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Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected ΔEx44 DMD mice to systemic delivery of AAV9-expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We found that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, we observed a loss of gene edited dystrophin expression. Analysis of on- and off-target editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.
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ObjectiveTo investigate the effect of Huangqisan pellets (HQS) on the phosphatidylinositol-3 kinase/protein kinase B/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway and autophagy in the kidney of diabetic nephropathy (DN) rats. MethodDN rat model was established through high-fat diet combined with intraperitoneal injection of streptozotocin (35 mg·kg-1). DN rats were randomly assigned into model group, irbesartan (0.027 g·kg-1) group, low-dose HQS (0.54 g·kg-1) group and high-dose HQS (1.08 g·kg-1) group. The levels of 24 h urinary total protein (UTP), serum albumin (Alb), serum creatinine (SCr), urea nitrogen (BUN), triglyceride (TG) and total cholesterol (TC) were measured after 12 weeks of continuous administration. The pathological changes of renal tissue were observed via hematoxylin-eosin (HE) staining. The expression of podocyte split diaphragm proteins nephrin and podocin in the renal tissue were detected by immunohistochemistry. The protein levels and phosphorylation of key proteins in PI3K/Akt/mTOR signaling pathway, as well as the expression of yeast Atg6 homolog (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3) in the renal tissue were analyzed by Western blot. ResultCompared with the control group, the model group showcased increased 24 h UTP, SCr, BUN, TG, and TC levels and decreased Alb level (P<0.01). After modeling, the rats showed granulosity of epithelial cells of renal tubules, thickening of capillary basement membrane, proliferation of mesangial cells, and sclerosis of glomerulus. Furthermore, modeling down-regulated the expression of nephrin and podocin in the podocyte hiatus of glomerulus (P<0.01) as well as the protein levels of p-PI3K, p-Akt, and p-mTOR and the autophagy markers LC3 and Beclin1 in renal tissue (P<0.01). Compared with model group, irbesartan and HQS decreased the 24 h UTP, Cr, BUN, TG, and TC levels, increased the Alb level, and alleviated the pathological damage of kidney. Moreover, they up-regulated the expression of Nephrin and Podocin in the podocyte hiatus of glomerulus, as well as the protein levels of p-PI3K, p-Akt, p-mTOR, LC3, and Beclin1 in renal tissue (P<0.05, P<0.01). ConclusionHQS may inhibit the PI3K/Akt/mTOR signaling pathway to enhance podocyte autophagy and protect the glomerulus, thus slowing down the development of DN.
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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.
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Exones , Eliminación de Gen , Edición Génica/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Animales , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/metabolismo , Miocitos Cardíacos/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
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.
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Proteína 9 Asociada a CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edición Génica , Nanopartículas/química , Especificidad de Órganos/genética , Ribonucleoproteínas/metabolismo , Animales , Cationes , ADN de Neoplasias/aislamiento & purificación , Distrofina/genética , Células HeLa , Humanos , Lípidos/química , Ratones Endogámicos C57BLRESUMEN
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.
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Sistemas CRISPR-Cas , Dependovirus/genética , Distrofina/genética , Edición Génica , Terapia Genética , Vectores Genéticos/genética , Distrofia Muscular de Duchenne/genética , Animales , Modelos Animales de Enfermedad , Exones , Dosificación de Gen , Expresión Génica , Marcación de Gen , Técnicas de Transferencia de Gen , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/terapia , Mutación , ARN Guía de Kinetoplastida/genética , Transducción GenéticaRESUMEN
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.
