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1.
Methods Mol Biol ; 2224: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33606217

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating X-linked muscle disorder affecting many children. The disease is caused by the lack of dystrophin production and characterized by muscle wasting. The most common causes of death are respiratory failure and heart failure. Antisense oligonucleotide-mediated exon skipping using a phosphorodiamidate morpholino oligomer (PMO) is a promising therapeutic approach for the treatment of DMD. In preclinical studies, dystrophic mouse models are commonly used for the development of therapeutic oligos. We employ a humanized model carrying the full-length human DMD transgene along with the complete knockout of the mouse Dmd gene. In this model, the effects of human-targeting AOs can be tested without cross-reaction between mouse sequences and human sequences (note that mdx, a conventional dystrophic mouse model, carries a nonsense point mutation in exon 23 and express the full-length mouse Dmd mRNA, which is a significant complicating factor). To determine if dystrophin expression is restored, the Western blotting analysis is commonly performed; however, due to the extremely large protein size of dystrophin (427 kDa), detection and accurate quantification of full-length dystrophin can be a challenge. Here, we present methodologies to systemically inject PMOs into humanized DMD model mice and determine levels of dystrophin restoration via Western blotting. Using a tris-acetate gradient SDS gel and semi-dry transfer with three buffers, including the Concentrated Anode Buffer, Anode Buffer, and Cathode Buffer, less than 1% normal levels of dystrophin expression are easily detectable. This method is fast, easy, and sensitive enough for the detection of dystrophin from both cultured muscle cells and muscle biopsy samples.


Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/genética , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Éxons/genética , Terapia Genética/métodos , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , RNA Mensageiro/genética , Transgenes/genética
3.
Expert Opin Investig Drugs ; 30(2): 167-176, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33393390

RESUMO

INTRODUCTION: Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the dystrophin (DMD) gene. Most patients die from respiratory failure or cardiomyopathy. There are significant unmet needs for treatments for DMD as the standard of care is principally limited to symptom relief through treatments including steroids. AREAS COVERED: This review summarizes safety and efficacy in promising areas of DMD therapeutics - small molecules, stop codon readthrough, gene replacement, and exon skipping - under clinical examination from 2015-2020 as demonstrated in the NIH Clinical Trials and PubMed search engines. EXPERT OPINION: Currently, steroids persist as the most accessible medicine for DMD. Stop-codon readthrough, gene replacement, and exon-skipping therapies all aim to restore dystrophin expression. Of these strategies, gene replacement therapy has recently gained momentum while exon-skipping retains great traction. The  FDA approval of three exon-skipping antisense oligonucleotides illustrate this regulatory momentum, though the effectiveness and sequence design of eteplirsen remain controversial. Cell-penetrating peptides promise to more efficaciously treat DMD-related cardiomyopathy.The recent success of antisense therapies, however, poses major regulatory challenges. To fully realize the benefits of exon-skipping, including cocktail oligonucleotide-mediated multiple exon-skipping and oligonucleotide drugs for very rare mutations, regulatory challenges need to be addressed in coordination with scientific advances.


Assuntos
Peptídeos Penetradores de Células/uso terapêutico , Distrofina/genética , Terapia Genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos/uso terapêutico , Animais , Peptídeos Penetradores de Células/efeitos adversos , Códon de Terminação , Desenvolvimento de Medicamentos , Drogas em Investigação/efeitos adversos , Drogas em Investigação/uso terapêutico , Éxons , Regulação da Expressão Gênica , Predisposição Genética para Doença , Terapia Genética/efeitos adversos , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Mutação , Oligonucleotídeos/efeitos adversos , Esteroides/efeitos adversos , Esteroides/uso terapêutico , Resultado do Tratamento
4.
PLoS One ; 15(12): e0244215, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33362201

