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1.
Int J Mol Sci ; 23(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36077211

RESUMO

Limb-girdle muscular dystrophies (LGMD) are clinically and genetically heterogenous presentations displaying predominantly proximal muscle weakness due to the loss of skeletal muscle fibers. Beta-sarcoglycanopathy (LGMDR4) results from biallelic molecular defects in SGCB and features pediatric onset with limb-girdle involvement, often complicated by respiratory and heart dysfunction. Here we describe a patient who presented at the age of 12 years reporting high creatine kinase levels and onset of cramps after strenuous exercise. Instrumental investigations, including a muscle biopsy, pointed towards a diagnosis of beta-sarcoglycanopathy. NGS panel sequencing identified two variants in the SGCB gene, one of which (c.243+1548T>C) was found to promote the inclusion of a pseudoexon between exons 2 and 3 in the SGCB transcript. Interestingly, we detected the same genotype in a previously reported LGMDR4 patient, deceased more than twenty years ago, who had escaped molecular diagnosis so far. After the delivery of morpholino oligomers targeting the pseudoexon in patient-specific induced pluripotent stem cells, we observed the correction of the physiological splicing and partial restoration of protein levels. Our findings prompt the analysis of the c.243+1548T>C variant in suspected LGMDR4 patients, especially those harbouring monoallelic SGCB variants, and provide a further example of the efficacy of antisense technology for the correction of molecular defects resulting in splicing abnormalities.


Assuntos
Distrofia Muscular do Cíngulo dos Membros , Sarcoglicanopatias , Criança , Humanos , Morfolinos/genética , Morfolinos/metabolismo , Músculo Esquelético/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Mutação , Sarcoglicanopatias/metabolismo
2.
J Am Chem Soc ; 144(37): 16819-16826, 2022 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-36073798

RESUMO

MicroRNAs play crucial and dynamic roles in vertebrate development and diseases. Some, like miR-430, are highly expressed during early embryo development and regulate hundreds of transcripts, which can make it difficult to study their role in the timing and location of specific developmental processes using conventional morpholino oligonucleotide (MO) knockdown or genetic deletion approaches. We demonstrate that light-activated circular morpholino oligonucleotides (cMOs) can be applied to the conditional control of microRNA function. We targeted miR-430 in zebrafish embryos to study its role in the development of the embryo body and the heart. Using 405 nm irradiation, precise spatial and temporal control over miR-430 function was demonstrated, offering insight into the cell populations and developmental timepoints involved in each process.


Assuntos
MicroRNAs , Peixe-Zebra , Animais , Embrião não Mamífero , MicroRNAs/genética , Morfolinos/farmacologia , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Proteínas de Peixe-Zebra/genética
3.
Bioorg Med Chem ; 72: 116995, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36095945

RESUMO

Aiming to develop novel tropomyosin receptor kinase A (TrkA) inhibitors, a scaffold hopping strategy was utilized by transforming the fused indazole of Entrectinib to phenyl triazole/thiazole skeleton to obtain compounds 7a-7 h and 13a-13 h. In the light of MTT assay, phenyl triazole derivatives 7a-7 h exhibited moderate anti-proliferative activities against KM-12 cells with the IC50 values of 1.78-17.51 µM, while phenyl thiazole derivatives 13a-13 h showed the weaker efficacy. Further structure-guided optimizations by combining the phenyl triazole skeleton with 3,5­difluorophenyl and 3-carbamoyl-4-piperazinylaniline moiety led to compounds 19a-19d and 20. Eventually, 19c bearing (2-(4-methylpiperazin-1-yl)phenyl)(morpholino)methanone moiety exhibited excellent anti-proliferative activity on TrkA-positive KM-12 cells with IC50 value of 0.17 µM. Meanwhile, compound 19c showed the inhibitory potency on TrkA with IC50 value of 1.6 nM, and displayed higher selectivity on TrkA over TrkB (IC50 = 12.3 nM) and TrkC (IC50 = 18.4 nM). The dedicated wound healing and colony formation assay indicated that the optimal compound 19c could suppress migration and significantly inhibit KM-12 cell colony formation in a dose-dependent manner. In addition, 19c could weakly induce apoptosis of KM-12 cell in immunofluorescent staining analysis. Taken together, the above results suggest 19c as a novel TrkA inhibitor worthy of further profiling.


