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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 -
Biomedicine & Pharmacotherapy =... Aug 2023K7 channels exert a pivotal role regulating vascular tone in several vascular beds. In this context, K7 channel agonists represent an attractive strategy for the...
K7 channels exert a pivotal role regulating vascular tone in several vascular beds. In this context, K7 channel agonists represent an attractive strategy for the treatment of pulmonary arterial hypertension (PAH). Therefore, in this study, we have explored the pulmonary vascular effects of the novel K7 channel agonist URO-K10. Consequently, the vasodilator and electrophysiological effects of URO-K10 were tested in rat and human pulmonary arteries (PA) and PA smooth muscle cells (PASMC) using myography and patch-clamp techniques. Protein expression was also determined by Western blot. Morpholino-induced knockdown of KCNE4 was assessed in isolated PA. PASMC proliferation was measured by BrdU incorporation assay. In summary, our data show that URO-K10 is a more effective relaxant of PA than the classical K7 activators retigabine and flupirtine. URO-K10 enhanced K currents in PASMC and its electrophysiological and relaxant effects were inhibited by the K7 channel blocker XE991. The effects of URO-K10 were confirmed in human PA. URO-K10 also exhibited antiproliferative effects in human PASMC. Unlike retigabine and flupirtine, URO-K10-induced pulmonary vasodilation was not affected by morpholino-induced knockdown of the KCNE4 regulatory subunit. Noteworthy, the pulmonary vasodilator efficacy of this compound was considerably increased under conditions mimicking the ionic remodelling (as an in vitro model of PAH) and in PA from monocrotaline-induced pulmonary hypertensive rats. Taking all together, URO-K10 behaves as a KCNE4-independent K7 channel activator with much increased pulmonary vascular effects compared to classical K7 channel activators. Our study identifies a promising new drug in the context of PAH.
Topics: Animals; Humans; Rats; KCNQ Potassium Channels; Morpholinos; Potassium Channels, Voltage-Gated; Vasodilator Agents
PubMed: 37295249
DOI: 10.1016/j.biopha.2023.114952 -
Scientific Reports Feb 2019Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of...
Incomplete fusion of the optic fissure leads to ocular coloboma, a congenital eye defect that affects up to 7.5 per 10,000 births and accounts for up to 10 percent of childhood blindness. The molecular and cellular mechanisms that facilitate optic fissure fusion remain elusive. We have profiled global gene expression during optic fissure morphogenesis by transcriptome analysis of tissue dissected from the margins of the zebrafish optic fissure and the opposing dorsal retina before (32 hours post fertilisation, hpf), during (48 hpf) and after (56 hpf) optic fissure fusion. Differential expression analysis between optic fissure and dorsal retinal tissue resulted in the detection of several known and novel developmental genes. The expression of selected genes was validated by qRT-PCR analysis and localisation investigated using in situ hybridisation. We discuss significantly overrepresented functional ontology categories in the context of optic fissure morphogenesis and highlight interesting transcripts from hierarchical clustering for subsequent analysis. We have identified netrin1a (ntn1a) as highly differentially expressed across optic fissure fusion, with a resultant ocular coloboma phenotype following morpholino antisense translation-blocking knockdown and downstream disruption of atoh7 expression. To support the identification of candidate genes in human studies, we have generated an online open-access resource for fast and simple quantitative querying of the gene expression data. Our study represents the first comprehensive analysis of the zebrafish optic fissure transcriptome and provides a valuable resource to facilitate our understanding of the complex aetiology of ocular coloboma.
Topics: Animals; Cluster Analysis; Coloboma; DNA-Binding Proteins; Disease Models, Animal; Embryo, Nonmammalian; Gene Expression Profiling; In Situ Hybridization, Fluorescence; Morpholinos; Netrin-1; Principal Component Analysis; Retina; Transcriptome; Zebrafish; Zebrafish Proteins
PubMed: 30733552
DOI: 10.1038/s41598-018-38379-5 -
Molecules (Basel, Switzerland) Aug 2023The aim of this study was to explore the mechanism of antitumor effect of ()-6-morpholino-9-(styrylsulfonyl)-9-purine (6-Morpholino-SPD) and...
