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1.
Molecules ; 24(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408997

RESUMO

One of the crucial aspects of screening antisense oligonucleotides destined for therapeutic application is confidence that the antisense oligomer is delivered efficiently into cultured cells. Efficient delivery is particularly vital for antisense phosphorodiamidate morpholino oligomers, which have a neutral backbone, and are known to show poor gymnotic uptake. Here, we report several methods to deliver these oligomers into cultured cells. Although 4D-Nucleofector™ or Neon™ electroporation systems provide efficient delivery and use lower amounts of phosphorodiamidate morpholino oligomer, both systems are costly. We show that some readily available transfection reagents can be used to deliver phosphorodiamidate morpholino oligomers as efficiently as the electroporation systems. Among the transfection reagents tested, we recommend Lipofectamine 3000™ for delivering phosphorodiamidate morpholino oligomers into fibroblasts and Lipofectamine 3000™ or Lipofectamine 2000™ for myoblasts/myotubes. We also provide optimal programs for nucleofection into various cell lines using the P3 Primary Cell 4D-Nucleofector™ X Kit (Lonza), as well as antisense oligomers that redirect expression of ubiquitously expressed genes that may be used as positive treatments for human and murine cell transfections.


Assuntos
Eletroporação/métodos , Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Interferência de RNA , Transfecção/métodos , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Cadeias alfa de Integrinas/antagonistas & inibidores , Cadeias alfa de Integrinas/genética , Cadeias alfa de Integrinas/metabolismo , Lipídeos/química , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/síntese química , Morfolinos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Oligonucleotídeos Antissenso/síntese química , Oligonucleotídeos Antissenso/metabolismo , Cultura Primária de Células , Proteínas do Complexo SMN/antagonistas & inibidores , Proteínas do Complexo SMN/genética , Proteínas do Complexo SMN/metabolismo
2.
Malar J ; 18(1): 294, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31462239

RESUMO

BACKGROUND: Insecticides are still at the core of insect pest and vector control programmes. Several lines of evidence indicate that ABC transporters are involved in detoxification processes against insecticides, including permethrin and other pyrethroids. In particular, the ABCG4 gene, a member of the G subfamily, has consistently been shown to be up-regulated in response to insecticide treatments in the mosquito malaria vector Anopheles stephensi (both adults and larvae). METHODS: To verify the actual involvement of this transmembrane protein in the detoxification process of permethrin, bioassays on larvae of An. stephensi, combining the insecticide with a siRNA, specifically designed for the inhibition of ABCG4 gene expression were performed. Administration to larvae of the same siRNA, labeled with a fluorescent molecule, was effected to investigate the systemic distribution of the inhibitory RNA into the larval bodies. Based on siRNA results, similar experiments using antisense Vivo-Morpholinos (Vivo-MOs) were effected. These molecules, compared to siRNA, are expected to guarantee a higher stability in environmental conditions and in the insect gut, and present thus a higher potential for future in-field applications. RESULTS: Bioassays using two different concentrations of siRNA, associated with permethrin, led to an increase of larval mortality, compared with results with permethrin alone. These outcomes confirm that ABCG4 transporter plays a role in the detoxification process against the selected insecticide. Moreover, after fluorescent labelling, it was shown the systemic dissemination of siRNA in different body districts of An. stephensi larvae, which suggest a potential systemic effect of the molecule. At the same time, results of Vivo-MO experiments were congruent with those obtained using siRNA, thus confirming the potential of ABCG4 inhibition as a strategy to increase permethrin susceptibility in mosquitoes. For the first time, Vivo-MOs were administered in water to larvae, with evidence for a biological effect. CONCLUSIONS: Targeting ABCG4 gene for silencing through both techniques resulted in an increased pyrethroid efficacy. These results open the way toward the possibility to exploit ABCG4 inhibition in the context of integrated programmes for the control An. stephensi mosquitoes and malaria transmission.