RESUMO

Duchenne muscular dystrophy (DMD) is a severe, progressive neuromuscular disorder caused by reading frame disrupting mutations in the DMD gene leading to absence of functional dystrophin. Antisense oligonucleotide (AON)-mediated exon skipping is a therapeutic approach aimed at restoring the reading frame at the pre-mRNA level, allowing the production of internally truncated partly functional dystrophin proteins. AONs work in a sequence specific manner, which warrants generating humanized mouse models for preclinical tests. To address this, we previously generated the hDMDdel52/mdx mouse model using transcription activator like effector nuclease (TALEN) technology. This model contains mutated murine and human DMD genes, and therefore lacks mouse and human dystrophin resulting in a dystrophic phenotype. It allows preclinical evaluation of AONs inducing the skipping of human DMD exons 51 and 53 and resulting in restoration of dystrophin synthesis. Here, we have further characterized this model genetically and functionally. We discovered that the hDMD and hDMDdel52 transgene is present twice per locus, in a tail-to-tail-orientation. Long-read sequencing revealed a partial deletion of exon 52 (first 25 bp), and a 2.3 kb inversion in intron 51 in both copies. These new findings on the genomic make-up of the hDMD and hDMDdel52 transgene do not affect exon 51 and/or 53 skipping, but do underline the need for extensive genetic analysis of mice generated with genome editing techniques to elucidate additional genetic changes that might have occurred. The hDMDdel52/mdx mice were also evaluated functionally using kinematic gait analysis. This revealed a clear and highly significant difference in overall gait between hDMDdel52/mdx mice and C57BL6/J controls. The motor deficit detected in the model confirms its suitability for preclinical testing of exon skipping AONs for human DMD at both the functional and molecular level.


Assuntos
Modelos Animais de Doenças , Distrofina/genética , Deleção de Genes , Distrofia Muscular de Duchenne/genética , Fenótipo , Transgenes , Animais , Fenômenos Biomecânicos , Distrofina/metabolismo , Éxons , Marcha , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/patologia
5.
PLoS One ; 15(9): e0239467, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970731

RESUMO

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcript molecules at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We instead multiplex within a single transcript, using probes targeted to the 5' and 3' regions of muscle dystrophin mRNA. Our approach shows this method can reveal transcriptional dynamics in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs in quantitative fashion (with the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggest this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.


Assuntos
Distrofina/genética , Expressão Gênica/genética , Hibridização In Situ/métodos , Animais , Distrofina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Multiplex/métodos , Músculos/metabolismo , RNA Mensageiro/genética , Transcrição Genética/genética
6.
PLoS One ; 15(9): e0239468, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32970732

RESUMO

Most Duchenne muscular dystrophy (DMD) cases are caused by deletions or duplications of one or more exons that disrupt the reading frame of DMD mRNA. Restoring the reading frame allows the production of partially functional dystrophin proteins, and result in less severe symptoms. Antisense oligonucleotide mediated exon skipping has been approved for DMD, but this strategy needs repeated treatment. CRISPR/Cas9 can also restore dystrophin reading frame. Although recent in vivo studies showed the efficacy of the single-cut reframing/exon skipping strategy, methods to find the most efficient single-cut sgRNAs for a specific mutation are lacking. Here we show that the insertion/deletion (INDEL) generating efficiency and the INDEL profiles both contribute to the reading frame restoring efficiency of a single-cut sgRNA, thus assays only examining INDEL frequency are not able to find the best sgRNAs. We therefore developed a GFP-reporter assay to evaluate single-cut reframing efficiency, reporting the combined effects of both aspects. We show that the GFP-reporter assay can reliably predict the performance of sgRNAs in myoblasts. This GFP-reporter assay makes it possible to efficiently and reliably find the most efficient single-cut sgRNA for restoring dystrophin expression.


Assuntos
Éxons/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Fases de Leitura/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Distrofina/genética , Distrofina/metabolismo , Genes Reporter/genética , Humanos , Mutação INDEL/genética , Distrofia Muscular de Duchenne/metabolismo , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/genética
7.
Georgian Med News ; (303): 79-85, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32841186