Assuntos
Antineoplásicos , Tiazóis , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Indazóis/farmacologia , Estrutura Molecular , Morfolinos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Tiazóis/farmacologia , Triazóis/farmacologia , Tropomiosina/farmacologia
4.
Hum Genomics ; 16(1): 41, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36123719

RESUMO

BACKGROUND: Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis. METHODS: Eighty-one HTX patients with complex congenital heart defects and 89 healthy children were enrolled, and we investigated the pathogenetic variants related to patients with HTX by exome sequencing. Zebrafish splice-blocking Morpholino oligo-mediated transient suppression assays were performed to confirm the potential pathogenicity of missense variants found in these patients with HTX. RESULTS: Three MMP21 heterozygous non-synonymous variants (c.731G > A (p.G244E), c.829C > T (p.L277F), and c.1459A > G (p.K487E)) were identified in three unrelated Chinese Han patients with HTX and complex congenital heart defects. Sanger sequencing confirmed that all variants were de novo. Cell transfection assay showed that none of the variants affect mRNA and protein expression levels of MMP21. Knockdown expression of mmp21 by splice-blocking Morpholino oligo in zebrafish embryos revealed a heart looping disorder, and mutant human MMP21 mRNA (c.731G > A, c.1459A > G, heterozygous mRNA (wild-type&c.731G > A), as well as heterozygous mRNA (wild-type& c.1459A > G) could not effectively rescue the heart looping defects. A patient with the MMP21 p.G244E variant was identified with other potential HTX-causing missense mutations, whereas the patient with the MMP21 p.K487E variant had no genetic mutations in other causative genes related to HTX. CONCLUSION: Our study highlights the role of the disruptive heterozygous MMP21 variant (p.K487E) in the etiology of HTX with complex cardiac malformations and expands the current mutation spectrum of MMP21 in HTX.


Assuntos
Síndrome de Heterotaxia , Animais , Criança , China , Síndrome de Heterotaxia/genética , Humanos , Morfolinos , RNA Mensageiro , Fatores de Risco , Peixe-Zebra/genética
5.
Biochim Biophys Acta Mol Basis Dis ; 1868(11): 166509, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35914653

RESUMO

Type 2 diabetes is associated with an inflammatory phenotype in the pancreatic islets. We previously demonstrated that proinflammatory cytokines potently activate the tryptophan/kynurenine pathway (TKP) in INS-1 cells and in normal rat islets. Here we examined: (1) the TKP enzymes expression in the diabetic GK islets; (2) the TKP enzymes expression profiles in the GK islets before and after the onset of diabetes; (3) The glucose-stimulated insulin secretion (GSIS) in vitro in GK islets after KMO knockdown using specific morpholino-oligonucleotides against KMO or KMO blockade using the specific inhibitor Ro618048; (4) The glucose tolerance and GSIS after acute in vivo exposure to Ro618048 in GK rats. We report a remarkable induction of the kmo gene in GK islets and in human islets exposed to proinflammatory conditions. It occurred prominently in beta cells. The increased expression and activity of KMO reflected an acquired adaptation. Both KMO knockdown and specific inhibitor Ro618048 enhanced GSIS in vitro in GK islets. Moreover, acute administration of Ro618048 in vivo improved glucose tolerance, GSIS and basal blood glucose levels in GK rats. These results demonstrate that targeting islet TKP is able to correct defective GSIS. KMO inhibition could represent a potential therapeutic strategy for type 2 diabetes.


Assuntos
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Animais , Glicemia/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase/metabolismo , Morfolinos , Ratos , Ratos Wistar , Triptofano/metabolismo
6.
Dev Neurobiol ; 82(6): 533-544, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35929227

RESUMO

Axonal connections between the two sides of the brain are essential for processing sensorimotor functions, especially in animals with bilateral symmetry. The anterior commissure and postoptic commissure are two crucial axonal projections that develop early in the zebrafish central nervous system. In this study, we characterized the function of collapsin response mediator protein 2 (CRMP2) and CRMP4 in patterning the development of the anterior and postoptic commissures by analyzing morpholino-knockdown zebrafish morphants and CRISPR/Cas9-edited gene-knockout mutants. We observed a loss of commissural structures or a significant reduction in axon bundles connecting the two hemispheres, but the defects could be largely recovered by co-injecting CRMP2 or CRMP4 mRNA. Loss of both CRMP2 and CRMP4 function resulted in a synergistic increase in the number of commissural defects. To elucidate the mechanism by which CRMP2 and CRMP4 provide guidance cues for the development of the anterior and postoptic commissures, we included neuropilin 1a (Nrp1a) morphants and double morphants (CRMP2/Nrp1a and CRMP4/Nrp1a) for analysis. Our experimental results indicated that CRMP2 and CRMP4 might mediate their activities through the common semaphorin 3/Nrp1a signaling pathway.