Assuntos
Anopheles/genética , Resistência a Inseticidas/genética , Inseticidas , Morfolinos/administração & dosagem , Piretrinas , RNA Antissenso/genética , Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Bioensaio , Larva/genética , Malária/prevenção & controle , Morfolinos/genética , Controle de Mosquitos , Mosquitos Vetores , Interferência de RNA , RNA Interferente Pequeno
3.
Methods Cell Biol ; 151: 323-334, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30948016

RESUMO

MicroRNAs (miRNAs) are small non-coding RNAs that repress the translation and reduce the stability of target mRNAs in animal cells. Post-transcriptional regulation mediated by miRNAs is a highly conserved mechanism utilized by organisms throughout phylogeny to fine tune gene expression. We document the approaches used to study the function of a single miRNA and miRNA regulation of biological pathways in the sea urchin embryo. The protocols that are described include selection of miRNA inhibitors, test of miRNA direct targets, and the use of target protector morpholinos to evaluate the impact of miRNA inhibition on its targets. Using the described techniques and strategies, the sea urchin researcher will be able to validate a miRNA's direct targets and evaluate how inhibition of the miRNA affects developmental processes. These results will contribute to our understanding of the regulatory roles of miRNAs in development.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Ouriços-do-Mar/genética , Animais , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Morfolinos/genética , RNA Mensageiro/genética , Ouriços-do-Mar/crescimento & desenvolvimento , Transdução de Sinais/genética , Fatores de Transcrição/genética
4.
Chem Commun (Camb) ; 55(35): 5139-5142, 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-30977478

RESUMO

The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.


Assuntos
Morfolinos/química , RNA Interferente Pequeno/química , Animais , Apolipoproteínas B/genética , Proteínas Argonauta/genética , Inativação Gênica , Humanos , Camundongos , Modelos Moleculares , Morfolinos/síntese química , Morfolinos/genética , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética
5.
Nat Commun ; 10(1): 1083, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842454

RESUMO

VEGFA signaling controls physiological and pathological angiogenesis and hematopoiesis. Although many context-dependent signaling pathways downstream of VEGFA have been uncovered, vegfa transcriptional regulation in vivo remains unclear. Here, we show that the ETS transcription factor, Etv6, positively regulates vegfa expression during Xenopus blood stem cell development through multiple transcriptional inputs. In agreement with its established repressive functions, Etv6 directly inhibits expression of the repressor foxo3, to prevent Foxo3 from binding to and repressing the vegfa promoter. Etv6 also directly activates expression of the activator klf4; reflecting a genome-wide paucity in ETS-binding motifs in Etv6 genomic targets, Klf4 then recruits Etv6 to the vegfa promoter to activate its expression. These two mechanisms (double negative gate and feed-forward loop) are classic features of gene regulatory networks specifying cell fates. Thus, Etv6's dual function, as a transcriptional repressor and activator, controls a major signaling pathway involved in endothelial and blood development in vivo.


Assuntos
Proteína Forkhead Box O3/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Repressoras/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/fisiologia , Animais , Embrião não Mamífero , Endotélio/embriologia , Endotélio/metabolismo , Proteína Forkhead Box O3/antagonistas & inibidores , Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Redes Reguladoras de Genes/fisiologia , Fatores de Transcrição Kruppel-Like/antagonistas & inibidores , Fatores de Transcrição Kruppel-Like/genética , Morfolinos/genética , Oligonucleotídeos Antissenso/genética , Proteínas Proto-Oncogênicas c-ets/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-ets/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais/fisiologia , Somitos/embriologia , Somitos/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética
6.
Mar Biotechnol (NY) ; 21(1): 52-64, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30443836

RESUMO

The protein level of muscle-specific human NogoA is abnormally upregulated in amyotrophic lateral sclerosis (ALS) mice and patients. On the other hand, while the presence of miR-206 in muscle cells delays onset and death in ALS, the relationship between these two phenomena remains unclear. Mammalian NogoA protein, also known as Reticulon 4a (Rtn4a), plays an important role in inhibiting the outgrowth of motor neurons. Our group previously identified zebrafish rtn4al as the target gene of miR-206 and found that knockdown of miR-206 increases rtn4al mRNA and Rtn4al protein in zebrafish embryos. It can be concluded from these results that neurite outgrowth of motor neurons is inhibited by Rtn4a1, which is entirely consistent with overexpression of either human NogoA or zebrafish homolog Rtn4al. Since an animal model able to express NogoA/rtn4al at the mature stage is unavailable, we generated a zebrafish transgenic line, Tg(Zα:TetON-Rtn4al), which conditionally and specifically overexpresses Rtn4al in the muscle tissue. After doxycycline induction, adult zebrafish displayed denervation at neuromuscular junction during the first week, then muscle disintegration and split myofibers during the third week, and, finally, significant weight loss in the sixth week. These results suggest that this zebrafish transgenic line, representing the inducible overexpression of Rtn4a1 in muscle, may provide an alternative animal model with which to study ALS because it exhibits ALS-like phenotype.