RESUMO

The purpose of the study is to examine in depth and analyze the clinical and some paraclinical characteristics for a family history of Duchenne muscular dystrophy. We analyzed the follow up clinical and laboratory data of Duchenne muscular dystrophy in two brothers-german, aged 16 and 14, respectively. The patients underwent a standardized examination, involving studying the medical case history, general clinical data, determining Sheldon's somatotype and the constitutional type, the detailed neurological status examination, testing a personality type, laboratory and instrumental examinations. Through the laboratory examination we determined the general blood test indicators, total serum protein levels, total cholesterol, the ALAT, ASAT, CPK levels, the indicators of the immunogram, myositis profile and the genetic markers of the disease. The instrumental examination included the ultrasound of the abdominal organs, muscles, as well as echo-cardiography, electroneuromyography. A complete examination fragment of 42 patients with myodystrophies is presented. The paper presents the neurological examination results, the genetic study data and the CPK level indicators in the representatives of Duchenne muscular dystrophy family history. The given family history of Duchenne muscular dystrophy showed two brothers-german to have differences both in the defective dystrophin gene exons at Xp21 and in the disease clinical picture. Thus, patient A., who is an elder brother was detected to have exon 47, 48, 50 and 52 deletion, and patient B., who is a younger brother, was found to have exon 45-43 deletion. The presented family history of Duchenne muscular dystrophy acknowledges the fact that the clinical, genetic, biochemical and other characteristics in patients with dystrophinopathies warrant further comprehensive investigations in order to update diagnostic and prognostic techniques, considering the great medical and social significance of this disabling pathology. However, the onset age of the disease, the clinical course, and the changes in the CPK level were different. Due to the muscle ultrasound both patients were detected to have degenerative changes in the proximal upper and lower limbs.


Assuntos
Distrofia Muscular de Duchenne/genética , Miosite , Idade de Início , Idoso , Distrofina/genética , Éxons , Humanos , Masculino
8.
PLoS One ; 15(8): e0236689, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785240

RESUMO

OBJECTIVE: To compare the effects of photobiomodulation therapy (PBMT) and pharmacological therapy (glucocorticoids and non-steroidal anti-inflammatory drugs) applied alone and in different combinations in mdx mice. METHODS: The animals were randomized and divided into seven experimental groups treated with placebo, PBMT, prednisone, non-steroidal anti-inflammatory drug (NSAIDs), PBMT plus prednisone and PBMT plus NSAID. Wild type animals were used as control. All treatments were performed during 14 consecutive weeks. Muscular morphology, protein expression of dystrophin and functional performance were assessed at the end of the last treatment. RESULTS: Both treatments with prednisone and PBMT applied alone or combined, were effective in preserving muscular morphology. In addition, the treatments with PBMT (p = 0.0005), PBMT plus prednisone (p = 0.0048) and PBMT plus NSAID (p = 0.0021) increased dystrophin gene expression compared to placebo-control group. However, in the functional performance the PBMT presented better results compared to glucocorticoids (p<0.0001). In contrast, the use of NSAIDs did not appear to add benefits to skeletal muscle tissue in mdx mice. CONCLUSION: We believe that the promising and optimistic results about the PBMT in skeletal muscle of mdx mice may in the future contribute to this therapy to be considered a safe alternative for patients with Duchenne Muscular Dystrophy (DMD) in a washout period (between treatment periods with glucocorticoids), allowing them to remain receiving effective and safe treatment in this period, avoiding at this way periods without administration of any treatment.


Assuntos
Distrofina/genética , Terapia com Luz de Baixa Intensidade , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/efeitos da radiação , Distrofia Muscular de Duchenne/terapia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Terapia Combinada , Modelos Animais de Doenças , Progressão da Doença , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Glucocorticoides/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Prednisona/farmacologia
9.
PLoS One ; 15(8): e0237803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813700

RESUMO

A nonsense mutation adds a premature stop signal that hinders any further translation of a protein-coding gene, usually resulting in a null allele. To investigate the possible exceptions, we used the DMD gene as an ideal model. First, because dystrophin absence causes Duchenne muscular dystrophy (DMD), while its reduction causes Becker muscular dystrophy (BMD). Second, the DMD gene is X-linked and there is no second allele that can interfere in males. Third, databases are accumulating reports on many mutations and phenotypic data. Finally, because DMD mutations may have important therapeutic implications. For our study, we analyzed large databases (LOVD, HGMD and ClinVar) and literature and revised critically all data, together with data from our internal patients. We totally collected 2593 patients. Positioning these mutations along the dystrophin transcript, we observed a nonrandom distribution of BMD-associated mutations within selected exons and concluded that the position can be predictive of the phenotype. Nonsense mutations always cause DMD when occurring at any point in fifty-one exons. In the remaining exons, we found milder BMD cases due to early 5' nonsense mutations, if reinitiation can occur, or due to late 3' nonsense when the shortened product retains functionality. In the central part of the gene, all mutations in some in-frame exons, such as in exons 25, 31, 37 and 38 cause BMD, while mutations in exons 30, 32, 34 and 36 cause DMD. This may have important implication in predicting the natural history and the efficacy of therapeutic use of drug-stimulated translational readthrough of premature termination codons, also considering the action of internal natural rescuers. More in general, our survey confirm that a nonsense mutation should be not necessarily classified as a null allele and this should be considered in genetic counselling.


