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Nucleic Acid Therapeutics Feb 2019The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the... (Review)
Review
Cell-Penetrating Peptide Conjugates of Steric Blocking Oligonucleotides as Therapeutics for Neuromuscular Diseases from a Historical Perspective to Current Prospects of Treatment.
The review starts with a historical perspective of the achievements of the Gait group in synthesis of oligonucleotides (ONs) and their peptide conjugates toward the award of the 2017 Oligonucleotide Therapeutic Society Lifetime Achievement Award. This acts as a prelude to the rewarding collaborative studies in the Gait and Wood research groups aimed toward the enhanced delivery of charge neutral ON drugs and the development of a series of Arg-rich cell-penetrating peptides called Pip (peptide nucleic acid/phosphorodiamidate morpholino oligonucleotide [PNA/PMO] internalization peptides) as conjugates of such ONs. In this review we concentrate on these developments toward the treatment of the neuromuscular diseases Duchenne muscular dystrophy and spinal muscular atrophy toward a platform technology for the enhancement of cellular and in vivo delivery suitable for widespread use as neuromuscular and neurodegenerative ON drugs.
Topics: Cell-Penetrating Peptides; Humans; Morpholinos; Muscular Atrophy, Spinal; Muscular Dystrophy, Duchenne; Neuromuscular Diseases; Peptide Nucleic Acids
PubMed: 30307373
DOI: 10.1089/nat.2018.0747 -
Molecular Biology of the Cell Feb 2019The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet...
The precise spatial and temporal expression of genes is essential for proper organismal development. Despite their importance, however, many developmental genes have yet to be identified. We have determined that Fer1l6, a member of the ferlin family of genes, is a novel factor in zebrafish development. We find that Fer1l6 is expressed broadly in the trunk and head of zebrafish larvae and is more restricted to gills and female gonads in adult zebrafish. Using both genetic mutant and morpholino knockdown models, we found that loss of Fer1l6 led to deformation of striated muscle tissues, delayed development of the heart, and high morbidity. Further, expression of genes associated with muscle cell proliferation and differentiation were affected. Fer1l6 was also detected in the C2C12 cell line, and unlike other ferlin homologues, we found Fer1l6 expression was independent of the myoblast-to-myotube transition. Finally, analysis of cell and recombinant protein-based assays indicate that Fer1l6 colocalizes with syntaxin 4 and vinculin, and that the putative C2 domains interact with lipid membranes. We conclude that Fer1l6 has diverged from other vertebrate ferlins to play an essential role in zebrafish skeletal and cardiac muscle development.
Topics: Animals; Cells, Cultured; Gene Expression Regulation, Developmental; Membrane Lipids; Mice; Morpholinos; Muscle Development; Muscles; Mutation; Qa-SNARE Proteins; Transcription, Genetic; Vesicular Transport Proteins; Vinculin; Zebrafish; Zebrafish Proteins
PubMed: 30516436
DOI: 10.1091/mbc.E18-06-0401 -
Developmental Biology Jul 2021Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered...
Echinoderms are important experimental models for analyzing embryonic development, but a lack of spatial and temporal control over gene perturbations has hindered developmental studies using these animals. Morpholino antisense oligonucleotides (MOs) have been used successfully by the echinoderm research community for almost two decades, and MOs remain the most widely used tool for acute gene knockdowns in these organisms. Echinoderm embryos develop externally and are optically transparent, making them ideally-suited to many light-based approaches for analyzing and manipulating development. Studies using zebrafish embryos have demonstrated the effectiveness of photoactivatable (caged) MOs for conditional gene knockdowns. Here we show that caged MOs, synthesized using nucleobase-caged monomers, provide light-regulated control over gene expression in sea urchin embryos. Our work provides the first robust approach for conditional gene silencing in this prominent model system.
Topics: Animals; Embryo, Nonmammalian; Embryonic Development; Gene Expression; Gene Expression Regulation; Gene Knockdown Techniques; Gene Silencing; Morpholinos; Oligonucleotides, Antisense; Sea Urchins
PubMed: 33684434
DOI: 10.1016/j.ydbio.2021.02.014 -
Methods in Cell Biology 2016The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse...
