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1.
Lancet Respir Med ; 10(4): 403-420, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35364035

RESUMO

The life-limiting complications of Duchenne muscular dystrophy (DMD) include loss of lung function and progressive cardiomyopathy; when patients are treated with assisted ventilation, cardiac function becomes the main determinant of survival. Therapy for DMD is changing rapidly, with the emergence of new genetic and molecular therapeutic options, the proliferation of which has fostered the perception that DMD is a potentially curable disease. However, data for respiratory and cardiac outcomes are scarce and available evidence is not uniformly positive. Patients who share a dystrophin (DMD) genotype can have highly divergent cardiorespiratory phenotypes; genetic modifiers of DMD gene expression are a probable cause of respiratory and cardiac phenotypic variability and discordance. In this Personal View, we provide an overview of new and emerging DMD therapies, highlighting the limitations of current research and considering strategies to incorporate cardiorespiratory assessments into clinical trials. We explore how genetic modifiers could be used to predict cardiorespiratory natural history and how manipulation of such modifiers might represent a promising therapeutic strategy. Finally, we examine the changing role of respiratory physicians, cardiologists, and intensive care clinicians on the frontline of a challenging new clinical landscape.


Assuntos
Distrofia Muscular de Duchenne , Genótipo , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Fenótipo
3.
PLoS One ; 17(4): e0254274, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35436319

RESUMO

PURPOSE: Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. METHODS: Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated. RESULTS: Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice. CONCLUSION: Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation.


Assuntos
Distrofia Muscular Animal , Distrofia Muscular de Duchenne , Animais , Terapia Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Contração Muscular/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/terapia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia
4.
Acta Neuropathol Commun ; 10(1): 60, 2022 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-35468843

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by mutations in the Dystrophin gene and for which there is currently no cure. To bridge the gap between preclinical and therapeutic evaluation studies, we have generated a rat model for DMD that carries an exon 52 deletion (R-DMDdel52) causing a complete lack of dystrophin protein. Here we show that R-DMDdel52 animals recapitulated human DMD pathophysiological trajectory more faithfully than the mdx mouse model. We report that R-DMDdel52 rats displayed progressive and severe skeletal muscle loss associated with fibrotic deposition, fat infiltration and fibre type switch. Early fibrosis was also apparent in the cardiac muscle. These histological modifications led to severe muscle, respiratory and cardiac functional impairments leading to premature death around 1 year. Moreover, DMD muscle exhibited systemic inflammation with a mixed M1/M2 phenotype. A comparative single cell RNAseq analysis of the diaphragm muscle was performed, revealing cellular populations alteration and molecular modifications in all muscle cell types. We show that DMD fibroadipogenic progenitors produced elevated levels of cartilage oligomeric matrix protein, a glycoprotein responsible for modulating homeostasis of extracellular matrix, and whose increased concentration correlated with muscle fibrosis both in R-DMDdel52 rats and human patients. Fibrosis is a component of tissue remodelling impacting the whole musculature of DMD patients, at the tissue level but most importantly at the functional level. We therefore propose that this specific biomarker can optimize the prognostic monitoring of functional improvement of patients included in clinical trials.


Assuntos
Distrofia Muscular de Duchenne , Animais , Biomarcadores , Proteína de Matriz Oligomérica de Cartilagem/uso terapêutico , Distrofina/metabolismo , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/terapia , Ratos
5.
J Am Heart Assoc ; 11(8): e024722, 2022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35411787