Assuntos
Semaforinas , Peixe-Zebra , Animais , Morfolinos/metabolismo , Morfolinos/farmacologia , Neuropilinas/metabolismo , Prosencéfalo/metabolismo , RNA Mensageiro/metabolismo , Semaforina-3A/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Peixe-Zebra/metabolismo
7.
Proc Natl Acad Sci U S A ; 119(36): e2207956119, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36037350

RESUMO

Recent advances in drug development have seen numerous successful clinical translations using synthetic antisense oligonucleotides (ASOs). However, major obstacles, such as challenging large-scale production, toxicity, localization of oligonucleotides in specific cellular compartments or tissues, and the high cost of treatment, need to be addressed. Thiomorpholino oligonucleotides (TMOs) are a recently developed novel nucleic acid analog that may potentially address these issues. TMOs are composed of a morpholino nucleoside joined by thiophosphoramidate internucleotide linkages. Unlike phosphorodiamidate morpholino oligomers (PMOs) that are currently used in various splice-switching ASO drugs, TMOs can be synthesized using solid-phase oligonucleotide synthesis methodologies. In this study, we synthesized various TMOs and evaluated their efficacy to induce exon skipping in a Duchenne muscular dystrophy (DMD) in vitro model using H2K mdx mouse myotubes. Our experiments demonstrated that TMOs can efficiently internalize and induce excellent exon 23 skipping potency compared with a conventional PMO control and other widely used nucleotide analogs, such as 2'-O-methyl and 2'-O-methoxyethyl ASOs. Notably, TMOs performed well at low concentrations (5-20 nM). Therefore, the dosages can be minimized, which may improve the drug safety profile. Based on the present study, we propose that TMOs represent a new, promising class of nucleic acid analogs for future oligonucleotide therapeutic development.


Assuntos
Terapia Genética , Morfolinos , Distrofia Muscular de Duchenne , Splicing de RNA , Animais , Modelos Animais de Doenças , Terapia Genética/métodos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/genética , Morfolinos/farmacologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos/genética , Oligonucleotídeos/farmacologia , Oligonucleotídeos/uso terapêutico , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , RNA Mensageiro
8.
Dev Biol ; 490: 117-124, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35917936

RESUMO

The impact of new technology can be appreciated by how broadly it is used. Investigators that previously relied only on pharmacological approaches or the use of morpholino antisense oligonucleotide (MASO) technologies are now able to apply CRISPR-Cas9 to study biological problems in their model organism of choice much more effectively. The transitions to new CRISPR-based approaches could be enhanced, first, by standardized protocols and education in their applications. Here we summarize our results for optimizing the CRISPR-Cas9 technology in a sea urchin and a sea star, and provide advice on how to set up CRISPR-Cas9 experiments and interpret the results in echinoderms. Our goal through these protocols and sharing examples of success by other labs is to lower the activation barrier so that more laboratories can apply CRISPR-Cas9 technologies in these important animals.


Assuntos
Sistemas CRISPR-Cas , Ouriços-do-Mar , Animais , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Morfolinos/genética , RNA Guia/genética , Ouriços-do-Mar/genética
9.
J Vis Exp ; (186)2022 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-36036621