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Distrofina/genética , Terapia Genética/métodos , Microscopía Intravital/métodos , Músculo Esquelético/diagnóstico por imagen , Distrofia Muscular de Duchenne/terapia , Animales , Sistemas CRISPR-Cas/genética , Dependovirus/genética , Modelos Animales de Enfermedad , Distrofina/metabolismo , Exones/genética , Edición Génica/métodos , Genes Reporteros/genética , Vectores Genéticos/química , Vectores Genéticos/genética , Humanos , Luciferasas/química , Luciferasas/genética , Mediciones Luminiscentes , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/diagnóstico por imagen , Distrofia Muscular de Duchenne/genética , Mutación , Resultado del TratamientoRESUMEN
The adult mammalian heart has limited capacity for regeneration following injury, whereas the neonatal heart can readily regenerate within a short period after birth. To uncover the molecular mechanisms underlying neonatal heart regeneration, we compared the transcriptomes and epigenomes of regenerative and nonregenerative mouse hearts over a 7-d time period following myocardial infarction injury. By integrating gene expression profiles with histone marks associated with active or repressed chromatin, we identified transcriptional programs underlying neonatal heart regeneration, and the blockade to regeneration in later life. Our results reveal a unique immune response in regenerative hearts and a retained embryonic cardiogenic gene program that is active during neonatal heart regeneration. Among the unique immune factors and embryonic genes associated with cardiac regeneration, we identified Ccl24, which encodes a cytokine, and Igf2bp3, which encodes an RNA-binding protein, as previously unrecognized regulators of cardiomyocyte proliferation. Our data provide insights into the molecular basis of neonatal heart regeneration and identify genes that can be modulated to promote heart regeneration.
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Animales Recién Nacidos/fisiología , Corazón/fisiología , Código de Histonas/fisiología , Regeneración/fisiología , Transcriptoma/fisiología , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Modelos Animales de Enfermedad , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Lesiones Cardíacas/genética , Lesiones Cardíacas/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Infarto del Miocardio/genética , Regeneración/genética , Transcriptoma/genéticaRESUMEN
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.
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Sistemas CRISPR-Cas , Distrofina/genética , Exones , Distrofia Muscular de Duchenne/genética , Eliminación de Secuencia , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Edición Génica , Expresión Génica , Marcación de Gen , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Distrofia Muscular de Duchenne/tratamiento farmacológico , Miocitos Cardíacos/metabolismo , ARN Guía de Kinetoplastida , Transducción GenéticaRESUMEN
The ability to efficiently modify the genome using CRISPR technology has rapidly revolutionized biology and genetics and will soon transform medicine. Duchenne muscular dystrophy (DMD) represents one of the first monogenic disorders that has been investigated with respect to CRISPR-mediated correction of causal genetic mutations. DMD results from mutations in the gene encoding dystrophin, a scaffolding protein that maintains the integrity of striated muscles. Thousands of different dystrophin mutations have been identified in DMD patients, who suffer from a loss of ambulation followed by respiratory insufficiency, heart failure, and death by the third decade of life. Using CRISPR to bypass DMD mutations, dystrophin expression has been efficiently restored in human cells and mouse models of DMD. Here, we review recent progress toward the development of possible CRISPR therapies for DMD and highlight opportunities and potential obstacles in attaining this goal.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Distrofina/genética , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Animales , Sistemas CRISPR-Cas/genética , Edición Génica , Predisposición Genética a la Enfermedad/epidemiología , Humanos , Ratones , Distrofia Muscular de Duchenne/epidemiología , Distrofia Muscular de Duchenne/fisiopatología , Mutación , Prevalencia , Pronóstico , Medición de Riesgo , Índice de Severidad de la Enfermedad , Resultado del TratamientoRESUMEN
Twist transcription factors function as ancestral regulators of mesodermal cell fates in organisms ranging from Drosophila to mammals. Through lineage tracing of Twist2 (Tw2)-expressing cells with tamoxifen-inducible Tw2-CreERT2 and tdTomato (tdTO) reporter mice, we discovered a unique cell population that progressively contributes to cardiomyocytes (CMs), endothelial cells, and fibroblasts in the adult heart. Clonal analysis confirmed the ability of Tw2-derived tdTO+ (Tw2-tdTO+) cells to form CMs in vitro. Within the adult heart, Tw2-tdTO+ CMs accounted for â¼13% of total CMs, the majority of which resulted from fusion of Tw2-tdTO+ cells with existing CMs. Tw2-tdTO+ cells also contribute to cardiac remodeling after injury. We conclude that Tw2-tdTO+ cells participate in lifelong maintenance of cardiac function, at least in part through de novo formation of CMs and fusion with preexisting CMs, as well as in the genesis of other cellular components of the adult heart.