RESUMO

Background Duchenne and Becker muscular dystrophy are progressive disorders associated with cardiac mortality. Guidelines recommend routine surveillance; we assess cardiac resource use and identify gaps in care delivery. Methods and Results Male patients, aged 1 to 18 years, with Duchenne and Becker muscular dystrophy between January 2013 and December 2017 were identified in the IBM MarketScan Research Database. The cohort was divided into <10 and 10 to 18 years of age. The primary outcome was rate of annual health care resource per person year. Resource use was assessed for place of service, cardiac testing, and medications. Adjusted incidence rate ratios (IRRs) were estimated using a Poisson regression model. Medication use was measured by proportion of days covered. There were 1386 patients with a median follow-up time of 3.0 years (interquartile range, 1.9-4.7 years). Patients in the 10 to 18 years group had only 0.40 (95% CI, 0.35-0.45) cardiology visits per person year and 0.66 (95% CI, 0.62-0.70) echocardiography/magnetic resonance imaging per person year. Older patients had higher rates of inpatient admissions (IRR, 1.46; 95% CI, 1.03-2.09), outpatient cardiology visits (IRR, 2.0; 95% CI, 1.66-2.40), cardiac imaging (IRR, 1.59; 95% CI, 1.40-1.80), and Holter monitoring (IRR, 3.33; 95% CI, 2.35-4.73). A proportion of days covered >80% for angiotensin-converting enzyme inhibitors/angiotensin receptor blockers was observed in 13.6% (419/3083) of total person years among patients in the 10 to 18 years group. Conclusions Children 10 to 18 years of age have higher rates of cardiac resource use compared with those <10 years of age. However, rates in both age groups fall short of guidelines. Opportunities exist to identify barriers to resource use and optimize cardiac care for patients with Duchenne and Becker muscular dystrophy.


Assuntos
Distrofia Muscular de Duchenne , Adolescente , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Criança , Atenção à Saúde , Ecocardiografia , Humanos , Imageamento por Ressonância Magnética , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/epidemiologia , Distrofia Muscular de Duchenne/terapia , Estados Unidos/epidemiologia
6.
Int J Mol Sci ; 23(8)2022 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-35457088

RESUMO

Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.


Assuntos
Distrofia Muscular de Duchenne , Linhagem Celular , Distrofina/genética , Éxons/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos/genética , Oligonucleotídeos Antissenso/genética
8.
Int J Mol Sci ; 23(5)2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35270040

RESUMO

Dystrophinopathy is caused by mutations in the dystrophin gene, which lead to progressive muscle degeneration, necrosis, and finally, death. Recently, golden retrievers have been suggested as a useful animal model for studying human dystrophinopathy, but the model has limitations due to difficulty in maintaining the genetic background using conventional breeding. In this study, we successfully generated a dystrophin mutant dog using the CRISPR/Cas9 system and somatic cell nuclear transfer. The dystrophin mutant dog displayed phenotypes such as elevated serum creatine kinase, dystrophin deficiency, skeletal muscle defects, an abnormal electrocardiogram, and avoidance of ambulation. These results indicate that donor cells with CRISPR/Cas9 for a specific gene combined with the somatic cell nuclear transfer technique can efficiently produce a dystrophin mutant dog, which will help in the successful development of gene therapy drugs for dogs and humans.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Animais , Sistemas CRISPR-Cas/genética , Cães , Distrofina/genética , Distrofina/metabolismo , Edição de Genes , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Técnicas de Transferência Nuclear
9.
PLoS One ; 17(3): e0265879, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35333888

RESUMO

BACKGROUND: Duchenne muscular dystrophy (DMD) is a rare, severely debilitating, and fatal neuromuscular disease characterized by progressive muscle degeneration. Like in many orphan diseases, randomized controlled trials are uncommon in DMD, resulting in the need to indirectly compare treatment effects, for example by pooling individual patient-level data from multiple sources. However, to derive reliable estimates, it is necessary to ensure that the samples considered are comparable with respect to factors significantly affecting the clinical progression of the disease. To help inform such analyses, the objective of this study was to review and synthesise published evidence of prognostic indicators of disease progression in DMD. We searched MEDLINE (via Ovid), Embase (via Ovid) and the Cochrane Library (via Wiley) for records published from inception up until April 23 2021, reporting evidence of prognostic indicators of disease progression in DMD. Risk of bias was established with the grading system of the Centre for Evidence-Based Medicine (CEBM). RESULTS: Our search included 135 studies involving 25,610 patients from 18 countries across six continents (Africa, Asia, Australia, Europe, North America and South America). We identified a total of 23 prognostic indicators of disease progression in DMD, namely age at diagnosis, age at onset of symptoms, ataluren treatment, ATL1102, BMI, cardiac medication, DMD genetic modifiers, DMD mutation type, drisapersen, edasalonexent, eteplirsen, glucocorticoid exposure, height, idebenone, lower limb surgery, orthoses, oxandrolone, spinal surgery, TAS-205, vamorolone, vitlolarsen, ventilation support, and weight. Of these, cardiac medication, DMD genetic modifiers, DMD mutation type, and glucocorticoid exposure were designated core prognostic indicators, each supported by a high level of evidence and significantly affecting a wide range of clinical outcomes. CONCLUSION: This study provides a current summary of prognostic indicators of disease progression in DMD, which will help inform the design of comparative analyses and future data collection initiatives in this patient population.