Assuntos
Esclerose Amiotrófica Lateral/genética , Modelos Animais de Doenças , Neurônios Motores/metabolismo , Proteínas da Mielina/genética , Junção Neuromuscular/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Animais Geneticamente Modificados , Doxiciclina/farmacologia , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas da Mielina/agonistas , Proteínas da Mielina/antagonistas & inibidores , Proteínas da Mielina/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Proteínas Nogo/agonistas , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/agonistas , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
7.
Mol Ther ; 27(1): 76-86, 2019 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-30448197

RESUMO

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.


Assuntos
Éxons/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Cães , Distrofina/genética , Distrofina/metabolismo , Morfolinos/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Oligonucleotídeos Antissenso/genética , Fases de Leitura/genética
8.
FASEB J ; 33(3): 3613-3622, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30475641

RESUMO

Centrosomal proteins play critical roles in ciliogenesis. Mutations in many centrosomal proteins have been documented to contribute to developmental defects and cilium-related diseases. Centrosomal protein fibroblast growth factor receptor 1 oncogene partner-related protein of 20 kDa (FOR20) is crucial for ciliogenesis in mammalian cells and the unicellular eukaryote Paramecium; however, the biologic significance of FOR20 in vertebrate development remains unclear. We cloned the zebrafish homolog of the for20 gene and found that for20 mRNA is enriched in ciliated tissues during early zebrafish development. Knockdown of for20 by morpholino oligonucleotides in zebrafish results in multiple ciliary phenotypes, including curved body, hydrocephaly, pericardial edema, kidney cysts, and left-right asymmetry defects. for20 morphants show reduced number and length of cilia in Kupffer's vesicle and pronephric ducts. High-speed video microscopy reveals that cilia in most for20 morphants are consistently paralyzed or beat arrhythmically. To confirm the ciliary phenotypes of for20 morphants, we used the CRISPR/Cas9 system to disrupt for20 gene in zebrafish. for20 mutants exhibit multiple ciliary phenotypes resembling the defects in for20 morphants. All of these phenotypes in for20 morphants and mutants are significantly reversed by exogenous expression of for20 mRNA. Taken together, these data suggest that FOR20 is required for cilium-mediated processes during zebrafish embryogenesis.-Xie, S., Jin, J., Xu, Z., Huang, Y., Zhang, W., Zhao, L., Lo, L. J., Peng, J., Liu, W., Wang, F., Shu, Q., Zhou, T. Centrosomal protein FOR20 is essential for cilia-dependent development in zebrafish embryos.


Assuntos
Centrossomo/fisiologia , Cílios/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Padronização Corporal/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes/métodos , Morfolinos/genética , Mutação/genética , RNA Mensageiro/genética
9.
ACS Sens ; 4(1): 185-191, 2019 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-30592402

RESUMO

Morpholino Oligonucleotides (MOs), an uncharged DNA analogue, are functionalized with an acrylamide moiety and incorporated into polymer hydrogels as responsive cross-links for microRNA sequence detection. The MO cross-links can be selectively cleaved by a short target analyte single-stranded DNA (ssDNA) sequence based on microRNA, inducing a distinct swelling response measured optically. The MO cross-links offer significant improvement over DNA based systems through improved thermal stability, no salt requirement and 1000-fold improved sensitivity over a comparative biosensor, facilitating a wider range of sensing conditions. Analysis was also achieved using a mobile phone camera, demonstrating portability.


Assuntos
DNA de Cadeia Simples/análise , Hidrogéis/química , MicroRNAs/genética , Morfolinos/química , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA de Cadeia Simples/genética , Humanos , Limite de Detecção , Morfolinos/genética , Hibridização de Ácido Nucleico , Smartphone
10.
Elife ; 72018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30520734

RESUMO

A crucial step in cell differentiation is the silencing of developmental programs underlying multipotency. While much is known about how lineage-specific genes are activated to generate distinct cell types, the mechanisms driving suppression of stemness are far less understood. To address this, we examined the regulation of the transcriptional network that maintains progenitor identity in avian neural crest cells. Our results show that a regulatory circuit formed by Wnt, Lin28a and let-7 miRNAs controls the deployment and the subsequent silencing of the multipotency program in a position-dependent manner. Transition from multipotency to differentiation is determined by the topological relationship between the migratory cells and the dorsal neural tube, which acts as a Wnt-producing stem cell niche. Our findings highlight a mechanism that rapidly silences complex regulatory programs, and elucidate how transcriptional networks respond to positional information during cell differentiation.