The aim of this study was to explore the mechanism of antitumor effect of ()-6-morpholino-9-(styrylsulfonyl)-9-purine (6-Morpholino-SPD) and ()-6-amino-9-(styrylsulfonyl)-9-purine (6-Amino-SPD). The effects on apoptosis induction, mitochondrial potential, and accumulation of ROS in treated K562 cells were determined by flow cytometry. The RT-PCR method was used to measure the expression of , , , and genes, as well as selected miRNAs. Western blot analysis was used to determine the expression of Akt, cytochrome c, and caspase 3. The results demonstrate the potential of the tested derivatives as effective antitumor agents with apoptotic-inducing properties. In leukemic cells treated with 6-Amino-SPD, increased expression of and genes was observed, indicating involvement of the intrinsic mitochondrial pathway in the induction of apoptosis. Conversely, leukemic cells treated with 6-Morpholino-SPD showed reduced expression of these genes. The observed downregulation of miR-21 by 6-Morpholino-SPD may contribute to the induction of apoptosis and disruption of mitochondrial function. In addition, both derivatives exhibited increased expression of and genes, suggesting activation of the Akt/HIF pathway. However, the exact mechanism and its relations to the observed overexpression of miR-210 need further investigation. The acceptable absorption and distribution properties predicted by ADMET analysis suggest favorable pharmacokinetic properties for these derivatives.
Topics: Humans; Caspase 3; Morpholinos; Cytochromes c; Proto-Oncogene Proteins c-akt; Leukemia; MicroRNAs
PubMed: 37630388
DOI: 10.3390/molecules28166136 -
Journal of Neuromuscular Diseases 2022Studies 4658-201/202 (201/202) evaluated treatment effects of eteplirsen over 4 years in patients with Duchenne muscular dystrophy and confirmed exon-51 amenable genetic... (Comparative Study)
Comparative Study Randomized Controlled Trial
A Combined Prospective and Retrospective Comparison of Long-Term Functional Outcomes Suggests Delayed Loss of Ambulation and Pulmonary Decline with Long-Term Eteplirsen Treatment.
BACKGROUND
Studies 4658-201/202 (201/202) evaluated treatment effects of eteplirsen over 4 years in patients with Duchenne muscular dystrophy and confirmed exon-51 amenable genetic mutations. Chart review Study 4658-405 (405) further followed these patients while receiving eteplirsen during usual clinical care.
OBJECTIVE
To compare long-term clinical outcomes of eteplirsen-treated patients from Studies 201/202/405 with those of external controls.
METHODS
Median total follow-up time was approximately 6 years of eteplirsen treatment. Outcomes included loss of ambulation (LOA) and percent-predicted forced vital capacity (FVC%p). Time to LOA was compared between eteplirsen-treated patients and standard of care (SOC) external controls and was measured from eteplirsen initiation in 201/202 or, in the SOC group, from the first study visit. Comparisons were conducted using univariate Kaplan-Meier analyses and log-rank tests, and multivariate Cox proportional hazards models with regression adjustment for baseline characteristics. Annual change in FVC%p was compared between eteplirsen-treated patients and natural history study patients using linear mixed models with repeated measures.
RESULTS
Data were included from all 12 patients in Studies 201/202 and the 10 patients with available data from 405. Median age at LOA was 15.16 years. Eteplirsen-treated patients experienced a statistically significant longer median time to LOA by 2.09 years (5.09 vs. 3.00 years, p < 0.01) and significantly attenuated rates of pulmonary decline vs. natural history patients (FVC%p change: -3.3 vs. -6.0 percentage points annually, p < 0.0001).
CONCLUSIONS
Study 405 highlights the functional benefits of eteplirsen on ambulatory and pulmonary function outcomes up to 7 years of follow-up in comparison to external controls.