Assuntos
Distrofia Muscular de Duchenne , Progressão da Doença , Glucocorticoides/uso terapêutico , Humanos , Morfolinas , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Piperidinas , Prognóstico , Pirróis
10.
Sci Rep ; 12(1): 3756, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260651

RESUMO

Among the mutations arising in the DMD gene and causing Duchenne Muscular Dystrophy (DMD), 10-15% are multi-exon duplications. There are no current therapeutic approaches with the ability to excise large multi-exon duplications, leaving this patient cohort without mutation-specific treatment. Using CRISPR/Cas9 could provide a valid alternative to achieve targeted excision of genomic duplications of any size. Here we show that the expression of a single CRISPR/Cas9 nuclease targeting a genomic region within a DMD duplication can restore the production of wild-type dystrophin in vitro. We assessed the extent of dystrophin repair following both constitutive and transient nuclease expression by either transducing DMD patient-derived myoblasts with integrating lentiviral vectors or electroporating them with CRISPR/Cas9 expressing plasmids. Comparing genomic, transcript and protein data, we observed that both continuous and transient nuclease expression resulted in approximately 50% dystrophin protein restoration in treated myoblasts. Our data demonstrate that a high transient expression profile of Cas9 circumvents its requirement of continuous expression within the cell for targeting DMD duplications. This proof-of-concept study therefore helps progress towards a clinically relevant gene editing strategy for in vivo dystrophin restoration, by highlighting important considerations for optimizing future therapeutic approaches.


Assuntos
Sistemas CRISPR-Cas , Distrofia Muscular de Duchenne , Sistemas CRISPR-Cas/genética , Distrofina/genética , Distrofina/metabolismo , Endonucleases/genética , Edição de Genes/métodos , Terapia Genética/métodos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Mioblastos/metabolismo
11.
Health Qual Life Outcomes ; 20(1): 36, 2022 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-35241084

RESUMO

BACKGROUND: To describe the reduced health-related quality of life (HRQoL) of duchenne muscular dystrophy (DMD) patients and their caregiver burden and to present its relationship with disease progression. METHODS: This cross-sectional study assessed patient HRQoL with the 3-level version of the EuroQol-5D (EQ-5D-3L) and caregiver burden with the Work Productivity and Activity Impairment: General Health questionnaire. DMD patients and their caregivers were identified through Portuguese Neuromuscular Association (APN). RESULTS: A total of 46 DMD main caregivers, of eight ambulant and 38 non-ambulant patients, completed the questionnaires. Over half (58.7%) of all non-ambulant patients were on ventilation support, either full-time (15.2%) or non full-time (43.5%). Non-ambulant patients had a lower mean utility scores than ambulant patients (- 0.05 versus 0.51, p value < 0.001). Caregivers of non-ambulant patients reported a significant mean daily activity impairment as compared to caregivers of ambulant patients (68% versus 23%, p value < 0.001). Among non-ambulant patients, both utility scores and caregiver impairment appeared to deteriorate according to a higher need for ventilation support, however, these results were not statistically significant. CONCLUSIONS: These results emphasise the significant negative impact that DMD progression has on the patient HRQoL, as well as caregivers' ability to conduct their daily activities. Therapeutic options that stop or slow the disease progression could have a beneficial impact for both patients and caregivers.


