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Molecular Therapy : the Journal of the... Mar 2022Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly...
Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality. Recently approved SMA therapies have transformed a deadly disease into a survivable one, but these compounds show a wide spectrum of clinical response and effective rescue only in the early stages of the disease. Therefore, safe, symptomatic-suitable, non-invasive treatments with high clinical impact across different phenotypes are urgently needed. We conjugated antisense oligonucleotides with Morpholino (MO) chemistry, which increase SMN protein levels, to cell-penetrating peptides (CPPs) for better cellular distribution. Systemically administered MOs linked to r6 and (RXRRBR)XB peptides crossed the blood-brain barrier and increased SMN protein levels remarkably, causing striking improvement of survival, neuromuscular function, and neuropathology, even in symptomatic SMA animals. Our study demonstrates that MO-CPP conjugates can significantly expand the therapeutic window through minimally invasive systemic administration, opening the path for clinical applications of this strategy.
Topics: Animals; Cell-Penetrating Peptides; Disease Models, Animal; Humans; Morpholinos; Muscular Atrophy, Spinal; Oligonucleotides, Antisense; Phenotype
PubMed: 34808387
DOI: 10.1016/j.ymthe.2021.11.012 -
Methods in Molecular Biology (Clifton,... 2023The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic...
The mutation c.-32-13T>G in the GAA gene impacts normal exon 2 splicing and is found in two-thirds of late-onset Pompe disease cases. We have explored a therapeutic strategy using splice modulating phosphorodiamidate morpholino oligomers to enhance GAA exon 2 inclusion in the mature mRNA of patients carrying this common mutation. We performed in silico analysis of the GAA gene transcript for potential splicing silencers and designed oligomers targeting motifs predicted to enhance exon 2 retention in the mature mRNA. Two patient-derived fibroblasts were obtained from Coriell Institute for Medical Research, and seven fibroblast strains from unrelated patients were supplied by Westmead Hospital in Sydney, Australia. Both fibroblasts and forced-myogenic cells were treated with optimized phosphorodiamidate morpholino oligomers supplied by Sarepta Therapeutics. Total RNA and protein were extracted from the cells after incubation with phosphorodiamidate morpholino oligomers, and RT-PCR and RT-qPCR were performed to confirm exon 2 inclusion is enhanced. Acid α-glucosidase activity and expression levels were also assessed to confirm therapeutic potential.
Topics: Humans; Adult; Glycogen Storage Disease Type II; Morpholinos; RNA Splicing; Mutation; RNA, Messenger
PubMed: 36401034
DOI: 10.1007/978-1-0716-2772-3_14 -
Human Gene Therapy Methods Jun 2019Duchenne muscular dystrophy (DMD) is a severe type of X-linked recessive degenerative muscle disease caused by mutations in the dystrophin () gene on the X chromosome.... (Review)
Review
Duchenne muscular dystrophy (DMD) is a severe type of X-linked recessive degenerative muscle disease caused by mutations in the dystrophin () gene on the X chromosome. The gene is complex, consisting of 79 exons, and mutations cause changes in the mRNA so that the reading frame is altered, and the muscle-specific isoform of the dystrophin protein is either absent or truncated with variable residual function. The emerging CRISPR-Cas9-mediated genome editing technique is being developed as a potential therapeutic approach to treat DMD because it can permanently replace the mutated dystrophin gene with the normal gene. Prenatal DNA testing can inform whether the female fetus is a carrier of DMD, and the male fetus has inherited a mutation from his mother (50% chance of both). This article summarizes the present status of current and future treatments for DMD.
