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1.
J Clin Invest ; 134(1)2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-37856214

RESUMO

Cardiovascular diseases are the most common cause of worldwide morbidity and mortality, highlighting the necessity for advanced therapeutic strategies. Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is a prominent inducer of various cardiac disorders, which is mediated by 2 oxidation-sensitive methionine residues within the regulatory domain. We have previously shown that ablation of CaMKIIδ oxidation by CRISPR-Cas9 base editing enables the heart to recover function from otherwise severe damage following ischemia/reperfusion (IR) injury. Here, we extended this therapeutic concept toward potential clinical translation. We generated a humanized CAMK2D knockin mouse model in which the genomic sequence encoding the entire regulatory domain was replaced with the human sequence. This enabled comparison and optimization of two different editing strategies for the human genome in mice. To edit CAMK2D in vivo, we packaged the optimized editing components into an engineered myotropic adeno-associated virus (MyoAAV 2A), which enabled efficient delivery at a very low AAV dose into the humanized mice at the time of IR injury. CAMK2D-edited mice recovered cardiac function, showed improved exercise performance, and were protected from myocardial fibrosis, which was otherwise observed in injured control mice after IR. Our findings identify a potentially effective strategy for cardioprotection in response to oxidative damage.


Assuntos
Cardiomiopatias , Doenças Cardiovasculares , Camundongos , Animais , Humanos , Sistemas CRISPR-Cas , Edição de Genes , Coração , Cardiomiopatias/genética , Doenças Cardiovasculares/genética
2.
Cardiovasc Res ; 120(1): 56-68, 2024 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-37890031

RESUMO

AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.


Assuntos
Processamento Alternativo , Células-Tronco Pluripotentes Induzidas , Animais , Humanos , Camundongos , Conectina/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos Knockout , Miócitos Cardíacos/metabolismo , RNA/metabolismo , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
3.
Circulation ; 148(19): 1490-1504, 2023 11 07.
Artigo em Inglês | MEDLINE | ID: mdl-37712250

RESUMO

BACKGROUND: Cardiovascular diseases are the main cause of worldwide morbidity and mortality, highlighting the need for new therapeutic strategies. Autophosphorylation and subsequent overactivation of the cardiac stress-responsive enzyme CaMKIIδ (Ca2+/calmodulin-dependent protein kinase IIδ) serves as a central driver of multiple cardiac disorders. METHODS: To develop a comprehensive therapy for heart failure, we used CRISPR-Cas9 adenine base editing to ablate the autophosphorylation site of CaMKIIδ. We generated mice harboring a phospho-resistant CaMKIIδ mutation in the germline and subjected these mice to severe transverse aortic constriction, a model for heart failure. Cardiac function, transcriptional changes, apoptosis, and fibrosis were assessed by echocardiography, RNA sequencing, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and standard histology, respectively. Specificity toward CaMKIIδ gene editing was assessed using deep amplicon sequencing. Cellular Ca2+ homeostasis was analyzed using epifluorescence microscopy in Fura-2-loaded cardiomyocytes. RESULTS: Within 2 weeks after severe transverse aortic constriction surgery, 65% of all wild-type mice died, and the surviving mice showed dramatically impaired cardiac function. In contrast to wild-type mice, CaMKIIδ phospho-resistant gene-edited mice showed a mortality rate of only 11% and exhibited substantially improved cardiac function after severe transverse aortic constriction. Moreover, CaMKIIδ phospho-resistant mice were protected from heart failure-related aberrant changes in cardiac gene expression, myocardial apoptosis, and subsequent fibrosis, which were observed in wild-type mice after severe transverse aortic constriction. On the basis of identical mouse and human genome sequences encoding the autophosphorylation site of CaMKIIδ, we deployed the same editing strategy to modify this pathogenic site in human induced pluripotent stem cells. It is notable that we detected a >2000-fold increased specificity for editing of CaMKIIδ compared with other CaMKII isoforms, which is an important safety feature. While wild-type cardiomyocytes showed impaired Ca2+ transients and an increased frequency of arrhythmias after chronic ß-adrenergic stress, CaMKIIδ-edited cardiomyocytes were protected from these adverse responses. CONCLUSIONS: Ablation of CaMKIIδ autophosphorylation by adenine base editing may offer a potential broad-based therapeutic concept for human cardiac disease.


