scholarly journals Development of a high-throughput screen to identify small molecule enhancers of sarcospan for the treatment of Duchenne muscular dystrophy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Cynthia Shu ◽  
Ariana N. Kaxon-Rupp ◽  
Judd R. Collado ◽  
Robert Damoiseaux ◽  
Rachelle H. Crosbie
Keyword(s):  
Gene Expression ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Calcium Channel ◽  
Muscle Cell ◽  
High Throughput ◽  
Promoter Activity ◽  
Muscle Cells ◽  
Wild Type ◽  
Protein Levels

Abstract Background Duchenne muscular dystrophy (DMD) is caused by loss of sarcolemma connection to the extracellular matrix. Transgenic overexpression of the transmembrane protein sarcospan (SSPN) in the DMD mdx mouse model significantly reduces disease pathology by restoring membrane adhesion. Identifying SSPN-based therapies has the potential to benefit patients with DMD and other forms of muscular dystrophies caused by deficits in muscle cell adhesion. Methods Standard cloning methods were used to generate C2C12 myoblasts stably transfected with a fluorescence reporter for human SSPN promoter activity. Assay development and screening were performed in a core facility using liquid handlers and imaging systems specialized for use with a 384-well microplate format. Drug-treated cells were analyzed for target gene expression using quantitative PCR and target protein expression using immunoblotting. Results We investigated the gene expression profiles of SSPN and its associated proteins during myoblast differentiation into myotubes, revealing an increase in expression after 3 days of differentiation. We created C2C12 muscle cells expressing an EGFP reporter for SSPN promoter activity and observed a comparable increase in reporter levels during differentiation. Assay conditions for high-throughput screening were optimized for a 384-well microplate format and a high-content imager for the visualization of reporter levels. We conducted a screen of 3200 compounds and identified seven hits, which include an overrepresentation of L-type calcium channel antagonists, suggesting that SSPN gene activity is sensitive to calcium. Further validation of a select hit revealed that the calcium channel inhibitor felodipine increased SSPN transcript and protein levels in both wild-type and dystrophin-deficient myotubes, without increasing differentiation. Conclusions We developed a stable muscle cell line containing the promoter region of the human SSPN protein fused to a fluorescent reporter. Using the reporter cells, we created and validated a scalable, cell-based assay that is able to identify compounds that increase SSPN promoter reporter, transcript, and protein levels in wild-type and dystrophin-deficient muscle cells.

scholarly journals CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

2020 ◽  
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Kasia Dzierlega ◽  
Yiqing Huang ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Animal Models ◽  
Mechanical Stress ◽  
Muscle Cell ◽  
Muscle Cells ◽  
Neuromuscular Disorder ◽  
Reading Frame ◽  
Advantages And Disadvantages ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose quite well, each comes with their own limitations. To help overcome this, clustered regularly interspaced short palindromic repeats (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

scholarly journals High-throughput screening identifies modulators of sarcospan that stabilize muscle cells and exhibit activity in the mouse model of Duchenne muscular dystrophy

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Cynthia Shu ◽  
Liubov Parfenova ◽  
Ekaterina Mokhonova ◽  
Judd R. Collado ◽  
Robert Damoiseaux ◽  
...  
Keyword(s):  
Gene Expression ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Cell Membrane ◽  
Mouse Model ◽  
Cell Surface ◽  
Protein Expression ◽  
High Throughput ◽  
High Throughput Screening ◽  
Deficient Mouse

Abstract Background Duchenne muscular dystrophy (DMD) is a degenerative muscle disease caused by mutations in the dystrophin gene. Loss of dystrophin prevents the formation of a critical connection between the muscle cell membrane and the extracellular matrix. Overexpression of sarcospan (SSPN) in the mouse model of DMD restores the membrane connection and reduces disease severity, making SSPN a promising therapeutic target for pharmacological upregulation. Methods Using a previously described cell-based promoter reporter assay of SSPN gene expression (hSSPN-EGFP), we conducted high-throughput screening on libraries of over 200,000 curated small molecules to identify SSPN modulators. The hits were validated in both hSSPN-EGFP and hSSPN-luciferase reporter cells. Hit selection was conducted on dystrophin-deficient mouse and human myotubes with assessments of (1) SSPN gene expression using quantitative PCR and (2) SSPN protein expression using immunoblotting and an ELISA. A membrane stability assay using osmotic shock was used to validate the functional effects of treatment followed by cell surface biotinylation to label cell surface proteins. Dystrophin-deficient mdx mice were treated with compound, and muscle was subjected to quantitative PCR to assess SSPN gene expression. Results We identified and validated lead compounds that increased SSPN gene and protein expression in dystrophin-deficient mouse and human muscle cells. The lead compound OT-9 increased cell membrane localization of compensatory laminin-binding adhesion complexes and improved membrane stability in DMD myotubes. We demonstrated that the membrane stabilizing benefit is dependent on SSPN. Intramuscular injection of OT-9 in the mouse model of DMD increased SSPN gene expression. Conclusions This study identifies a pharmacological approach to treat DMD and sets the path for the development of SSPN-based therapies.

scholarly journals Mass Spectrometric Profiling of Extraocular Muscle and Proteomic Adaptations in the mdx-4cv Model of Duchenne Muscular Dystrophy

Life ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 595
Author(s):  
Stephen Gargan ◽  
Paul Dowling ◽  
Margit Zweyer ◽  
Jens Reimann ◽  
Michael Henry ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Biochemical Properties ◽  
Mass Spectrometric ◽  
Cellular Stress Response ◽  
Diaphragm Muscle ◽  
Spectrometric Analysis ◽  
Mitochondrial Enzymes ◽  
Wild Type ◽  
Potential Marker

Extraocular muscles (EOMs) represent a specialized type of contractile tissue with unique cellular, physiological, and biochemical properties. In Duchenne muscular dystrophy, EOMs stay functionally unaffected in the course of disease progression. Therefore, it was of interest to determine their proteomic profile in dystrophinopathy. The proteomic survey of wild type mice and the dystrophic mdx-4cv model revealed a broad spectrum of sarcomere-associated proteoforms, including components of the thick filament, thin filament, M-band and Z-disk, as well as a variety of muscle-specific markers. Interestingly, the mass spectrometric analysis revealed unusual expression levels of contractile proteins, especially isoforms of myosin heavy chain. As compared to diaphragm muscle, both proteomics and immunoblotting established isoform MyHC14 as a new potential marker in wild type EOMs, in addition to the previously identified isoforms MyHC13 and MyHC15. Comparative proteomics was employed to establish alterations in the protein expression profile between normal EOMs and dystrophin-lacking EOMs. The analysis of mdx-4cv EOMs identified elevated levels of glycolytic enzymes and molecular chaperones, as well as decreases in mitochondrial enzymes. These findings suggest a process of adaptation in dystrophin-deficient EOMs via a bioenergetic shift to more glycolytic metabolism, as well as an efficient cellular stress response in EOMs in dystrophinopathy.

scholarly journals In vivo genome editing in mouse restores dystrophin expression in Duchenne muscular dystrophy patient muscle fibers

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Menglong Chen ◽  
Hui Shi ◽  
Shixue Gou ◽  
Xiaomin Wang ