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1.
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
2.
Ann Neurol ; 84(6): 814-828, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30427554

RESUMO

OBJECTIVE: Galloway-Mowat syndrome (GAMOS) is a neural and renal disorder, characterized by microcephaly, brain anomalies, and early onset nephrotic syndrome. Biallelic mutations in WDR73 and the 4 subunit genes of the KEOPS complex are reported to cause GAMOS. Furthermore, an identical homozygous NUP107 (nucleoporin 107kDa) mutation was identified in 4 GAMOS-like families, although biallelic NUP107 mutations were originally identified in steroid-resistant nephrotic syndrome. NUP107 and NUP133 (nucleoporin 133kDa) are interacting subunits of the nuclear pore complex in the nuclear envelope during interphase, and these proteins are also involved in centrosome positioning and spindle assembly during mitosis. METHODS: Linkage analysis and whole exome sequencing were performed in a previously reported GAMOS family with brain atrophy and steroid-resistant nephrotic syndrome. RESULTS: We identified a homozygous NUP133 mutation, c.3335-11T>A, which results in the insertion of 9bp of intronic sequence between exons 25 and 26 in the mutant transcript. NUP133 and NUP107 interaction was impaired by the NUP133 mutation based on an immunoprecipitation assay. Importantly, focal cortical dysplasia type IIa was recognized in the brain of an autopsied patient and focal segmental glomerulosclerosis was confirmed in the kidneys of the 3 examined patients. A nup133-knockdown zebrafish model exhibited microcephaly, fewer neuronal cells, underdeveloped glomeruli, and fusion of the foot processes of the podocytes, which mimicked human GAMOS features. nup133 morphants could be rescued by human wild-type NUP133 mRNA but not by mutant mRNA. INTERPRETATION: These data indicate that the biallelic NUP133 loss-of-function mutation causes GAMOS. Ann Neurol 2018;84:814-828.


Assuntos
Predisposição Genética para Doença/genética , Hérnia Hiatal/genética , Microcefalia/genética , Antígenos de Histocompatibilidade Menor/genética , Mutação/genética , Nefrose/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Animais , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Encéfalo/patologia , Pré-Escolar , Saúde da Família , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hérnia Hiatal/diagnóstico por imagem , Hérnia Hiatal/patologia , Humanos , Lactente , Japão , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Linfócitos/metabolismo , Linfócitos/ultraestrutura , Masculino , Microcefalia/diagnóstico por imagem , Microcefalia/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Antígenos de Histocompatibilidade Menor/ultraestrutura , Morfolinos/administração & dosagem , Mutagênese Sítio-Dirigida , Nefrose/diagnóstico por imagem , Nefrose/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Fosfopiruvato Hidratase/metabolismo , Adulto Jovem , Peixe-Zebra
3.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
12.
Methods Mol Biol ; 1828: 481-487, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171561

RESUMO

Abnormal splicing of the chloride channel 1 (CLCN1) gene causes myotonic dystrophy type 1 (DM1). Therefore, controlling the alternative splicing process of this gene by antisense oligonucleotides can be a promising treatment for DM1. In this study, we describe an efficient phosphorodiamidate morpholino oligomer (PMO) delivery method by ultrasound-mediated bubble liposomes, which is a known gene delivery tool with ultrasound exposure, to treat skeletal muscles in a DM1 mouse model, HSALR. Effective delivery of PMO using this technique can help control the alternative splicing of the Clcn1 gene via exon skipping and enhance the expression of Clcn1 protein in skeletal muscles and the amelioration of myotonia. Thus, exon skipping by PMO delivery with ultrasound-mediated BLs may be feasible in myotonic dystrophy model mice.