Assuntos
Proteínas Aviárias/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Crista Neural/metabolismo , Neurônios/metabolismo , Proteínas Wnt/genética , Dedos de Zinco/genética , Animais , Proteínas Aviárias/metabolismo , Diferenciação Celular , Movimento Celular , Embrião de Galinha , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , MicroRNAs/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Crista Neural/citologia , Crista Neural/crescimento & desenvolvimento , Neurônios/citologia , Neuropeptídeos/biossíntese , Neuropeptídeos/genética , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Transcrição Genética , Proteínas Wnt/antagonistas & inibidores , Proteínas Wnt/metabolismo , Via de Sinalização Wnt
11.
Neural Dev ; 13(1): 22, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30219101

RESUMO

BACKGROUND: Proper patterning of dendritic and axonal arbors is a critical step in the formation of functional neuronal circuits. Developing circuits rely on an array of molecular cues to shape arbor morphology, but the underlying mechanisms guiding the structural formation and interconnectivity of pre- and postsynaptic arbors in real time remain unclear. Here we explore how Down syndrome cell adhesion molecule (DSCAM) differentially shapes the dendritic morphology of central neurons and their presynaptic retinal ganglion cell (RGC) axons in the developing vertebrate visual system. METHODS: The cell-autonomous role of DSCAM, in tectal neurons and in RGCs, was examined using targeted single-cell knockdown and overexpression approaches in developing Xenopus laevis tadpoles. Axonal arbors of RGCs and dendritic arbors of tectal neurons were visualized using real-time in vivo confocal microscopy imaging over the course of 3 days. RESULTS: In the Xenopus visual system, DSCAM immunoreactivity is present in RGCs, cells in the optic tectum and the tectal neuropil at the time retinotectal synaptic connections are made. Downregulating DSCAM in tectal neurons significantly increased dendritic growth and branching rates while inducing dendrites to take on tortuous paths. Overexpression of DSCAM, in contrast, reduced dendritic branching and growth rate. Functional deficits mediated by tectal DSCAM knockdown were examined using visually guided behavioral assays in swimming tadpoles, revealing irregular behavioral responses to visual stimulus. Functional deficits in visual behavior also corresponded with changes in VGLUT/VGAT expression, markers of excitatory and inhibitory transmission, in the tectum. Conversely, single-cell DSCAM knockdown in the retina revealed that RGC axon arborization at the target is influenced by DSCAM, where axons grew at a slower rate and remained relatively simple. In the retina, dendritic arbors of RGCs were not affected by the reduction of DSCAM expression. CONCLUSIONS: Together, our observations implicate DSCAM in the control of both pre- and postsynaptic structural and functional connectivity in the developing retinotectal circuit, where it primarily acts as a neuronal brake to limit and guide postsynaptic dendrite growth of tectal neurons while it also facilitates arborization of presynaptic RGC axons cell autonomously.


Assuntos
Moléculas de Adesão Celular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/citologia , Sinapses/metabolismo , Vias Visuais/citologia , Vias Visuais/crescimento & desenvolvimento , Proteínas de Xenopus/metabolismo , Animais , Aprendizagem da Esquiva/fisiologia , Axônios/metabolismo , Moléculas de Adesão Celular/genética , Dendritos/metabolismo , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Processamento de Imagem Assistida por Computador , Microscopia Confocal , Morfolinos/genética , Morfolinos/metabolismo , Morfolinos/farmacologia , Neurônios/metabolismo , Estimulação Luminosa/efeitos adversos , Retina/citologia , Retina/crescimento & desenvolvimento , Colículos Superiores/citologia , Colículos Superiores/crescimento & desenvolvimento , Sinapses/efeitos dos fármacos , Transfecção , Proteínas Vesiculares de Transporte de Glutamato/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis
12.
Methods Mol Biol ; 1828: 151-163, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171540