RESUMO

The morpholino oligomer-based knockdown system has been used to identify the function of various gene products through loss or reduced expression. Morpholinos (MOs) have the advantage in biological stability over DNA oligos because they are not susceptible to enzymatic degradation. For optimal effectiveness, MOs are injected into 1-4 cell stage embryos. The temporal efficacy of knockdown is variable, but MOs are believed to lose their effects due to dilution eventually. Morpholino dilution and injection amount should be closely controlled to minimize the occurrence of off-target effects while maintaining on-target efficacy. Additional complementary tools, such as CRISPR/Cas9 should be performed against the target gene of interest to generate mutant lines and to confirm the morphant phenotype with these lines. This article will demonstrate how to design, prepare, and microinject a translation-blocking morpholino against hand2 into the yolk of 1-4 cell stage zebrafish embryos to knockdown hand2 function and rescue these "morphants" by co-injection of mRNA encoding the corresponding cDNA. Subsequently, the efficacy of the morpholino microinjections is assessed by first verifying the presence of morpholino in the yolk (co-injected with phenol red) and then by phenotypic analysis. Moreover, cardiac functional analysis to test for knockdown efficacy will be discussed. Finally, assessing the effect of morpholino-induced blockage of gene translation via western blotting will be explained.


Assuntos
Oligonucleotídeos Antissenso , Peixe-Zebra , Animais , Embrião não Mamífero , Técnicas de Silenciamento de Genes , Morfolinos/genética , Morfolinos/farmacologia , Oligonucleotídeos Antissenso/genética , Fenótipo , RNA Mensageiro/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
10.
J Org Chem ; 87(15): 9466-9478, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35839125

RESUMO

Phosphorodiamidate morpholino oligonucleotides (PMOs) constitute 3 out of the 11 FDA-approved oligonucleotide-based drugs in the last 6 years. PMOs can effectively silence disease-causing genes and modify splicing. However, PMO synthesis has remained challenging for a variety of reasons: inefficient deprotection and coupling methods and instability of monomers. Here, we report the development of a suitable combination of resin supports, deblocking and coupling reagents for synthesizing PMOs using either trityl or Fmoc-protected chlorophosphoramidate monomers. The synthesized PMOs using both the methods on a solid support have been validated for gene silencing in a zebrafish model. The protocol was successfully transferred into an automated DNA synthesizer to make several sequences of PMOs, demonstrating for the first time the adaptation of regular PMOs in a commercial DNA synthesizer. Moreover, PMOs with longer than 20-mer sequences, including FDA-approved Eteplirsen (30-mer), were achieved in >20% overall yield that is superior to previous reports. Hybridization study shows that PMOs exhibit a higher binding affinity toward complementary DNA relative to the DNA/DNA duplex (>6 °C). Additionally, the introduction of Fmoc chemistry into PMOs opens up the possibility for PMO synthesis in commercial peptide synthesizers for future development.


Assuntos
Oligonucleotídeos Antissenso , Peixe-Zebra , Animais , DNA , Morfolinos/genética , Splicing de RNA
11.
Muscle Nerve ; 66(3): 262-269, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35715998

RESUMO

INTRODUCTION/AIMS: Pulmonary decline is a major issue in patients with Duchenne muscular dystrophy (DMD). Eteplirsen is a United States-approved treatment for patients with DMD and exon 51 skip-amenable mutations. Previous analyses have shown that eteplirsen is associated with a statistically significant attenuation of pulmonary decline. In this study we evaluate the effect of eteplirsen treatment from newly available data sources on pulmonary function over time in patients with DMD. METHODS: We used a post hoc pooled analysis to compare the percentage of predicted forced vital capacity (FVC%p) and projected time with pulmonary function milestones in patients with DMD and exon 51 skip-amenable mutations receiving eteplirsen (Studies 204 and 301) or standard of care (SoC; Cooperative International Neuromuscular Research Group Duchenne Natural History Study). A mixed model for repeated-measures framework was applied to evaluate the impact of eteplirsen. RESULTS: An average annual rate of FVC%p decline for eteplirsen-treated patients was estimated to be 3.47%, a statistically significant attenuation from the 5.95% rate of decline estimated in SoC patients (P = .0001). Using linear extrapolations of the model-estimated decline in FVC%p, the attenuation in FVC%p decline for eteplirsen-treated patients corresponded to a delay of 5.72 years in time to needing continuous ventilation, 3.31 years in time to needing nighttime ventilation, and 2.11 years in time to needing a cough assist device compared with SoC patients. DISCUSSION: The attenuation of FVC%p decline suggests that eteplirsen-treated patients had statistically significant and clinically meaningful attenuations in pulmonary decline compared with SoC patients.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Pulmão , Morfolinos/farmacologia , Capacidade Vital
12.
Bioconjug Chem ; 33(7): 1311-1318, 2022 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-35737901