Topics: Animals; CRISPR-Cas Systems; Exons; Gene Editing; Humans; Morpholinos; Muscular Dystrophy, Duchenne; Oxadiazoles
PubMed: 31062609
DOI: 10.1089/hgtb.2018.187 -
Current Genetics Dec 2017Antibiotic resistance is a worldwide public health problem (Bush et al. in Nat Rev Microbiol 9:894-896, 2011). The lack of effective therapies against resistant bacteria... (Review)
Review
Antibiotic resistance is a worldwide public health problem (Bush et al. in Nat Rev Microbiol 9:894-896, 2011). The lack of effective therapies against resistant bacteria globally leads to prolonged treatments, increased mortality, and inflating health care costs (Oz et al. in Mol Biol Evol 31:2387-2401, 2014; Martinez in Science 321:365-367, 2008; Lipsitch et al. in Proc Natl Acad Sci USA 97:1938-1943, 2000; Taubes in Science 321:356-361, 2008; Laxminarayan et al. in Lancet, 2016; Laxminarayan et al. in Lancet Infect Dis 13:1057-1098, 2013). Current efforts towards a solution of this problem can be boiled down to two main strategies: (1) developing of new antimicrobial agents and (2) searching for smart strategies that can restore or preserve the efficacy of existing antimicrobial agents. In this short review article, we discuss the need for evolvable antimicrobial agents, focusing on a new antimicrobial technology that utilizes peptide-conjugated phosphorodiamidate morpholino oligomers to inhibit the growth of pathogenic bacteria by targeting bacterial genes.
Topics: Anti-Bacterial Agents; Drug Resistance, Microbial; Escherichia coli; Escherichia coli Proteins; Evolution, Molecular; Gene Expression Regulation, Bacterial; Gene Silencing; Klebsiella pneumoniae; Lipoproteins; Membrane Transport Proteins; Microbial Sensitivity Tests; Morpholinos; Peptides; Salmonella enterica
PubMed: 28497241
DOI: 10.1007/s00294-017-0703-x -
Methods in Molecular Biology (Clifton,... 2017The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the... (Review)
Review
The control processes that underlie the progression of development can be summarized in maps of gene regulatory networks (GRNs). A critical step in their assembly is the systematic perturbation of network candidates. In sea urchins the most important method for interfering with expression in a gene-specific way is application of morpholino antisense oligonucleotides (MOs). MOs act by binding to their sequence complement in transcripts resulting in a block in translation or a change in splicing and thus result in a loss of function. Despite the tremendous success of this technology, recent comparisons to mutants generated by genome editing have led to renewed criticism and challenged its reliability. As with all methods based on sequence recognition, MOs are prone to off-target binding that may result in phenotypes that are erroneously ascribed to the loss of the intended target. However, the slow progression of development in sea urchins has enabled extremely detailed studies of gene activity in the embryo. This wealth of knowledge paired with the simplicity of the sea urchin embryo enables careful analysis of MO phenotypes through a variety of methods that do not rely on terminal phenotypes. This article summarizes the use of MOs in probing GRNs and the steps that should be taken to assure their specificity.
Topics: Animals; Base Pairing; Gene Expression Regulation; Gene Knockdown Techniques; Gene Regulatory Networks; Gene Targeting; Gene Transfer Techniques; Morpholinos; Mutation; Oligonucleotides, Antisense; Phenotype; Sea Urchins
PubMed: 28364236
DOI: 10.1007/978-1-4939-6817-6_8 -
Journal of Cell Science Mar 2024Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they...
Cell shape changes mainly rely on the remodeling of the actin cytoskeleton. Multiciliated cells (MCCs) of the mucociliary epidermis of Xenopus laevis embryos, as they mature, dramatically reshape their apical domain to grow cilia, in coordination with the underlying actin cytoskeleton. Crumbs (Crb) proteins are multifaceted transmembrane apical polarity proteins known to recruit actin linkers and promote apical membrane growth. Here, we identify the homeolog Crb3.L as an important player for the migration of centrioles or basal bodies (collectively centrioles/BBs) and apical domain morphogenesis in MCCs. Crb3.L is present in cytoplasmic vesicles close to the ascending centrioles/BBs, where it partially colocalizes with Rab11a. Crb3.L morpholino-mediated depletion in MCCs caused abnormal migration of centrioles/BBs, a reduction of their apical surface, disorganization of their apical actin meshwork and defective ciliogenesis. Rab11a morpholino-mediated depletion phenocopied Crb3.L loss-of-function in MCCs. Thus, the control of centrioles/BBs migration by Crb3.L might be mediated by Rab11a-dependent apical trafficking. Furthermore, we show that both phospho-activated ERM (pERM; Ezrin-Radixin-Moesin) and Crb3.L are recruited to the growing apical domain of MCCs, where Crb3.L likely anchors pERM, allowing actin-dependent expansion of the apical membrane.