The zebrafish has been a powerful model in forward genetic screens to identify genes essential for organogenesis and embryonic development. Conversely, using reverse genetics to investigate specific gene function requires phenotypic analysis of complete gene inactivation. Despite the availability and efficacy of morpholinos, the lack of tractable and efficient knockout technologies has impeded reverse genetic studies in the zebrafish, particularly in adult animals. The recent development of genome-editing technologies such as CRISPR/Cas9 greatly widened the scope of loss-of-function studies in the zebrafish, allowing for the rapid phenotypic assessment of gene silencing in embryos, the generation of knockout lines, and large-scale reverse genetic screens. Tissue-specific gene inactivation would be ideal for these studies given the caveats of whole-embryo gene silencing, yet spatial control of gene targeting remains a challenge. In this chapter, we focus on tissue-specific gene inactivation using the CRISPR/Cas9 technology. We first explain the rationale for this technique, including some of its potential applications to tackle important biological issues and the inability of current technologies to address these issues. We then present a method to target genes in a tissue-specific manner in the zebrafish. Finally, we discuss technical difficulties and limitations of this method as well as possible future developments.
Topics: Animals; CRISPR-Cas Systems; Embryonic Development; Gene Editing; Gene Targeting; Genetic Engineering; Morpholinos; Organ Specificity; Zebrafish
PubMed: 27443926
DOI: 10.1016/bs.mcb.2016.03.004 -
Biology Open Mar 2021LIM homeobox 9 (Lhx9) is a member of the LIM homeodomain transcription factor family, which expresses and functions in various vertebrate tissues, such as the gonads...
LIM homeobox 9 (Lhx9) is a member of the LIM homeodomain transcription factor family, which expresses and functions in various vertebrate tissues, such as the gonads and pineal gland. Previous studies on in zebrafish have mainly focused on the brain. However, little is known about the expression pattern of during embryogenesis. Here, we detected expression in zebrafish embryos using whole-mount hybridization and found expressed in heart, pectoral fin, and retina during their development in zebrafish. We then detailed the expression of in retinal development. To further investigate the function of Lhx9 in retinogenesis, we performed morpholino (MO) knockdown experiments and found that upon knockdown by MO, larvae presented normal eye development, retinal neural development, differentiation, proliferation, apoptosis, and responses to light stimulus. We not only elaborated the expression pattern of in zebrafish embryogenesis, but we also demonstrated that knockdown by morpholino does not affect the zebrafish retinal development, and our study provides data for further understanding of the role of Lhx9 in zebrafish retinal development.
Topics: Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Embryonic Development; Gene Expression Regulation, Developmental; Gene Knockdown Techniques; LIM-Homeodomain Proteins; Morpholinos; Organogenesis; Retina; Zebrafish
PubMed: 33579692
DOI: 10.1242/bio.056382 -
Biosensors & Bioelectronics Jul 2021SARS-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...
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.
Topics: Biosensing Techniques; COVID-19; COVID-19 Testing; Gold; Graphite; Humans; Limit of Detection; Metal Nanoparticles; Morpholinos; RNA, Viral; SARS-CoV-2; Sensitivity and Specificity
PubMed: 33823464
DOI: 10.1016/j.bios.2021.113206 -
Biochimica Et Biophysica Acta.... Dec 2019Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell...
Downregulation of hepatic stem cell factor by Vivo-Morpholino treatment inhibits mast cell migration and decreases biliary damage/senescence and liver fibrosis in Mdr2 mice.
UNLABELLED
Primary sclerosing cholangitis (PSC) is characterized by increased mast cell (MC) infiltration, biliary damage and hepatic fibrosis. Cholangiocytes secrete stem cell factor (SCF), which is a chemoattractant for c-kit expressed on MCs. We aimed to determine if blocking SCF inhibits MC migration, biliary damage and hepatic fibrosis.
METHODS
FVB/NJ and Mdr2 mice were treated with Mismatch or SCF Vivo-Morpholinos. We measured (i) SCF expression and secretion; (ii) hepatic damage; (iii) MC migration/activation and histamine signaling; (iv) ductular reaction and biliary senescence; and (v) hepatic fibrosis. In human PSC patients, SCF expression and secretion were measured. In vitro, cholangiocytes were evaluated for SCF expression and secretion. Biliary proliferation/senescence was measured in cholangiocytes pretreated with 0.1% BSA or the SCF inhibitor, ISK03. Cultured HSCs were stimulated with cholangiocyte supernatant and activation measured. MC migration was determined with cholangiocytes pretreated with BSA or ISK03 loaded into the bottom of Boyden chambers and MCs into top chamber.