Assuntos
Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Camundongos , Humanos , Animais , Edição de Genes , Sistemas CRISPR-Cas , Camundongos Knockout , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Fosforilação , Fibrose , Adenina , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo
4.
Nat Commun ; 14(1): 4333, 2023 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-37468485

RESUMO

Skeletal muscle fibers express distinct gene programs during development and maturation, but the underlying gene regulatory networks that confer stage-specific myofiber properties remain unknown. To decipher these distinctive gene programs and how they respond to neural activity, we generated a combined multi-omic single-nucleus RNA-seq and ATAC-seq atlas of mouse skeletal muscle development at multiple stages of embryonic, fetal, and postnatal life. We found that Myogenin, Klf5, and Tead4 form a transcriptional complex that synergistically activates the expression of muscle genes in developing myofibers. During myofiber maturation, the transcription factor Maf acts as a transcriptional switch to activate the mature fast muscle gene program. In skeletal muscles of mutant mice lacking voltage-gated L-type Ca2+ channels (Cav1.1), Maf expression and myofiber maturation are impaired. These findings provide a transcriptional atlas of muscle development and reveal genetic links between myofiber formation, maturation, and contraction.


Assuntos
Fibras Musculares Esqueléticas , Músculo Esquelético , Camundongos , Animais , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Diferenciação Celular
5.
J Clin Invest ; 133(13)2023 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-37395273

RESUMO

Mutations in genes encoding nuclear envelope proteins lead to diseases known as nuclear envelopathies, characterized by skeletal muscle and heart abnormalities, such as Emery-Dreifuss muscular dystrophy (EDMD). The tissue-specific role of the nuclear envelope in the etiology of these diseases has not been extensively explored. We previously showed that global deletion of the muscle-specific nuclear envelope protein NET39 in mice leads to neonatal lethality due to skeletal muscle dysfunction. To study the potential role of the Net39 gene in adulthood, we generated a muscle-specific conditional knockout (cKO) of Net39 in mice. cKO mice recapitulated key skeletal muscle features of EDMD, including muscle wasting, impaired muscle contractility, abnormal myonuclear morphology, and DNA damage. The loss of Net39 rendered myoblasts hypersensitive to mechanical stretch, resulting in stretch-induced DNA damage. Net39 was downregulated in a mouse model of congenital myopathy, and restoration of Net39 expression through AAV gene delivery extended life span and ameliorated muscle abnormalities. These findings establish NET39 as a direct contributor to the pathogenesis of EDMD that acts by protecting against mechanical stress and DNA damage.


Assuntos
Distrofia Muscular de Emery-Dreifuss , Animais , Camundongos , Estresse Mecânico , Distrofia Muscular de Emery-Dreifuss/metabolismo , Núcleo Celular/metabolismo , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Lamina Tipo A/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
6.
Nat Med ; 29(2): 401-411, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36797478

RESUMO

The most common form of genetic heart disease is hypertrophic cardiomyopathy (HCM), which is caused by variants in cardiac sarcomeric genes and leads to abnormal heart muscle thickening. Complications of HCM include heart failure, arrhythmia and sudden cardiac death. The dominant-negative c.1208G>A (p.R403Q) pathogenic variant (PV) in ß-myosin (MYH7) is a common and well-studied PV that leads to increased cardiac contractility and HCM onset. In this study we identify an adenine base editor and single-guide RNA system that can efficiently correct this human PV with minimal bystander editing and off-target editing at selected sites. We show that delivery of base editing components rescues pathological manifestations of HCM in induced pluripotent stem cell cardiomyocytes derived from patients with HCM and in a humanized mouse model of HCM. Our findings demonstrate the potential of base editing to treat inherited cardiac diseases and prompt the further development of adenine base editor-based therapies to correct monogenic variants causing cardiac disease.


Assuntos
Cardiomiopatia Hipertrófica , Miócitos Cardíacos , Humanos , Animais , Camundongos , Edição de Genes , Miocárdio , Arritmias Cardíacas , Mutação
7.
Sci Transl Med ; 14(672): eade1633, 2022 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-36417486