Assuntos
Éxons , Técnicas de Transferência de Genes , Morfolinos/genética , Distrofia Miotônica/genética , Processamento de RNA , Animais , Modelos Animais de Doenças , Lipossomos , Camundongos , Camundongos Transgênicos , Morfolinos/administração & dosagem , Transfecção , Ondas Ultrassônicas
13.
Methods Mol Biol ; 1828: 497-502, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171563

RESUMO

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal-dominant disorder characterized by progressive heterotopic ossification. More than 95% of cases are caused by a recurrent mutation (617G>A; R206H) of ACVR1/ALK2, a bone morphogenetic protein (BMP) type I receptor. Recent studies revealed that ACVR1R206H induces heterotopic ossification by aberrant activation in response to activin A. Because ACVR1R206H is a hyperactive receptor, a promising therapeutic strategy is to decrease the activity of ACVR1 in patients. Here, we describe a method to reduce ACVR1 expression in FOP patient cells by exon skipping in ACVR1 mRNAs using phosphorodiamidate morpholino oligomers (PMOs). This strategy can be applied to the screen to select antisense oligomers to knockdown not only ACVR1 but also genes which cause other autosomal-dominant genetic diseases.


Assuntos
Receptores de Ativinas Tipo I/genética , Éxons , Marcação de Genes , Morfolinos/genética , Miosite Ossificante/genética , Processamento de RNA , Biomarcadores , Regulação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Morfolinos/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Methods Mol Biol ; 1828: 553-564, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30171567

RESUMO

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches available for the treatment of several neuromuscular disorders, including Duchenne muscular dystrophy. The main weakness of this treatment arises from the low efficiency and sporadic nature of delivery of the neutrally charged PMO into muscle fibers, the mechanism of which is unknown.Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibers took up PMO more efficiently during myotube formation. Interestingly, through in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. Next, we tested the therapeutic potential of PMO in laminin-alpha2 (laminin-α2) chain-null dy 3K/dy 3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following skipping of the mutated exon 4 in dy 3K/dy 3K mice, which prolonged the life span of the animals slightly. These findings support the theory that PMO entry into fibers is dependent on the developmental stage in myogenesis rather than on dystrophinless muscle membranes, and provide a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and evaluation of the efficacy of exon skipping in the laminin-α2-deficient dy 3K/dy 3K mouse model both in vitro and in vivo.


Assuntos
Éxons , Laminina/deficiência , Distrofias Musculares/genética , Oligonucleotídeos Antissenso/genética , Processamento de RNA , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Morfolinos/administração & dosagem , Morfolinos/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Oligonucleotídeos Antissenso/administração & dosagem
16.
Sci Transl Med ; 10(437)2018 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-29669851

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene encoding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t ). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.


Assuntos
Éxons/genética , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Oligonucleotídeos Antissenso/uso terapêutico , Administração Intravenosa , Adolescente , Criança , Pré-Escolar , Distrofina/genética , Humanos , Masculino , Morfolinos/administração & dosagem , Morfolinos/uso terapêutico , Oligonucleotídeos Antissenso/administração & dosagem , Reação em Cadeia da Polimerase Via Transcriptase Reversa
17.
Prog Neuropsychopharmacol Biol Psychiatry ; 84(Pt A): 181-189, 2018 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-29496512

RESUMO

Polymorphisms in the gene coding for the adhesion G-protein coupled receptor LPHN3 are a risk factor for attention-deficit/hyperactivity disorder (ADHD). Transient down-regulation of latrophilin3.1 (lphn3.1), the zebrafish LPHN3 homologue, causes hyperactivity. Zebrafish injected with a lphn3.1-specific morpholino are hyperactive and display an impairment in dopaminergic neuron development. In the present study we used lphn3.1 morphants to further characterize the changes to dopaminergic signaling that trigger hyperactivity. We applied dopamine agonists (Apomorphine, Quinpirole, SKF-38393) and antagonists (Haloperidol, Eticlopride, SCH-23390) to Lphn3.1 morpholino-injected or control-injected animals. The percentage of change in locomotor activity was then determined at three different time periods (10-20 min, 30-40 min and 60-70 min). Our results show that drugs targeting dopamine receptors appear to elicit similar effects on locomotion in zebrafish larvae and mammals. In addition, we observed that lphn3.1 morphants have an overall hyposensitivity to dopamine agonists and antagonists compared to control fish. These results are compatible with a model whereby dopaminergic neurotransmission is saturated in lphn3.1 morphants.