RESUMO

The secondary structures of cell-penetrating peptides (CPPs) influence their properties including their cell-membrane permeability, tolerability to proteases, and intracellular distribution. Herein, we developed helix-stabilized arginine-rich peptides containing α,α-disubstituted α-amino acids and their conjugates with antisense phosphorodiamidate morpholino oligomers (PMOs), to investigate the relationships among the helicity of the peptides, cellular uptake, and antisense activity of the peptide-conjugated PMOs. We demonstrated that helical CPPs can efficiently deliver the conjugated PMO into cells compared with nonhelical CPPs and that their antisense activities are synergistically enhanced in the presence of an endosomolytic reagent or an endosomal escape domain peptide.


Assuntos
Peptídeos Penetradores de Células , Transporte Biológico , Permeabilidade da Membrana Celular , Morfolinos , Oligonucleotídeos Antissenso/química
13.
Bioconjug Chem ; 33(5): 907-917, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35486710

RESUMO

Cell-penetrating peptides (CPPs) are structurally diverse sophisticated tools endowed with high arginine content, amphipathicity, and well-adopted suitable secondary structures. Despite its capability of breaching the lipid barriers, CPP has major limitations such as in vivo metabolic instability, poor bioavailability, and reduced endosomal escape tendency, which are yet to be improved. In this context, we first have introduced a new class of cellular transporter having a guanidinium-functionalized δ-azaproline (δ-azp)-containing peptide where the δ-azp structurally resembles the "proline" amino acid having an additional "N" at the δ-position. This non-natural peptidic backbone was found to impart proteolytic stability, as reported earlier by our group. Herein, we report the synthesis of a flexible azaproline-tetraguanidinium transporter named FAT along with a revised scalable methodology for δ-azp compared to our previously reported procedure. FAT shows a random-coil-like structure as determined by CD spectroscopy, and is hence structurally different from the polyproline PPII helix. Direct translocation is predicted to be the possible mode of the cellular entrance of FAT into CHO cells when the "Bodipy" fluorophore is covalently attached as the cargo. Simultaneously, two other macromolecular therapeutics, e.g., proapoptotic domain peptide (PAD, a 14-mer peptide) and programmed death ligand 1 (PDL1) morpholino (a 25-mer antisense oligo), were successfully conjugated with FAT and delivered into human carcinoma cells, and their efficacy was analyzed by MTT assay and western blot technique, respectively. Having obtained promising results in internalizing different types of cargos, FAT could be envisaged as a potential drug delivery agent as an alternative to natural CPPs for future application.


Assuntos
Carcinoma , Peptídeos Penetradores de Células , Animais , Antígeno B7-H1 , Peptídeos Penetradores de Células/química , Cricetinae , Cricetulus , Guanidina , Humanos , Morfolinos
14.
J Cell Sci ; 135(9)2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35393618

RESUMO

In the trunk of developing zebrafish embryos, adjacent myotome blocks transmit contractile force via myoseptal junctions (MJs), which are dynamic structures that connect the actin cytoskeleton of skeletal muscle cells to extracellular matrix components via transmembrane protein complexes in the sarcolemma. Here, we report that the endolysosomal ion channel, two-pore channel type 1 (TPC1, encoded by tpcn1), generates highly localized non-propagating Ca2+ transients that play a distinct and required role in the capture and attachment of superficial slow skeletal muscle cells at MJs. Use of antisense morpholinos or CRISPR/Cas9 gene editing to disrupt tpcn1 gene expression resulted in abnormal MJ phenotypes, including slow skeletal muscle cells detaching from or crossing the myosepta. We also report that TPC1-decorated endolysosomes are dynamically associated with MJs in a microtubule-dependent manner, and that attenuating tpcn1 expression or TPC1 function disrupted endolysosomal trafficking and resulted in an abnormal distribution of ß-dystroglycan (encoded by dag1; a key transmembrane component of the dystrophin-associated protein complex). Taken together, our data suggest that localized TPC1-generated Ca2+ signals facilitate essential endolysosomal trafficking and membrane contact events, which help form and maintain MJs following the onset of slow skeletal muscle cell contractile activity. This article has an associated First Person interview with the first author of the paper.