RESUMO

Mutations in RNA binding motif protein 20 (RBM20) are a common cause of familial dilated cardiomyopathy (DCM). Many RBM20 mutations cluster within an arginine/serine-rich (RS-rich) domain, which mediates nuclear localization. These mutations induce RBM20 mis-localization to form aberrant ribonucleoprotein (RNP) granules in the cytoplasm of cardiomyocytes and abnormal alternative splicing of cardiac genes, contributing to DCM. We used adenine base editing (ABE) and prime editing (PE) to correct pathogenic p.R634Q and p.R636S mutations in the RS-rich domain in human isogenic induced pluripotent stem cell (iPSC)-derived cardiomyocytes. Using ABE to correct RBM20R634Q human iPSCs, we achieved 92% efficiency of A-to-G editing, which normalized alternative splicing of cardiac genes, restored nuclear localization of RBM20, and eliminated RNP granule formation. In addition, we developed a PE strategy to correct the RBM20R636S mutation in iPSCs and observed A-to-C editing at 40% efficiency. To evaluate the potential of ABE for DCM treatment, we also created Rbm20R636Q mutant mice. Homozygous (R636Q/R636Q) mice developed severe cardiac dysfunction, heart failure, and premature death. Systemic delivery of ABE components containing ABEmax-VRQR-SpCas9 and single-guide RNA by adeno-associated virus serotype 9 in these mice restored cardiac function as assessed by echocardiography and extended life span. As seen by RNA sequencing analysis, ABE correction rescued the cardiac transcriptional profile of treated R636Q/R636Q mice, compared to the abnormal gene expression seen in untreated mice. These findings demonstrate the potential of precise correction of genetic mutations as a promising therapeutic approach for DCM.


Assuntos
Cardiomiopatia Dilatada , Humanos , Camundongos , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Mutação/genética , Miócitos Cardíacos/metabolismo , Genômica
8.
J Clin Invest ; 132(22)2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36377660

RESUMO

Mutations in nuclear envelope proteins (NEPs) cause devastating genetic diseases, known as envelopathies, that primarily affect the heart and skeletal muscle. A mutation in the NEP LEM domain-containing protein 2 (LEMD2) causes severe cardiomyopathy in humans. However, the roles of LEMD2 in the heart and the pathological mechanisms responsible for its association with cardiac disease are unknown. We generated knockin (KI) mice carrying the human c.T38>G Lemd2 mutation, which causes a missense amino acid exchange (p.L13>R) in the LEM domain of the protein. These mice represent a preclinical model that phenocopies the human disease, as they developed severe dilated cardiomyopathy and cardiac fibrosis leading to premature death. At the cellular level, KI/KI cardiomyocytes exhibited disorganization of the transcriptionally silent heterochromatin associated with the nuclear envelope. Moreover, mice with cardiac-specific deletion of Lemd2 also died shortly after birth due to heart abnormalities. Cardiomyocytes lacking Lemd2 displayed nuclear envelope deformations and extensive DNA damage and apoptosis linked to p53 activation. Importantly, cardiomyocyte-specific Lemd2 gene therapy via adeno-associated virus rescued cardiac function in KI/KI mice. Together, our results reveal the essentiality of LEMD2 for genome stability and cardiac function and unveil its mechanistic association with human disease.


Assuntos
Cardiomiopatias , Membrana Nuclear , Humanos , Camundongos , Animais , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Dano ao DNA , Miócitos Cardíacos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo
9.
Mol Ther Nucleic Acids ; 29: 525-537, 2022 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-36035749

RESUMO

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the X-linked dystrophin (DMD) gene. Exon deletions flanking exon 51, which disrupt the dystrophin open reading frame (ORF), represent one of the most common types of human DMD mutations. Previously, we used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) gene editing to restore the reading frame of exon 51 in mice and dogs with exon 50 deletions. Due to genomic sequence variations between species, the single guide RNAs (sgRNAs) used for DMD gene editing are often not conserved, impeding direct clinical translation of CRISPR-Cas therapeutic gene-editing strategies. To circumvent this potential obstacle, we generated a humanized DMD mouse model by replacing mouse exon 51 with human exon 51, followed by deletion of mouse exon 50, which disrupted the dystrophin ORF. Systemic CRISPR-Cas9 gene editing using an sgRNA that targets human exon 51 efficiently restored dystrophin expression and ameliorated pathologic hallmarks of DMD, including histopathology and grip strength in this mouse model. This unique DMD mouse model with the human genomic sequence allows in vivo assessment of clinically relevant gene editing strategies as well as other therapeutic approaches and represents a significant step toward therapeutic translation of CRISPR-Cas9 gene editing for correction of DMD.

