animal that is amenable to forward genetics approaches. To uncover unknown developmental regulatory mechanisms in vertebrates, we conducted chemical mutagenesis screening and identified a novel mutation, kanazutsi (kzt). This mutation is recessive, and its homozygotes are embryonic lethal. Mutant embryos suffered from a variety of morphological defects, such as head flattening, pericardial edema, circulation defects, disrupted patterns of melanophore distribution, dwarf eyes, a defective jaw, and extensive apoptosis in the head, which indicates that the main affected tissues are derived from neural crest cells (NCCs). The expression of tissue-specific markers in kzt mutants showed that the early specification of NCCs was normal, but their later differentiation was severely affected. The mutation was mapped to chromosome 3 by linkage analyses, near cytoglobin 1 (cygb1), the product of which is a globin-family respiratory protein. cygb1 expression was activated during somitogenesis in somites and cranial NCCs in wild-type embryos but was significantly downregulated in mutant embryos, despite the normal primary structure of the gene product. The kzt mutation was phenocopied by cygb1 knockdown with low-dose morpholino oligos and was partially rescued by cygb1 overexpression. Both severe knockdown and null mutation of cygb1, established by the CRISPR/Cas9 technique, resulted in far more severe defects at early stages. Thus, it is highly likely that the downregulation of cygb1 is responsible for many, if not all, of the phenotypes of the kzt mutation. These results reveal a requirement for globin family proteins in vertebrate embryos, particularly in the differentiation and subsequent development of NCCs.

Loss of PIGK function causes severe infantile encephalopathy and extensive neuronal apoptosis.

PIGK gene, encoding a key component of glycosylphosphatidylinositol (GPI) transamidase, was recently reported to be associated with inherited GPI deficiency disorders (IGDs). However, little is known about the specific downstream effects of PIGK on neurodevelopment due to the rarity of the disease and the lack of in vivo study. Here, we described 2 patients in a Chinese family presented with profound global developmental delay, severe hypotonia, seizures, and postnatal progressive global brain atrophy including hemisphere, cerebellar and corpus callosum atrophy. Two novel compound heterozygous variants in PIGK were identified via genetic analysis, which was proved to cause significant decrease of PIGK protein and reduced cell surface presence of GPI-APs in the patients. To explore the role of Pigk on embryonic and neuronal development, we constructed Pigk knock-down zebrafish and knock-in mouse models. Zebrafish injected with a small dose of morpholino oligonucleotides displayed severe developmental defects including small eyes, deformed head, curly spinal cord, and unconsumed yolk sac. Primary motor neuronal dysplasia and extensive neural cell apoptosis were further observed. Meanwhile, the mouse models, carrying the two variants respectively homologous with the patients, both resulted in complete embryonic lethality of the homozygotes, which suggested the intolerable effect caused by amino acid substitution of Asp204 as well as the truncated mutation. Our findings provide the in vivo evidence for the essential role of PIGK during the embryonic and neuronal development. Based on these data, we propose a basis for further study of pathological and molecular mechanisms of PIGK-related neurodevelopmental defects.