Assuntos
Distrofia Muscular de Duchenne , Qualidade de Vida , Fardo do Cuidador , Cuidadores , Estudos Transversais , Humanos , Distrofia Muscular de Duchenne/terapia , Assistência ao Paciente , Portugal , Inquéritos e Questionários
12.
Elife ; 112022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-35324428

RESUMO

Neuromuscular electrical stimulation (NMES) allows activation of muscle fibers in the absence of voluntary force generation. NMES could have the potential to promote muscle homeostasis in the context of muscle disease, but the impacts of NMES on diseased muscle are not well understood. We used the zebrafish Duchenne muscular dystrophy (dmd) mutant and a longitudinal design to elucidate the consequences of NMES on muscle health. We designed four neuromuscular stimulation paradigms loosely based on weightlifting regimens. Each paradigm differentially affected neuromuscular structure, function, and survival. Only endurance neuromuscular stimulation (eNMES) improved all outcome measures. We found that eNMES improves muscle and neuromuscular junction morphology, swimming, and survival. Heme oxygenase and integrin alpha7 are required for eNMES-mediated improvement. Our data indicate that neuromuscular stimulation can be beneficial, suggesting that the right type of activity may benefit patients with muscle disease.


Assuntos
Distrofia Muscular de Duchenne , Animais , Estimulação Elétrica , Humanos , Músculo Esquelético/fisiologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Junção Neuromuscular/fisiologia , Peixe-Zebra
13.
Methods Mol Biol ; 2434: 217-233, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213020

RESUMO

Several exon skipping antisense oligonucleotides (eteplirsen, golodirsen, viltolarsen, and casimersen) have been approved for the treatment of Duchenne muscular dystrophy, but many more are in development targeting an array of different DMD exons. Preclinical screening of the new oligonucleotide sequences is routinely performed using patient-derived cell cultures, and evaluation of their efficacy may be performed at RNA and/or protein level. While several methods to assess exon skipping and dystrophin expression in cell culture have been developed, the choice of methodology often depends on the availability of specific research equipment.In this chapter, we describe and indicate the relevant bibliography of all the methods that may be used in this evaluation and describe in detail the protocols routinely followed at our institution, one to evaluate the efficacy of skipping at RNA level (nested PCR) and the other the restoration of protein expression (myoblot ), which provide good results using equipment largely available to most research laboratories.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Distrofina/genética , Distrofina/metabolismo , Éxons/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico
14.
Methods Mol Biol ; 2434: 235-243, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35213021

RESUMO

Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.


Assuntos
Células-Tronco Pluripotentes Induzidas , Distrofia Muscular de Duchenne , Processamento Alternativo , Éxons , Humanos , Fibras Musculares Esqueléticas , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , RNA , Splicing de RNA
15.
Int J Mol Sci ; 23(4)2022 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-35216132

RESUMO

Duchenne muscular dystrophy (DMD) is the most common and cureless muscle pediatric genetic disease, which is caused by the lack or the drastically reduced expression of dystrophin. Experimental therapeutic approaches for DMD have been mainly focused in recent years on attempts to restore the expression of dystrophin. While significant progress was achieved, the therapeutic benefit of treated patients is still unsatisfactory. Efficiency in gene therapy for DMD is hampered not only by incompletely resolved technical issues, but likely also due to the progressive nature of DMD. It is indeed suspected that some of the secondary pathologies, which are evolving over time in DMD patients, are not fully corrected by the restoration of dystrophin expression. We recently identified perturbations of the mevalonate pathway and of cholesterol metabolism in DMD patients. Taking advantage of the mdx model for DMD, we then demonstrated that some of these perturbations are improved by treatment with the cholesterol-lowering drug, simvastatin. In the present investigation, we tested whether the combination of the restoration of dystrophin expression with simvastatin treatment could have an additive beneficial effect in the mdx model. We confirmed the positive effects of microdystrophin, and of simvastatin, when administrated separately, but detected no additive effect by their combination. Thus, the present study does not support an additive beneficial effect by combining dystrophin restoration with a metabolic normalization by simvastatin.


Assuntos
Distrofia Muscular de Duchenne/tratamento farmacológico , Distrofia Muscular de Duchenne/terapia , Sinvastatina/administração & dosagem , Animais , Modelos Animais de Doenças , Terapia Genética/métodos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/efeitos dos fármacos
16.
Int J Mol Sci ; 23(3)2022 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-35163475