RESUMO

Antisense oligonucleotide induced exon skipping emerges as a promising therapeutic strategy for patients suffering from a devastating muscle disorder Duchenne muscular dystrophy (DMD). Systemic administration of antisense phosphorodiamidate morpholino oligomers (PMOs) targeting exons 6 and 8 in dystrophin mRNA of the canine X-linked muscular dystrophy model in Japan (CXMDJ) that lacks exon 7, restored dystrophin expression throughout skeletal muscle and ameliorated skeletal muscle pathology and function. However, the antisense PMO regime used in CXMDJ could not be considered for a direct application to DMD patients so far, because this type of mutation is quite rare. We have identified a DMD patient with an exon 7 deletion; and tried a direct translation of the antisense PMOs used in dog models to the DMD patient's cells. We converted fibroblasts obtained from CXMDJ dogs and from the DMD patient to myotubes by MyoD transduction using fluorescence-activated cell sorting (FACS). We subsequently designed antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 and administered them as a cocktail to the in vitro generated dog or human myotubes. In both cases, we observed comparable skipping efficacy of exons 6 and 8 and restoration of dystrophin protein. The accompanying skipping of exon 9, which does not alter the reading frame, varied according to the cell origin. The antisense PMOs originally administered to the CXMDJ dog model were capable of inducing multi-exon skipping of the dystrophin gene on the FACS-aided MyoD-transduced fibroblasts derived from an exon 7-deleted DMD patient. These data support the suitability of dog as a laboratory model for DMD because the similarity of dystrophin sequences allowed a successful translation of the dog's PMOs to DMD patients cells.


Assuntos
Distrofina/genética , Éxons , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/genética , Processamento de RNA , Animais , Células Cultivadas , Cães , Fibroblastos/metabolismo , Terapia Genética , Vetores Genéticos/genética , Humanos , Morfolinos/administração & dosagem , Distrofia Muscular de Duchenne/terapia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/metabolismo , Oligonucleotídeos Antissenso/administração & dosagem , Retroviridae/genética , Transdução Genética
13.
Methods Mol Biol ; 1828: 263-273, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171547

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder due to the lack of dystrophin production. The disease is characterized by muscle wasting, with the most common causes of death being respiratory failure or heart failure. Recently, exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is used as an FDA approved treatment for DMD. Peptide-conjugated PMOs (PPMOs) are used to increase exon skipping efficacy in the heart and are a promising therapy for DMD. Researchers have previously relied on high-performance liquid chromatography (HPLC) or liquid chromatography-mass spectrometry (LC/MS) methods for detecting PPMO uptake, but an enzyme-linked immunosorbent assay (ELISA) has been shown to have greater sensitivity. Here, we present methodologies to determine the uptake efficiency of a PPMO into the heart and efficacy of exon 51 skipping by a PPMO injected retro-orbitally into a humanized DMD mouse model via ELISA and RT-PCR, respectively.


Assuntos
Distrofina/genética , Regulação da Expressão Gênica , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Éxons , Humanos , Camundongos , Camundongos Transgênicos , Morfolinos/administração & dosagem , Distrofia Muscular de Duchenne/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
14.
Methods Mol Biol ; 1828: 275-292, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171548

RESUMO

Exon-skipping therapy is an emerging approach that uses synthetic DNA-like molecules called antisense oligonucleotides (ASOs) to splice out frame-disrupting parts of mRNA, restore the reading frame, and produce truncated yet functional proteins. Phosphorodiamidate morpholino oligomer (PMO) is one of the safest among therapeutic ASOs for patients and has recently been approved under the accelerated approval program by the US Food and Drug Administration (FDA) as the first ASO-based drug for Duchenne muscular dystrophy (DMD). Multi-exon skipping utilizing ASOs can theoretically treat 80-90% of patients with DMD. Here, we describe the systemic delivery of a cocktail of ASOs to skip exon 51 and exons 45-55 in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting, and the evaluation of their efficacies in vivo.