Assuntos
Cálcio , Peixe-Zebra , Animais , Cálcio/metabolismo , Distroglicanas/metabolismo , Humanos , Morfolinos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
15.
Stem Cells Dev ; 31(11-12): 278-295, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35469439

RESUMO

Cellular metabolism plays both an active and passive role in embryonic development, pluripotency, and cell-fate decisions. However, little is known regarding the role of metabolism in regulating the recently described "formative" pluripotent state. The pluripotent developmental continuum features a metabolic switch from a bivalent metabolism (both glycolysis and oxidative phosphorylation) in naive cells, to predominantly glycolysis in primed cells. We investigated the role of pyruvate kinase muscle isoforms 1/2 (PKM1/2) in naive, formative, and primed mouse embryonic stem cells through modulation of PKM1/2 messenger RNA transcripts using steric blocking morpholinos that downregulate PKM2 and upregulate PKM1. We have examined these effects in naive, formative, and primed cells by quantifying the effects of PKM1/2 modulation on pluripotent and metabolic transcripts and by measuring shifts in the population frequencies of cells expressing naive and primed cell surface markers by flow cytometry. Our results demonstrate that modulating PKM1 and PKM2 levels alters the transition from the naive state into a primed pluripotent state by enhancing the proportion of the affected cells seen in the "formative" state. Therefore, we conclude that PKM1/2 actively contributes to mechanisms that oversee early stem pluripotency and their progression toward a primed pluripotent state.


Assuntos
Células-Tronco Pluripotentes , Piruvato Quinase , Animais , Diferenciação Celular/genética , Camundongos , Morfolinos/metabolismo , Músculos , Células-Tronco Pluripotentes/metabolismo , Isoformas de Proteínas , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
16.
EMBO Rep ; 23(6): e53955, 2022 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-35393769

RESUMO

Duchenne muscular dystrophy (DMD) is a systemic progressive muscular disease caused by frame-disrupting mutations in the DMD gene. Although exon-skipping antisense oligonucleotides (AOs) are clinically approved and can correct DMD, insufficient muscle delivery limits efficacy. If AO activity can be enhanced by safe dietary supplements, clinical trials for efficacy can be undertaken rapidly to benefit patients. We showed previously that intravenous glycine enhanced phosphorodiamidate morpholino oligomer (PMO) delivery to peripheral muscles in mdx mice. Here, we demonstrate that the combination of oral glycine and metformin with intravenous PMO enhances PMO activity, dystrophin restoration, extends lifespan, and improves body-wide function and phenotypic rescue of dystrophin /utrophin double knock-out (DKO) mice without any overt adverse effects. The DKO mice treated with the combination without altering the approved administration protocol of PMO show improved cardio-respiratory and behavioral functions. Metformin and glycine individually are ineffective in DMD patients, but the combination of PMO with clinically-approved oral glycine and metformin might improve the efficacy of the treatment also in DMD patients. Our data suggest that this combination therapy might be an attractive therapy for DMD and potentially other muscle diseases requiring systemic treatment with AOs.


Assuntos
Distrofina , Metformina , Animais , Distrofina/genética , Terapia Genética/métodos , Glicina/uso terapêutico , Humanos , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/genética , Morfolinos/uso terapêutico , Músculo Esquelético , Utrofina/genética
17.
Methods Mol Biol ; 2442: 425-443, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35320539

RESUMO

Techniques for disrupting gene expression are invaluable tools for the analysis of the biological role of a gene product. Because of its genetic tractability and multiple advantages over conventional mammalian models, the zebrafish (Danio rerio) is recognized as a powerful system for gaining new insight into diverse aspects of human health and disease. Among the multiple mammalian gene families for which the zebrafish has shown promise as an invaluable model for functional studies, the galectins have attracted great interest due to their participation in early development, regulation of immune homeostasis, and recognition of microbial pathogens. Galectins are ß-galactosyl-binding lectins with a characteristic sequence motif in their carbohydrate recognition domains (CRDs), that constitute an evolutionary conserved family ubiquitous in eukaryotic taxa. Galectins are emerging as key players in the modulation of many important pathological processes, which include acute and chronic inflammatory diseases, autoimmunity and cancer, thus making them potential molecular targets for innovative drug discovery. Here, we provide a review of the current methods available for the manipulation of gene expression in the zebrafish, with a focus on gene knockdown [morpholino (MO)-derived antisense oligonucleotides] and knockout (CRISPR-Cas) technologies.