Assuntos
Transtorno do Deficit de Atenção com Hiperatividade/metabolismo , Dopamina/metabolismo , Locomoção/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Larva , Locomoção/efeitos dos fármacos , Morfolinos/administração & dosagem , Receptores Dopaminérgicos/metabolismo , Fatores de Tempo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
18.
Sheng Li Xue Bao ; 70(1): 47-51, 2018 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-29492514

RESUMO

In vivo electroporation of morpholinos (MOs) into the retina of adult zebrafish is an efficient method to study gene function related to retinal disease and regeneration. However, the currently reported methods are complicated with low MO transfer efficiency and high probability to cause collateral damage. The present study was aimed to optimize the existing MO electroporation methods. Two major changes were made to MO electroporation procedure in zebrafish retina. One was to coat the inner side of the electrode with ultrasonic gel. The other was to replace the commonly used round electrode with novel rectangular one. The results showed that the use of ultrasonic gel reduced collateral damage caused by retinal electroporation and simplified the experimental procedure. The rectangular electrode significantly increased transfection efficiency of MO electroporation. In particular, knocking down the expression of Ascl1a in the retina by using our method significantly inhibited the generation of retinal progenitor cells. These results suggest our method is the optimization of the current MO electroporation methods and may be a better alternative for relevant researchers.


Assuntos
Eletroporação , Morfolinos/administração & dosagem , Retina , Animais , Técnicas de Silenciamento de Genes , Células-Tronco/citologia , Transfecção , Peixe-Zebra
19.
ACS Infect Dis ; 4(5): 806-814, 2018 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-29461800

RESUMO

The Burkholderia cepacia complex is a group of Gram-negative bacteria that are opportunistic pathogens in immunocompromised individuals, such as those with cystic fibrosis (CF) or chronic granulomatous disease (CGD). Burkholderia are intrinsically resistant to many antibiotics and the lack of antibiotic development necessitates novel therapeutics. Peptide-conjugated phosphorodiamidate morpholino oligomers are antisense molecules that inhibit bacterial mRNA translation. Targeting of PPMOs to the gene acpP, which is essential for membrane synthesis, lead to defects in the membrane and ultimately bactericidal activity. Exploration of additional PPMO sequences identified the ATG and Shine-Dalgarno sites as the most efficacious for targeting acpP. The CF lung is a complex microenvironment, but PPMO inhibition was still efficacious in an artificial model of CF sputum. PPMOs had low toxicity in human CF cells at doses that were antibacterial. PPMOs also reduced the bacterial burden in the lungs of immunocompromised CyBB mice, a model of CGD. Finally, the use of multiple PPMOs was efficacious in inhibiting the growth of both Burkholderia and Pseudomonas in an in vitro model of coinfection. Due to the intrinsic resistance of Burkholderia to traditional antibiotics, PPMOs represent a novel and viable approach to the treatment of Burkholderia infections.


Assuntos
Infecções por Burkholderia/microbiologia , Burkholderia/genética , Oligonucleotídeos Antissenso/genética , Pneumonia Bacteriana/microbiologia , Animais , Antibacterianos/administração & dosagem , Infecções por Burkholderia/terapia , Complexo Burkholderia cepacia/genética , Fibrose Cística/complicações , Modelos Animais de Doenças , Camundongos , Testes de Sensibilidade Microbiana , Morfolinos/administração & dosagem , Morfolinos/química , Morfolinos/genética , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/química , Pneumonia Bacteriana/terapia
20.
Methods Mol Biol ; 1742: 205-211, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29330802

RESUMO

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.


Assuntos
Engenharia Genética/instrumentação , Microinjeções/métodos , Peixe-Zebra/embriologia , Animais , Sistemas CRISPR-Cas , Desenvolvimento Embrionário , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Modelos Animais , Morfolinos/administração & dosagem , Oligonucleotídeos Antissenso/administração & dosagem , RNA Guia/administração & dosagem , Ribonucleoproteínas/administração & dosagem , Tionucleotídeos , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Zigoto/crescimento & desenvolvimento
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