Assuntos
Códon sem Sentido/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação/genética , Inquéritos e Questionários , Sequência de Aminoácidos , Sequência de Bases , Distrofina/química , Éxons/genética , Humanos , Fenótipo
10.
Life Sci ; 257: 118069, 2020 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-32659370

RESUMO

AIM: Up-regulation of inflammasome proteins was reported in dystrophin-deficient muscles. However, it remains to be determined whether inflammasome activation plays a role in the pathogenesis of Duchenne muscular dystrophy. This study was therefore set out to investigate whether genetic disruption of the inflammasome pathway impacts the disease progression in mdx mice. MAIN METHODS: Mice deficient in both dystrophin and ASC (encoded by Pycard [PYD And CARD Domain Containing]) were generated. The impact of ASC deficiency on muscular dystrophy of mdx mice were assessed by measurements of serum cytokines, Western blot, real-time PCR and histopathological staining. KEY FINDINGS: The pro-inflammatory cytokines such as TNF-α, IL-6, KC/GRO and IL-10 were markedly increased in the sera of 8-week-old mdx mice compared to WT. Western blotting showed that P2X7, caspase-1, ASC and IL-18 were upregulated. Disruption of ASC and dystrophin expression in the mdx/ASC-/- mice was verified by Western blot analysis. Histopathological analysis did not find significant alterations in the muscular dystrophy phenotype in mdx/ASC-/- mice as compared to mdx mice. SIGNIFICANCE: Taken together, our results show that disruption of the central adaptor ASC of the inflammasome is insufficient to alleviate muscular dystrophy phenotype in mdx mice.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Distrofina/genética , Inflamassomos/metabolismo , Distrofia Muscular de Duchenne/fisiopatologia , Animais , Citocinas/sangue , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Camundongos Knockout , Distrofia Muscular de Duchenne/genética , Regulação para Cima
11.
Proc Natl Acad Sci U S A ; 117(28): 16456-16464, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32616572

RESUMO

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcription dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.


Assuntos
Códon sem Sentido/genética , Distrofina/genética , Distrofia Muscular de Duchenne/genética , RNA Mensageiro/genética , Animais , Códon sem Sentido/metabolismo , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , RNA Mensageiro/metabolismo
12.
AJNR Am J Neuroradiol ; 41(7): 1271-1278, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32616576

RESUMO

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy is an X-linked disorder characterized by progressive muscle weakness and prominent nonmotor manifestations, such as a low intelligence quotient and neuropsychiatric disturbance. We investigated WM integrity in patients with Duchenne muscular dystrophy using DTI. MATERIALS AND METHODS: Fractional anisotropy and mean, axial, and radial diffusivity (DTI measures) were used to assess WM microstructural integrity along with neuropsychological evaluation in patients with Duchenne muscular dystrophy (n = 60) and controls (n = 40). Exon deletions in the DMD gene were confirmed using multiplex ligation-dependent probe amplification. Patients were classified into proximal (DMD Dp140+) and distal (DMD Dp140-) subgroups based on the location of the exon deletion and expression of short dystrophin Dp140 isoform. WM integrity was examined using whole-brain Tract-Based Spatial Statistics and atlas-based analysis of DTI data. The Pearson correlation was performed to investigate the possible relationship between neuropsychological scores and DTI metrics. RESULTS: The mean ages of Duchenne muscular dystrophy and control participants were 8.0 ± 1.2 years and 8.2 ± 1.4 years, respectively. The mean age at disease onset was 4.1 ± 1.8 years, and mean illness duration was 40.8 ± 25.2 months. Significant differences in neuropsychological scores were observed between the proximal and distal gene-deletion subgroups, with more severe impairment in the distal-deletion subgroup (P < .05). Localized fractional anisotropy changes were seen in the corpus callosum, parietal WM, and fornices in the patient subgroup with Dp140+, while widespread changes were noted in the Dp140- subgroup. The Dp140+ subgroup showed increased axial diffusivity in multiple WM regions relative to the Dp140- subgroup. No significant correlation was observed between clinical and neuropsychological scores and diffusion metrics. CONCLUSIONS: Widespread WM differences are evident in patients with Duchenne muscular dystrophy relative to healthy controls. Distal mutations in particular are associated with extensive WM abnormalities and poor neuropsychological profiles.