RESULTS
Biliary SCF expression and SCF serum levels increase in human PSC. Cholangiocytes, but not hepatocytes, from SCF Mismatch Mdr2 mice have increased SCF expression and secretion. Inhibition of SCF in Mdr2 mice reduced (i) hepatic damage; (ii) MC migration; (iii) histamine and SCF serum levels; and (iv) ductular reaction/biliary senescence/hepatic fibrosis. In vitro, cholangiocytes express and secrete SCF. Blocking biliary SCF decreased MC migration, biliary proliferation/senescence, and HSC activation.
CONCLUSION
Cholangiocytes secrete increased levels of SCF inducing MC migration, contributing to biliary damage/hepatic fibrosis. Targeting MC infiltration may be an option to ameliorate PSC progression.
Topics: ATP Binding Cassette Transporter, Subfamily B; Animals; Biliary Tract; Cell Movement; Cellular Senescence; Cholangitis, Sclerosing; Down-Regulation; Female; Gene Deletion; Humans; Liver Cirrhosis; Male; Mast Cells; Mice; Morpholinos; Stem Cell Factor; ATP-Binding Cassette Sub-Family B Member 4
PubMed: 31521820
DOI: 10.1016/j.bbadis.2019.165557 -
The American Journal of Pathology May 2020Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor...
Histamine binds to one of the four G-protein-coupled receptors expressed by large cholangiocytes and increases large cholangiocyte proliferation via histamine-2 receptor (H2HR), which is increased in patients with primary sclerosing cholangitis (PSC). Ranitidine decreases liver damage in Mdr2 (ATP binding cassette subfamily B member 4 null) mice. We targeted hepatic H2HR in Mdr2 mice using vivo-morpholino. Wild-type and Mdr2 mice were treated with mismatch or H2HR vivo-morpholino by tail vein injection for 1 week. Liver damage, mast cell (MC) activation, biliary H2HR, and histamine serum levels were studied. MC markers were determined by quantitative real-time PCR for chymase and c-kit. Intrahepatic biliary mass was detected by cytokeratin-19 and F4/80 to evaluate inflammation. Biliary senescence was determined by immunofluorescence and senescence-associated β-galactosidase staining. Hepatic fibrosis was evaluated by staining for desmin, Sirius Red/Fast Green, and vimentin. Immunofluorescence for transforming growth factor-β, vascular endothelial growth factor-A/C, and cAMP/ERK expression was performed. Transforming growth factor-β and vascular endothelial growth factor-A secretion was measured in serum and/or cholangiocyte supernatant. Treatment with H2HR vivo-morpholino in Mdr2-mice decreased hepatic damage; H2HR protein expression and MC presence or activation; large intrahepatic bile duct mass, inflammation and senescence; and fibrosis, angiogenesis, and cAMP/phospho-ERK expression. Inhibition of H2HR signaling ameliorates large ductal PSC-induced damage. The H2HR axis may be targeted in treating PSC.
Topics: Animals; Bile Ducts; Cholangitis, Sclerosing; Mast Cells; Mice; Mice, Knockout; Morpholinos; Receptors, Histamine H2
PubMed: 32142732
DOI: 10.1016/j.ajpath.2020.01.013 -
Nature Reviews. Genetics Jan 2017Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or... (Review)
Review
Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method.
Topics: Animals; CRISPR-Cas Systems; Gene Silencing; Genotype; Humans; Models, Animal; Morpholinos; Mutagenesis; Mutation; Phenotype; RNA Interference; Species Specificity
PubMed: 27795562
DOI: 10.1038/nrg.2016.118 -
Cell Nov 2021The mitochondrial genome encodes proteins central to mitochondrial function; however, transcript-specific mechanistic studies of mitochondrial gene products have been...
The mitochondrial genome encodes proteins central to mitochondrial function; however, transcript-specific mechanistic studies of mitochondrial gene products have been difficult because of challenges in their experimental manipulation. Cruz-Zaragoza et al. provide a solution to this challenge, introducing an elegant system for efficient translational silencing of transcripts in human mitochondria.
Topics: Gene Expression; Humans; Mitochondria; Mitochondrial Proteins; Morpholinos; Organelles
PubMed: 34767774
DOI: 10.1016/j.cell.2021.10.019