Topics: Adolescent; Child; Disease Progression; Humans; Male; Mobility Limitation; Morpholinos; Muscular Dystrophy, Duchenne; Outcome Assessment, Health Care; Prospective Studies; Registries; Retrospective Studies; Time Factors; Vital Capacity; Walk Test
PubMed: 34420980
DOI: 10.3233/JND-210665 -
Developmental Cell May 2019It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response... (Review)
Review
It has recently been reported that a common side effect of translation-blocking morpholino antisense oligonucleotides is the induction of a set of innate immune response genes in Xenopus embryos and that splicing-blocking morpholinos lead to unexpected off-target mis-splicing events. Here, we present an analysis of all publicly available Xenopus RNA sequencing (RNA-seq) data in a reexamination of the effects of translation-blocking morpholinos on the innate immune response. Our analysis does not support the authors' general conclusion, which was based on a limited number of RNA-seq datasets. Moreover, the strong induction of an immune response appears to be specific to the tbxt/tbxt2 morpholinos. The more comprehensive study presented here indicates that using morpholinos for targeted gene knockdowns remains of considerable value for the rapid identification of gene function.
Topics: Animals; Embryonic Development; Gene Knockdown Techniques; Immunity, Innate; Morpholinos; Oligonucleotides, Antisense; RNA Splicing; T-Box Domain Proteins; Transcriptome; Xenopus; Xenopus Proteins; Xenopus laevis
PubMed: 31112700
DOI: 10.1016/j.devcel.2019.04.019 -
EMBO Molecular Medicine Apr 2024Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show...
Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXO). EXO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.
Topics: Animals; Mice; Dystrophin; Mice, Inbred mdx; Exosomes; Morpholinos; Muscular Dystrophy, Duchenne; Oligonucleotides
PubMed: 38448545
DOI: 10.1038/s44321-024-00049-7 -
Biochemical and Biophysical Research... Apr 2021The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and...
The chemical structure of oligonucleotide analogues dictates the conformation of oligonucleotide analogue oligomers, their ability to hybridize complementary DNA and RNA, their stability to degradation and their pharmacokinetic properties. In a study aimed at investigating new analogues featuring a neutral backbone, we explored the ability of oligomers containing a morpholino-peptide backbone to bind oligonucleotides. Circular Dichroism studies revealed the ability of our oligomers to interact with DNA, molecular modelling studies revealed the interaction responsible for complex stabilization.
Topics: Circular Dichroism; DNA; Models, Molecular; Molecular Conformation; Morpholinos; Peptides
PubMed: 33652207
DOI: 10.1016/j.bbrc.2021.02.087 -
PloS One 2018Snail2 is a zinc-finger transcription factor best known to repress expression of genes encoding cell adherence proteins to facilitate induction of the...
Snail2 is a zinc-finger transcription factor best known to repress expression of genes encoding cell adherence proteins to facilitate induction of the epithelial-to-mesenchymal transition. While this role has been best documented in the developmental migration of the neural crest and mesoderm, here we expand on previously reported preliminary findings that morpholino knock-down of snai2 impairs the generation of hematopoietic stem cells (HSCs) during zebrafish development. We demonstrate that snai2 morphants fail to initiate HSC specification and show defects in the somitic niche of migrating HSC precursors. These defects include a reduction in sclerotome markers as well as in the Notch ligands dlc and dld, which are known to be essential components of HSC specification. Accordingly, enforced expression of the Notch1-intracellular domain was capable of rescuing HSC specification in snai2 morphants. To parallel our approach, we obtained two mutant alleles of snai2. In contrast to the morphants, homozygous mutant embryos displayed no defects in HSC specification or in sclerotome development, and mutant fish survive into adulthood. However, when these homozygous mutants were injected with snai2 morpholino, HSCs were improperly specified. In summary, our morpholino data support a role for Snai2 in HSC development, whereas our mutant data suggest that Snai2 is dispensable for this process. Together, these findings further support the need for careful consideration of both morpholino and mutant phenotypes in studies of gene function.
Topics: Amino Acid Sequence; Animals; Cell Differentiation; Embryo, Nonmammalian; Forkhead Transcription Factors; Gene Expression Regulation, Developmental; Genotype; Hematopoietic Stem Cells; Morpholinos; Mutagenesis, Site-Directed; PAX9 Transcription Factor; Phenotype; Receptors, Notch; Signal Transduction; Snail Family Transcription Factors; Zebrafish; Zebrafish Proteins
PubMed: 30208064
DOI: 10.1371/journal.pone.0202747 -
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