Assuntos
Galectinas , Peixe-Zebra , Animais , Galectinas/metabolismo , Técnicas de Silenciamento de Genes , Mamíferos/genética , Morfolinos/genética , Morfolinos/metabolismo , RNA/metabolismo , Peixe-Zebra/metabolismo
18.
Br J Cancer ; 127(1): 43-55, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35277659

RESUMO

BACKGROUND: 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 tumour necrosis can be stimulated. The canonical Wnt inhibitor Dickkopf-1 (Dkk-1) enhances OS survival in part through upregulation of aldehyde-dehydrogenase-1A1 which neutralises reactive oxygen species originating from nutritional stress and chemotherapeutic challenge. METHODS: A vivo morpholino (DkkMo) was employed to block the expression of Dkk-1 in OS cells. Cell mitosis, gene expression and bone destruction were measured in vitro and in vivo in the presence and absence of doxorubicin (DRB). RESULTS: DkkMo reduced the expression of Dkk-1 and Aldh1a1, reduced expansion of OS tumours, preserved bone volume and architecture and stimulated tumour necrosis. This was observed in the presence or absence of DRB. CONCLUSION: These results indicate that administration of DkkMo with or without chemotherapeutics can substantially improve OS outcome with respect to tumour expansion and osteolytic corruption of bone in experimental OS model.


Assuntos
Neoplasias Ósseas , Osteossarcoma , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Morfolinos/genética , Morfolinos/farmacologia , Necrose , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Osteossarcoma/metabolismo
19.
Methods Mol Biol ; 2434: 129-141, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213014

RESUMO

Phosphorodiamidate morpholino oligomers (PMOs) offer great promise as therapeutic agents for translation blocking or splice modulation due to their high stability and affinity for target sequences. However, in spite of their neutral charge as compared to natural oligonucleotides or phosphorothioate analogs, they still show little permeability for cellular membranes, highlighting the need for effective cytosolic delivery strategies. In addition, the implementation of strategies for efficient cellular targeting is highly desirable to minimize side effects and maximize the drug dose at its site of action. Anthrax toxin is a three-protein toxin of which the pore-forming protein anthrax protective antigen (PA) can be redirected to a receptor of choice and lethal factor (LF), one of the two substrate proteins, can be coupled to various cargoes for efficient cytosolic cargo delivery. In this protocol, we describe the steps to produce the proteins and protein conjugates required for cytosolic delivery of PMOs through the cation-selective pore generated by anthrax protective antigen. The method relies on the introduction of a unique cysteine at the C-terminal end of a truncated LF (aa 1-254), high-yield expression of the (truncated) toxin proteins in E. coli, functionalization of a PMO with a maleimide group and coupling of the maleimide-functionalized PMO to the unique cysteine on LF by maleimide-thiol conjugation chemistry. Through co-administration of PA with LF-PMO conjugates, an efficient cytosolic delivery of PMOs can be obtained.


Assuntos
Antraz , Toxinas Bacterianas , Antígenos de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Escherichia coli/metabolismo , Humanos , Morfolinos/farmacologia , Oligonucleotídeos Antissenso/farmacologia
20.
Methods Mol Biol ; 2434: 191-205, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213018

RESUMO

Antisense oligonucleotides (AONs) are small synthetic molecules of therapeutic interest for a variety of human disease. Their ability to bind mRNA and affect its splicing gives AONs potential use for exon skipping therapies aimed at restoring the dystrophin transcript reading frame for Duchenne muscular dystrophy (DMD) patients. The neutrally charged phosphorodiamidate morpholino oligomers (PMOs) are a stable and relatively nontoxic AON modification. To assess exon skipping efficiency in vitro, it is important to deliver them to target cells. Here, we describe a method for the delivery of PMOs to myoblasts by electroporation. The described protocol for the Amaxa 4D X unit nucleofector system allows efficient processing of 16 samples in one nucleocuvette strip, aiding in high-throughput PMO efficacy screens.


Assuntos
Terapia Genética , Distrofia Muscular de Duchenne , Distrofina/genética , Distrofina/metabolismo , Eletroporação , Terapia Genética/métodos , Humanos , Morfolinos/genética , Morfolinos/uso terapêutico , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo
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