10.
J Clin Invest ; 132(11)2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35642635

RESUMO

Skeletal muscle fibers contain hundreds of nuclei, which increase the overall transcriptional activity of the tissue and perform specialized functions. Multinucleation occurs through myoblast fusion, mediated by the muscle fusogens Myomaker (MYMK) and Myomixer (MYMX). We describe a human pedigree harboring a recessive truncating variant of the MYMX gene that eliminates an evolutionarily conserved extracellular hydrophobic domain of MYMX, thereby impairing fusogenic activity. Homozygosity of this human variant resulted in a spectrum of abnormalities that mimicked the clinical presentation of Carey-Fineman-Ziter syndrome (CFZS), caused by hypomorphic MYMK variants. Myoblasts generated from patient-derived induced pluripotent stem cells displayed defective fusion, and mice bearing the human MYMX variant died perinatally due to muscle abnormalities. In vitro assays showed that the human MYMX variant conferred minimal cell-cell fusogenicity, which could be restored with CRISPR/Cas9-mediated base editing, thus providing therapeutic potential for this disorder. Our findings identify MYMX as a recessive, monogenic human disease gene involved in CFZS, and provide new insights into the contribution of myoblast fusion to neuromuscular diseases.


Assuntos
Síndrome de Möbius , Doenças Musculares , Animais , Humanos , Proteínas de Membrana/genética , Camundongos , Proteínas Musculares/genética , Doenças Musculares/genética , Síndrome de Pierre Robin
11.
Proc Natl Acad Sci U S A ; 118(23)2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34088848

RESUMO

Homeothermic vertebrates produce heat in cold environments through thermogenesis, in which brown adipose tissue (BAT) increases mitochondrial oxidation along with uncoupling of the electron transport chain and activation of uncoupling protein 1 (UCP1). Although the transcription factors regulating the expression of UCP1 and nutrient oxidation genes have been extensively studied, only a few other proteins essential for BAT function have been identified. We describe the discovery of FAM195A, a BAT-enriched RNA binding protein, which is required for cold-dependent thermogenesis in mice. FAM195A knockout (KO) mice display whitening of BAT and an inability to thermoregulate. In BAT of FAM195A KO mice, enzymes involved in branched-chain amino acid (BCAA) metabolism are down-regulated, impairing their response to cold. Knockdown of FAM195A in brown adipocytes in vitro also impairs expression of leucine oxidation enzymes, revealing FAM195A to be a regulator of BCAA metabolism and a potential target for metabolic disorders.


Assuntos
Adipócitos Marrons , Tecido Adiposo Marrom , Temperatura Baixa , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Termogênese , Aminoácidos de Cadeia Ramificada/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Linhagem Celular Transformada , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Camundongos Knockout
12.
Genes Dev ; 35(11-12): 835-840, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33985971

RESUMO

Myocardin, a potent coactivator of serum response factor (SRF), competes with ternary complex factor (TCF) proteins for SRF binding to balance opposing mitogenic and myogenic gene programs in cardiac and smooth muscle. Here we identify a cardiac lncRNA transcribed adjacent to myocardin, named CARDINAL, which antagonizes SRF-dependent mitogenic gene transcription in the heart. CARDINAL-deficient mice show ectopic TCF/SRF-dependent mitogenic gene expression and decreased cardiac contractility in response to age and ischemic stress. CARDINAL forms a nuclear complex with SRF and inhibits TCF-mediated transactivation of the promitogenic gene c-fos, suggesting CARDINAL functions as an RNA cofactor for SRF in the heart.


Assuntos
Regulação da Expressão Gênica/genética , Coração/fisiologia , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/metabolismo , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Fatores Etários , Animais , Modelos Animais de Doenças , Deleção de Genes , Fatores de Transcrição MEF2/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica/genética , Infarto do Miocárdio/genética , Infarto do Miocárdio/fisiopatologia , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Ativação Transcricional
13.
Nat Commun ; 12(1): 690, 2021 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-33514739

RESUMO

Lamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery-Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD.


Assuntos
Proteínas de Membrana/deficiência , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/patologia , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Animais , Linhagem Celular , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Lamina Tipo A/genética , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Músculo Esquelético/citologia , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , RNA-Seq , Estudos Retrospectivos
14.
Proc Natl Acad Sci U S A ; 117(47): 29691-29701, 2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33148801

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.