Topics: Actins; Morpholinos; Actin Cytoskeleton; Cell Membrane; Cilia
PubMed: 37840525
DOI: 10.1242/jcs.261046 -
The European Journal of Neuroscience Jul 2015The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the... (Review)
Review
The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience.
Topics: Animals; Brain; DNA; Gene Expression Regulation; History, 20th Century; History, 21st Century; Molecular Biology; Morpholinos; Mutagenesis; Neurosciences; RNA, Messenger; Zebrafish
PubMed: 25900095
DOI: 10.1111/ejn.12932 -
Investigative Ophthalmology & Visual... Dec 2018Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however,...
PURPOSE
Retinal degenerative diseases can progress to severe reductions of vision. In general, the changes are permanent in higher vertebrates, including humans; however, retinal regeneration can occur in lower vertebrates, such as amphibians and teleost fish. Progranulin is a secreted growth factor that is involved in normal development and wound-healing processes. We have shown that progranulin promotes the proliferation of retinal precursor cells in mouse retinas. The purpose of this study was to investigate the role played by granulin 1 (grn1) in the retinal regeneration in zebrafish.
METHODS
We injured the retina of zebrafish with needle puncturing, and the retinas were examined at different times after the injury. We also checked the proliferation and the expression of retinal regeneration-related genes after knockdown of grn1 by electroporation with morpholino oligonucleotides (MO) and intravitreal injection of recombinant grn1.
RESULTS
Our results showed that the level of grn1 was highly increased after retinal injury, and it was expressed in various types of retinal cells. A knockdown of grn1 reduced the proliferation of Müller glial cells in zebrafish eyes undergoing retinal regeneration. The knockdown of grn1 also reduced the expression of achaete-scute homolog 1a (ascl1a), an important factor in retinal regeneration. An intravitreal injection of recombinant grn1 led to a proliferation of Müller glial cells and an increase in the expression of retinal regeneration-related genes, such as ascl1a and lin28.
CONCLUSIONS
These findings suggested that grn1 should be considered as a target for stimulating the dedifferentiation of Müller glial cells and retinal regeneration.
Topics: Animals; Bromodeoxyuridine; Cell Count; Electroporation; Gene Silencing; Granulins; Immunohistochemistry; Morpholinos; RNA, Messenger; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Regeneration; Retina; Retinal Degeneration; Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 30577041
DOI: 10.1167/iovs.18-24828 -
Nature Dec 2018
Topics: Clinical Decision-Making; Cost-Benefit Analysis; Drug Approval; Genetic Therapy; Gross Domestic Product; Humans; Insurance, Health; Morpholinos; Muscular Dystrophy, Duchenne; Stakeholder Participation; Treatment Outcome; United States; United States Food and Drug Administration
PubMed: 30542179
DOI: 10.1038/d41586-018-07648-8 -
Biochemical and Biophysical Research... Dec 2021Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly...
Telomerase is a reverse transcriptase that catalyzes the addition of telomeric repeated DNA onto the 3' ends of linear chromosomes. Telomerase inhibition was broadly used for cancer therapeutics. Here, six antisense oligonucleotides were designed to regulate TERT mRNA alternative splicing and protein translation. To pursue a better stability in vitro, we chemically modified the oligonucleotides into phosphorothioate (PS) backbone and 2'-O-methoxyethyl (2'-MOE PS) version and phosphoroamidate morpholino oligomer (PMO) version. The oligonucleotides were transfected into HEK 293T cells and HeLa cells, and the mRNA expression, protein level and catalytic activity of telomerase were determined. We found the Int8 notably promoted hTERT mRNA exon 7-8 skipping, which greatly reduced telomerase activity, and the 5'-UTR treatment led to an obvious protein translation barrier and telomerase inhibition. These results demonstrate the potential of antisense oligonucleotide drugs targeting hTERT for antitumor therapy. Moreover, two specific antisense oligonucleotides were identified to be effective in reducing telomerase activity.
Topics: Alternative Splicing; Antineoplastic Agents; HEK293 Cells; HeLa Cells; Humans; Morpholinos; Oligonucleotides, Antisense; Phosphorothioate Oligonucleotides; Protein Biosynthesis; RNA, Messenger; Telomerase
PubMed: 34710826
DOI: 10.1016/j.bbrc.2021.10.034