Assuntos
Distrofina/genética , Éxons , Distrofia Muscular de Duchenne/genética , Processamento de RNA , Animais , Expressão Gênica , Marcação de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/administração & dosagem , Morfolinos/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Deleção de Sequência
15.
Methods Mol Biol ; 1828: 293-306, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171549

RESUMO

Morpholino oligomers have great therapeutic potential for treatment of a broad range of human diseases, including viral, bacterial, age-related, and genetic diseases, but they suffer from poor systemic delivery into cells. Although various approaches have been undertaken to address the delivery problem, it remains as the major barrier of morpholinos to be used as effective therapeutics. This slow development is in part due to the cost of materials and the animal models used for screening the efficacy and safety of those delivery approaches. The need to have an inexpensive vertebrate model for assessing in vivo delivery of morpholinos is evident. Therefore, we have produced a novel transgenic zebrafish model containing a dual reporter cassette for determination of in vivo delivery, bio-distribution, and safety of a morpholino. The levels of morpholino delivered to the cells in various tissues can be determined by changes in reporter gene expressions caused by morpholino-induced exon skipping. This chapter provides a description of the reagents, equipment, and procedure for successful retro-orbital injection of a peptide-conjugated morpholino into the blood stream of the adult zebrafish to cause targeted exon skipping in the heart of the zebrafish.


Assuntos
Técnicas de Transferência de Genes , Morfolinos/genética , Peixe-Zebra/genética , Animais , Éxons , Expressão Gênica , Genes Reporter , Modelos Animais , Morfolinos/administração & dosagem , Reação em Cadeia da Polimerase
16.
Methods Mol Biol ; 1828: 327-342, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171551

RESUMO

Exon-skipping antisense oligonucleotides (AOs) are promising treatments for muscle-related genetic ailments including Duchenne muscular dystrophy (DMD), but clinical translation is unfortunately hampered by insufficient systemic delivery. Here we describe that how one can employ a glucose-fructose injection mixture to improve muscle uptake and functional outcomes of DMD AOs in energy-deficient peripheral muscles of mdx mice. The potentiating effect of glucose-fructose on AOs in energy-deficient muscles offers a simple and economical method for enhancing AO potency, reducing screening costs for researchers and accelerating the translation of nucleic acid-based therapeutics in DMD and other muscular dystrophies.


Assuntos
Éxons , Frutose/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Processamento de RNA , Trifosfato de Adenosina/metabolismo , Animais , Clatrina/metabolismo , Distrofina/genética , Distrofina/metabolismo , Complexo de Proteínas Associadas Distrofina/genética , Complexo de Proteínas Associadas Distrofina/metabolismo , Endocitose , Metabolismo Energético , Frutose/metabolismo , Glucose/metabolismo , Humanos , Imuno-Histoquímica , Redes e Vias Metabólicas , Camundongos , Camundongos Endogâmicos mdx , Morfolinos/administração & dosagem , Morfolinos/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética
17.
Methods Mol Biol ; 1828: 355-363, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171553

RESUMO

Efficient intracellular delivery is critical to the successful application of synthetic antisense oligonucleotides (ASOs) to modulate gene expression. The conjugation of cell-penetrating peptides (CPPs) to ASOs has been shown to significantly improve their intracellular delivery. It is important, however, that formation of the covalent linkage between the peptide and oligonucleotide is efficient and orthogonal, to ensure high yields and a homogeneous product. Described herein are efficient and facile methodologies for the conjugation of peptides to ASOs, and their subsequent labeling with various moieties such as fluorescent dyes for intracellular tracking studies.


Assuntos
Corantes Fluorescentes , Técnicas de Transferência de Genes , Oligonucleotídeos , Peptídeos , Corantes Fluorescentes/química , Humanos , Estrutura Molecular , Morfolinos/administração & dosagem , Morfolinos/química , Morfolinos/genética , Oligonucleotídeos/química , Oligonucleotídeos Antissenso , Peptídeos/química , Coloração e Rotulagem
18.
Methods Mol Biol ; 1828: 365-379, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171554

RESUMO

Exon skipping is an emerging approach to treating Duchenne muscular dystrophy (DMD), one of the most common lethal genetic disorders. Exon skipping uses synthetic antisense oligonucleotides (AONs) to splice out frame-disrupting exon(s) of DMD mRNA to restore the reading frame of the gene products and produce truncated yet functional proteins. The FDA conditionally approved the first exon-skipping AON, called eteplirsen (brand name ExonDys51), targeting exon 51 of the DMD gene, in late 2016. Using a cocktail of AONs, multiple exons can be skipped, which can theoretically treat 80-90% of patients with DMD. Although the success of multiple exon skipping in a DMD dog model has made a significant impact on the development of therapeutics for DMD, unmodified AONs such as phosphorodiamidate morpholino oligomers (PMOs) have little efficacy in cardiac muscles. Here, we describe our technique of intravenous injection of a cocktail of peptide-conjugated PMOs (PPMOs) to skip multiple exons, exons 6 and 8, in both skeletal and cardiac muscles in dystrophic dogs and the evaluation of the efficacy and toxicity.