Assuntos
Encéfalo/diagnóstico por imagem , Distrofia Muscular de Duchenne/diagnóstico por imagem , Substância Branca/diagnóstico por imagem , Adolescente , Encéfalo/patologia , Criança , Imagem de Tensor de Difusão/métodos , Distrofina/genética , Feminino , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação , Neuroimagem/métodos , Substância Branca/patologia
13.
Circ Arrhythm Electrophysiol ; 13(7): e008241, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32536203

RESUMO

BACKGROUND: Mutations in the gene encoding the cardiac voltage-gated sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. METHODS: We used novel single-molecule localization microscopy and computational modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, the lateral membrane groove, and T-tubules in cardiomyocytes from wild-type (N=3), dystrophin-deficient (mdx; N=3) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV; N=3). We moreover assessed T-tubules sodium current by recording whole-cell sodium currents in control (N=5) and detubulated (N=5) wild-type cardiomyocytes. RESULTS: We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a nonuniform redistribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A T-tubules sodium current could, however, not be demonstrated. CONCLUSIONS: Nav1.5 mutations may site-specifically affect Nav1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.


Assuntos
Membrana Celular/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Imagem Individual de Molécula , Animais , Membrana Celular/ultraestrutura , Simulação por Computador , Distrofina/genética , Distrofina/metabolismo , Potenciais da Membrana , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Modelos Cardiovasculares , Miócitos Cardíacos/ultraestrutura , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Transporte Proteico
14.
Muscle Nerve ; 62(3): 369-376, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32564389

RESUMO

We performed an observational, natural history study of males with in-frame dystrophin gene deletions causing Becker muscular dystrophy (BMD). A prospective natural history study collected longitudinal medical, strength, and timed function assessments. Eighty-three participants with genetically confirmed BMD were enrolled (age range 5.6-75.4 years). Lower extremity function and the percentage of participants who retained ambulation declined across the age span. The largest single group of participants had in-frame deletions that corresponded to an out-of-frame deletion treated with an exon 45 skip to restore the reading frame. This group of 54 participants showed similarities in baseline motor functional assessments when compared to the group of all others in the study. A prospective natural history cohort with in-frame dystrophin gene deletions offers the potential to contribute to clinical trial readiness for BMD and to analyze therapeutic benefit of exon skipping for Duchenne muscular dystrophy.


Assuntos
Distrofina/genética , Distrofia Muscular de Duchenne/diagnóstico , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Progressão da Doença , Deleção de Genes , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Fenótipo , Estudos Prospectivos , Avaliação de Sintomas , Adulto Jovem
15.
Nat Commun ; 11(1): 3232, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591530

RESUMO

CRISPR-Cas9 has emerged as a powerful technology that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) to target and edit DNA. However, many therapeutic targets cannot currently be accessed due to the lack of carriers that can deliver RNPs systemically. Here, we report a generalizable methodology that allows engineering of modified lipid nanoparticles to efficiently deliver RNPs into cells and edit tissues including muscle, brain, liver, and lungs. Intravenous injection facilitated tissue-specific, multiplexed editing of six genes in mouse lungs. High carrier potency was leveraged to create organ-specific cancer models in livers and lungs of mice though facile knockout of multiple genes. The developed carriers were also able to deliver RNPs to restore dystrophin expression in DMD mice and significantly decrease serum PCSK9 level in C57BL/6 mice. Application of this generalizable strategy will facilitate broad nanoparticle development for a variety of disease targets amenable to protein delivery and precise gene correction approaches.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes , Nanopartículas/química , Especificidade de Órgãos/genética , Ribonucleoproteínas/metabolismo , Animais , Cátions , DNA de Neoplasias/isolamento & purificação , Distrofina/genética , Células HeLa , Humanos , Lipídeos/química , Camundongos Endogâmicos C57BL
16.
Cell Mol Life Sci ; 77(21): 4299-4313, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32350552