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Células Madre Multipotentes/metabolismo , Miocardio/metabolismo , Proteínas Represoras/biosíntesis , Proteína 1 Relacionada con Twist/biosíntesis , Animales , Drosophila melanogaster , Células Endoteliales/citología , Células Endoteliales/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Ratones Transgénicos , Células Madre Multipotentes/citología , Miocardio/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Represoras/genética , Proteína 1 Relacionada con Twist/genéticaRESUMEN
Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (DMD). Most of these mutations are clustered in "hotspots." There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame DMD exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the DMD gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.
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Edición Génica , Genoma Humano , Distrofia Muscular de Duchenne/genética , Mutación/genética , Miocardio/patología , Ingeniería de Tejidos/métodos , Secuencia de Bases , Distrofina/genética , Exones/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , ARN Guía de Kinetoplastida/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Tremendous effort has been made to improve stability and delivery efficacy of small RNA therapeutics. However, nearly all current nano-encapsulation carriers utilize the critical balance between only two interacting parameters: RNA-binding electrostatic interactions and nanoparticle-stabilizing hydrophobic interactions. We report the development of intercalation-meditated nucleic acid (IMNA) nanoparticles, which utilize intercalation as a third interaction to enhance small RNA delivery. This toolbox expansion of interaction parameters may inspire the use of additional forces in nanoparticle drug carriers to increase potency and stability.
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Sistemas de Liberación de Medicamentos , Sustancias Intercalantes/química , Nanopartículas/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Portadores de Fármacos , Silenciador del Gen , Células HeLa , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Nanopartículas/administración & dosificaciónRESUMEN
During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell-cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.
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Membrana Celular , Proteínas de la Membrana , Desarrollo de Músculos/fisiología , Proteínas Musculares , Mioblastos Esqueléticos , Animales , Fusión Celular , Línea Celular , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Musculares/química , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Mioblastos Esqueléticos/química , Mioblastos Esqueléticos/citología , Mioblastos Esqueléticos/metabolismo , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
AIM: To evaluate the physiologic change of retinal thickness during pregnancy. METHODS:Forty cases ( 80 eyes ) were included two groups:40 eyes ( 20 cases ) in healthy pregnant women group (including in the second and last trimester), and 40 eyes (20 cases) in healthy nonpregnant women group ( control group ) . The macular volume, average thickness, central subfield thickness and retinal thickness of other parafoveal areas were measured by optical coherence tomography scan. RESULTS: The macular volume was 10. 06±0. 41mm3 and 9. 87±0. 30mm3 in healthy pregnant women group and control group respectively. The average thickness was 279. 43±10. 86μm and 274. 25±8. 07μm in healthy pregnant women group and control group respectively. The central subfield thickness was 235. 15±15. 05μm and 233. 00±15. 81μm in healthy pregnant women group and control group espectively. Statistically significant difference was found in macular volume and average thickness (P CONCLUSION:The macular retinal thickness increases during pregnancy in the second and last trimester. The physiologic change of retinal thickness should be considered when evaluating pathologic retinal disease of pregnant women.
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A total of 24 biologically pure entophytic fungal strains were isolated from stems, leaves, and seed coats of Xylocarpus plants by repeated purification, and identified with Internal Transcribed Spacer (ITS) rDNA molecular method, which belonging to 14 genera, 11 families, 9 orders and 3 classes. There were differences in genus and species levels among three plant materials from different habitats and species, and it was found that the strains of Phomopsis and Colletotrichum existed in all three plant materials. In vitro assay of antitumor activity by MTT method revealed that the EtOAc extracts of 15 strains exhibited potent antitumor activity. These results suggest that it is of value for further investigation on the above fungal strains.
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Humanos , Antineoplásicos , Farmacología , Biodiversidad , Línea Celular Tumoral , Endófitos , Química , Clasificación , Genética , Hongos , Química , Clasificación , Genética , Células HCT116 , Meliaceae , Microbiología , FilogeniaRESUMEN
Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.