Assuntos
Degeneração Macular/genética , Distrofia Muscular Animal/genética , Distrofia Muscular de Duchenne/genética , Regeneração/genética , Transdução de Sinais/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Mutação/genética , Miofibrilas/genética , Análise de Sequência de RNA/métodos , Transcrição Gênica/genética , Transcriptoma/genética
15.
Mol Ther ; 28(9): 2044-2055, 2020 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-32892813

RESUMO

Duchenne muscular dystrophy (DMD), one of the most common neuromuscular disorders of children, is caused by the absence of dystrophin protein in striated muscle. Deletions of exons 43, 45, and 52 represent mutational "hotspot" regions in the dystrophin gene. We created three new DMD mouse models harboring deletions of exons 43, 45, and 52 to represent common DMD mutations. To optimize CRISPR-Cas9 genome editing using the single-cut strategy, we identified single guide RNAs (sgRNAs) capable of restoring dystrophin expression by inducing exon skipping and reframing. Intramuscular delivery of AAV9 encoding SpCas9 and selected sgRNAs efficiently restored dystrophin expression in these new mouse models, offering a platform for future studies of dystrophin gene correction therapies. To validate the therapeutic potential of this approach, we identified sgRNAs capable of restoring dystrophin expression by the single-cut strategy in cardiomyocytes derived from human induced pluripotent stem cells (iPSCs) with each of these hotspot deletion mutations. We found that the potential effectiveness of individual sgRNAs in correction of DMD mutations cannot be predicted a priori, highlighting the importance of sgRNA design and testing as a prelude for applying gene editing as a therapeutic strategy for DMD.


Assuntos
Éxons , Deleção de Genes , Edição de Genes/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Animais , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Dependovirus/genética , Modelos Animais de Doenças , Distrofina/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miócitos Cardíacos/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo
16.
Circ Res ; 126(1): 6-24, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31730408

RESUMO

RATIONALE: Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is evolving rapidly. Recently, second-generation CRISPR/Cas9 activation systems based on nuclease inactive dead (d)Cas9 fused to transcriptional transactivation domains were developed for directing specific guide (g)RNAs to regulatory regions of any gene of interest, to enhance transcription. The application of dCas9 to activate cardiomyocyte transcription in targeted genomic loci in vivo has not been demonstrated so far. OBJECTIVE: We aimed to develop a mouse model for cardiomyocyte-specific, CRISPR-mediated transcriptional modulation, and to demonstrate its versatility by targeting Mef2d and Klf15 loci (2 well-characterized genes implicated in cardiac hypertrophy and homeostasis) for enhanced transcription. METHODS AND RESULTS: A mouse model expressing dCas9 with the VPR transcriptional transactivation domains under the control of the Myh (myosin heavy chain) 6 promoter was generated. These mice innocuously expressed dCas9 exclusively in cardiomyocytes. For initial proof-of-concept, we selected Mef2d, which when overexpressed, led to hypertrophy and heart failure, and Klf15, which is lowly expressed in the neonatal heart. The most effective gRNAs were first identified in fibroblast (C3H/10T1/2) and myoblast (C2C12) cell lines. Using an improved triple gRNA expression system (TRISPR [triple gRNA expression construct]), up to 3 different gRNAs were transduced simultaneously to identify optimal conditions for transcriptional activation. For in vivo delivery of the validated gRNA combinations, we employed systemic administration via adeno-associated virus serotype 9. On gRNA delivery targeting Mef2d expression, we recapitulated the anticipated cardiac hypertrophy phenotype. Using gRNA targeting Klf15, we could enhance its transcription significantly, although Klf15 is physiologically silenced at that time point. We further confirmed specific and robust dCas9VPR on-target effects. CONCLUSIONS: The developed mouse model permits enhancement of gene expression by using endogenous regulatory genomic elements. Proof-of-concept in 2 independent genomic loci suggests versatile applications in controlling transcription in cardiomyocytes of the postnatal heart.


Assuntos
Sistemas CRISPR-Cas , Regulação da Expressão Gênica , Miocárdio/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Dependovirus/genética , Fibroblastos/metabolismo , Regulação da Expressão Gênica/genética , Genes Sintéticos , Vetores Genéticos/genética , Coração/crescimento & desenvolvimento , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição MEF2/biossíntese , Fatores de Transcrição MEF2/genética , Camundongos , Camundongos Transgênicos , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , Regiões Promotoras Genéticas , Domínios Proteicos , RNA Polimerase III/genética , RNA Guia de Cinetoplastídeos/genética
17.
Nat Commun ; 10(1): 4537, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31586095

RESUMO

Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by mutations in the dystrophin gene. To enable the non-invasive analysis of DMD gene correction strategies in vivo, we introduced a luciferase reporter in-frame with the C-terminus of the dystrophin gene in mice. Expression of this reporter mimics endogenous dystrophin expression and DMD mutations that disrupt the dystrophin open reading frame extinguish luciferase expression. We evaluated the correction of the dystrophin reading frame coupled to luciferase in mice lacking exon 50, a common mutational hotspot, after delivery of CRISPR/Cas9 gene editing machinery with adeno-associated virus. Bioluminescence monitoring revealed efficient and rapid restoration of dystrophin protein expression in affected skeletal muscles and the heart. Our results provide a sensitive non-invasive means of monitoring dystrophin correction in mouse models of DMD and offer a platform for testing different strategies for amelioration of DMD pathogenesis.