RESUMO

Noncoding RNAs (ncRNAs), such as miRNAs and long noncoding RNAs, are key regulators of gene expression at the post-transcriptional level and represent promising therapeutic targets and biomarkers for several human diseases, including Duchenne and Becker muscular dystrophies (DMD/BMD). A role for ncRNAs in the pathogenesis of muscular dystrophies has been suggested, even if it is still incompletely understood. Here, we discuss current progress leading towards the clinical utility of ncRNAs for DMD/BMD. Long and short noncoding RNAs are differentially expressed in DMD/BMD and have a mechanism of action via targeting mRNAs. A subset of muscle-enriched miRNAs, the so-called myomiRs (miR-1, miR-133, and miR-206), are increased in the serum of patients with DMD and in dystrophin-defective animal models. Interestingly, myomiRs might be used as biomarkers, given that their levels can be corrected after dystrophin restoration in dystrophic mice. Remarkably, further evidence demonstrates that ncRNAs also play a role in dystrophin expression; thus, their modulations might represent a potential therapeutic strategy with the aim of upregulating the dystrophin protein in combination with other oligonucleotides/gene therapy approaches.


Assuntos
Distrofia Muscular de Duchenne/genética , RNA não Traduzido/genética , Animais , Distrofina/genética , Regulação da Expressão Gênica , Terapia Genética , Humanos , MicroRNAs/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/patologia , Distrofia Muscular de Duchenne/terapia , Prognóstico , RNA Mensageiro/genética , Transcriptoma
18.
Sci Rep ; 10(1): 5683, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32231219

RESUMO

Duchenne muscular dystrophy (DMD) is a severe X-linked neuromuscular disorder that affects males. However, 8% of female carriers are symptomatic and underrepresented in research due to the lack of animal models. We generated a symptomatic mouse model of DMD carriers via injection of mdx (murine DMD) embryonic stem cells (ESCs) into wild-type (WT) blastocysts (mdx/WT chimera). mdx/WT chimeras developed cardiomyopathic features and dystrophic skeletal muscle phenotypes including elevated mononuclear invasion, central nucleation, fibrosis and declined forelimb grip strength. The disease was accompanied by connexin-43 (Cx43) aberrantly enhanced in both cardiac and skeletal muscles and remodeled in the heart. Genetic reduction of Cx43-copy number in mdx/WT-Cx43(+/-) chimeras protected them from both cardiac and skeletal muscle fiber damage. In dystrophic skeletal muscle, Cx43 expression was not seen in the fibers but in adjacent F4/80+ mononuclear cells. Ethidium Bromide uptake in purified F4/80+/CD11b+ mdx macrophages revealed functional activity of Cx43, which was inhibited by administration of Gap19 peptide mimetic, a Cx43 hemichannel-specific inhibitor. Thus, we suggest that Cx43 reduction in symptomatic DMD carrier mice leads to prevention of Cx43 remodeling in the heart and prevention of aberrant Cx43 hemichannel activity in the skeletal muscle macrophages neighboring Cx43 non-expressing fibers.


Assuntos
Conexina 43/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/fisiopatologia , Animais , Cardiomiopatias/metabolismo , Conexina 43/genética , Modelos Animais de Doenças , Distrofina/genética , Feminino , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/metabolismo , Força Muscular , Músculo Esquelético/metabolismo , Distrofia Muscular Animal/metabolismo
19.
Sci Adv ; 6(6): eaaz2736, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128386

RESUMO

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca2+ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca2+ channels may represent a promising approach to treat DMD and related muscle diseases.


Assuntos
Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Canais de Receptores Transientes de Potencial/agonistas , Animais , Biomarcadores , Biópsia , Modelos Animais de Doenças , Distrofina/genética , Imunofluorescência , Expressão Gênica , Camundongos , Camundongos Endogâmicos mdx , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Miocárdio/metabolismo , Miocárdio/patologia
20.
Sci Adv ; 6(8): eaay6812, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32128412

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9-mediated "single-cut" genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9-mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.


Assuntos
Sistemas CRISPR-Cas , Dependovirus/genética , Distrofina/genética , Edição de Genes , Terapia Genética , Vetores Genéticos/genética , Distrofia Muscular de Duchenne/genética , Animais , Modelos Animais de Doenças , Éxons , Dosagem de Genes , Expressão Gênica , Marcação de Genes , Técnicas de Transferência de Genes , Camundongos , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutação , RNA Guia/genética , Transdução Genética
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