RESUMO

Duchenne muscular dystrophy (DMD) is caused by loss-of-function mutations in the dystrophin gene on chromosome Xp21. Disruption of the dystrophin-glycoprotein complex (DGC) on the cell membrane causes cytosolic Ca2+ influx, resulting in protease activation, mitochondrial dysfunction, and progressive myofiber degeneration, leading to muscle wasting and fragility. In addition to the function of dystrophin in the structural integrity of myofibers, a novel function of asymmetric cell division in muscular stem cells (satellite cells) has been reported. Therefore, it has been suggested that myofiber instability may not be the only cause of dystrophic degeneration, but rather that the phenotype might be caused by multiple factors, including stem cell and myofiber functions. Furthermore, it has been focused functional regulation of satellite cells by intracellular communication of extracellular vesicles (EVs) in DMD pathology. Recently, a novel molecular mechanism of DMD pathogenesis-circulating RNA molecules-has been revealed through the study of target pathways modulated by the Neutral sphingomyelinase2/Neutral sphingomyelinase3 (nSMase2/Smpd3) protein. In addition, adeno-associated virus (AAV) has been clinically applied for DMD therapy owing to the safety and long-term expression of transduction genes. Furthermore, the EV-capsulated AAV vector (EV-AAV) has been shown to be a useful tool for the intervention of DMD, because of the high efficacy of the transgene and avoidance of neutralizing antibodies. Thus, we review application of AAV and EV-AAV vectors for DMD as novel therapeutic strategy.


Assuntos
Vesículas Extracelulares/virologia , Distrofia Muscular de Duchenne/terapia , Células Satélites de Músculo Esquelético/metabolismo , Esfingomielina Fosfodiesterase/genética , Animais , Ácidos Nucleicos Livres/genética , Dependovirus/genética , Vesículas Extracelulares/genética , Vesículas Extracelulares/transplante , Terapia Genética , Vetores Genéticos , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/imunologia , Transdução Genética
18.
Proc Natl Acad Sci U S A ; 119(9)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35193974

RESUMO

Duchenne muscular dystrophy (DMD) is primarily caused by out-of-frame deletions in the dystrophin gene. Exon skipping using phosphorodiamidate morpholino oligomers (PMOs) converts out-of-frame to in-frame mutations, producing partially functional dystrophin. Four single-exon skipping PMOs are approved for DMD but treat only 8 to 14% of patients each, and some exhibit poor efficacy. Alternatively, exons 45 to 55 skipping could treat 40 to 47% of all patients and is associated with improved clinical outcomes. Here, we report the development of peptide-conjugated PMOs for exons 45 to 55 skipping. Experiments with immortalized patient myotubes revealed that exons 45 to 55 could be skipped by targeting as few as five exons. We also found that conjugating DG9, a cell-penetrating peptide, to PMOs improved single-exon 51 skipping, dystrophin restoration, and muscle function in hDMDdel52;mdx mice. Local administration of a minimized exons 45 to 55-skipping DG9-PMO mixture restored dystrophin production. This study provides proof of concept toward the development of a more economical and effective exons 45 to 55-skipping DMD therapy.


Assuntos
Éxons , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/uso terapêutico , Peptídeos/química , Animais , Distrofina/biossíntese , Terapia Genética , Humanos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , Miocárdio/metabolismo , Oligonucleotídeos Antissenso/genética
19.
Genes (Basel) ; 13(2)2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35205302

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal genetic disease affecting children that is caused by a mutation in the gene encoding for dystrophin. In the absence of functional dystrophin, patients experience progressive muscle deterioration, leaving them wheelchair-bound by age 12 and with few patients surviving beyond their third decade of life as the disease advances and causes cardiac and respiratory difficulties. In recent years, an increasing number of antisense and gene therapies have been studied for the treatment of muscular dystrophy; however, few of these therapies focus on treating mutations arising in the N-terminal encoding region of the dystrophin gene. This review summarizes the current state of development of N-terminal antisense and gene therapies for DMD, mainly focusing on exon-skipping therapy for duplications and deletions, as well as microdystrophin therapy.


Assuntos
Distrofina , Distrofia Muscular de Duchenne , Criança , Distrofina/genética , Éxons/genética , Terapia Genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Mutação
20.
Int J Mol Sci ; 23(3)2022 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-35163754

RESUMO

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3500-5000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.


Assuntos
Distrofina/genética , Edição de Genes/métodos , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Distrofina/metabolismo , Mutação da Fase de Leitura , Humanos , Masculino , Distrofia Muscular de Duchenne/diagnóstico , Distrofia Muscular de Duchenne/genética , Fases de Leitura
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