Assuntos
Distrofina/genética , Éxons , Morfolinos/genética , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , Peptídeos , Processamento de RNA , Administração Intravenosa , Animais , Modelos Animais de Doenças , Cães , Distrofina/metabolismo , Expressão Gênica , Imuno-Histoquímica , Morfolinos/administração & dosagem , Morfolinos/química , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/fisiopatologia , Peptídeos/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Methods Mol Biol ; 1828: 439-454, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171558

RESUMO

Spinal muscular atrophy (SMA), the most common gentic cause of infantile death caused by mutations in the SMN1 gene, presents a unique case in the field of splice modulation therapy, where a gene (or lack of) is responsible for causing the disease phenotype but treatment is not focused around it. Antisense therapy targeting SMN2 which leads to SMN protein expression has been at the forefront of research when it comes to developing a feasible therapy for treating SMA. Recent FDA approval of an antisense-based drug with the 2'-methoxyethoxy (2'MOE) chemistry, called nusinersen (Spinraza), brought antisense drugs into the spotlight. The 2'MOE, although effective, has weaknesses such as the inability to cross the blood-brain barrier and the high cost of treatment. This propelled the research community to investigate new chemistries of antisense oligonucleotides (ASOs) that may be better in both treatment and cost efficiency. Here we describe two types of ASOs, phosphorodiamidate morpholino oligomers (PMOs) and locked nucleic acids (LNA)-DNA mixmers, being investigated as potential treatments for SMA, and methods used to test their efficacy, including quantitative RT-PCR, Western blotting, and immunofluorescence staining to detect SMN in nuclear gems/Cajal bodies, in type I SMA patient fibroblast cell lines.


Assuntos
Éxons , Regulação da Expressão Gênica , Atrofia Muscular Espinal/genética , Oligonucleotídeos Antissenso/genética , Processamento de RNA , Fibroblastos , Expressão Gênica , Marcação de Genes , Humanos , Morfolinos/administração & dosagem , Morfolinos/química , Morfolinos/genética , Neurônios Motores/metabolismo , Oligonucleotídeos , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
20.
Methods Mol Biol ; 1828: 467-477, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171560

RESUMO

The application of antisense oligonucleotides (AONs) to modify pre-messenger RNA splicing has great potential for treating genetic diseases. The strategies used to redirect splicing for therapeutic purpose involve the use of AONs complementary to splice motifs, enhancer or silencer sequences. AONs to block intronic splicing silencer motifs can efficiently augment exon 7 inclusion in survival motor neuron 2 (SMN2) gene and have demonstrated robust therapeutic effects in both preclinical studies and clinical trials in spinal muscular atrophy (SMA), which has led to a recently approved drug. AONs with phosphorodiamidate morpholino oligomer (PMO) backbone have shown target engagement with restoration of the defective protein in Duchenne muscular dystrophy (DMD) and their safety profile lead to a recent conditional approval for one DMD PMO drug. PMO AONs are also effective in correcting SMN2 exon 7 splicing and rescuing SMA transgenic mice. Here we provide the details of methods that our lab has used to evaluate PMO-mediated SMN2 exon 7 inclusion in the in vivo studies conducted in SMA transgenic mice. The methods comprise mouse experiment procedures, assessment of PMOs on exon 7 inclusion at RNA levels by reverse transcription (RT-) PCR and quantitative real-time PCR. In addition, we present methodology for protein quantification using western blot in mouse tissues, on neuropathology assessment of skeletal muscle (muscle pathology and neuromuscular junction staining) as well as behaviour test in the SMA mice (righting reflex).


Assuntos
Éxons , Regulação da Expressão Gênica , Morfolinos/genética , Atrofia Muscular Espinal/genética , Processamento de RNA , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Processamento Alternativo , Animais , Animais Recém-Nascidos , Biópsia , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Transgênicos , Morfolinos/administração & dosagem , Neurônios Motores/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Junção Neuromuscular/metabolismo , Reflexo/genética
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