Assuntos
Distrofina/genética , Terapia Genética/métodos , Microscopia Intravital/métodos , Músculo Esquelético/diagnóstico por imagem , Distrofia Muscular de Duchenne/terapia , Animais , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animais de Doenças , Distrofina/metabolismo , Éxons/genética , Edição de Genes/métodos , Genes Reporter/genética , Vetores Genéticos/química , Vetores Genéticos/genética , Humanos , Luciferases/química , Luciferases/genética , Medições Luminescentes , Masculino , Camundongos , Camundongos Transgênicos , Músculo Esquelético/patologia , Distrofia Muscular de Duchenne/diagnóstico por imagem , Distrofia Muscular de Duchenne/genética , Mutação , Resultado do Tratamento
18.
Sci Adv ; 5(3): eaav4324, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30854433

RESUMO

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.


Assuntos
Sistemas CRISPR-Cas , Distrofina/genética , Éxons , Distrofia Muscular de Duchenne/genética , Deleção de Sequência , Animais , Dependovirus/genética , Modelos Animais de Doenças , Edição de Genes , Expressão Gênica , Marcação de Genes , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Distrofia Muscular de Duchenne/tratamento farmacológico , Miócitos Cardíacos/metabolismo , RNA Guia de Cinetoplastídeos , Transdução Genética
19.
Cell Rep ; 23(13): 3701-3709, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29949755

RESUMO

Micropeptide regulator of ß-oxidation (MOXI) is a conserved muscle-enriched protein encoded by an RNA transcript misannotated as non-coding. MOXI localizes to the inner mitochondrial membrane where it associates with the mitochondrial trifunctional protein, an enzyme complex that plays a critical role in fatty acid ß-oxidation. Isolated heart and skeletal muscle mitochondria from MOXI knockout mice exhibit a diminished ability to metabolize fatty acids, while transgenic MOXI overexpression leads to enhanced ß-oxidation. Additionally, hearts from MOXI knockout mice preferentially oxidize carbohydrates over fatty acids in an isolated perfused heart system compared to wild-type (WT) animals. MOXI knockout mice also exhibit a profound reduction in exercise capacity, highlighting the role of MOXI in metabolic control. The functional characterization of MOXI provides insight into the regulation of mitochondrial metabolism and energy homeostasis and underscores the regulatory potential of additional micropeptides that have yet to be identified.


Assuntos
Ácidos Graxos/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Sequência de Aminoácidos , Animais , Ácidos Graxos/química , Humanos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mitocôndrias Cardíacas/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredução , Alinhamento de Sequência
20.
Proc Natl Acad Sci U S A ; 115(15): 3864-3869, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29581287

RESUMO

Regeneration of skeletal muscle in response to injury occurs through fusion of a population of stem cells, known as satellite cells, with injured myofibers. Myomixer, a muscle-specific membrane micropeptide, cooperates with the transmembrane protein Myomaker to regulate embryonic myoblast fusion and muscle formation. To investigate the role of Myomixer in muscle regeneration, we used CRISPR/Cas9-mediated genome editing to generate conditional knockout Myomixer alleles in mice. We show that genetic deletion of Myomixer in satellite cells using a tamoxifen-regulated Cre recombinase transgene under control of the Pax7 promoter abolishes satellite cell fusion and prevents muscle regeneration, resulting in severe muscle degeneration after injury. Satellite cells devoid of Myomixer maintain expression of Myomaker, demonstrating that Myomaker alone is insufficient to drive myoblast fusion. These findings, together with prior studies demonstrating the essentiality of Myomaker for muscle regeneration, highlight the obligatory partnership of Myomixer and Myomaker for myofiber formation throughout embryogenesis and adulthood.


Assuntos
Proteínas de Membrana/metabolismo , Músculo Esquelético/fisiopatologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Fusão Celular , Feminino , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/lesões , Músculo Esquelético/metabolismo , Regeneração , Células Satélites de Músculo Esquelético/citologia
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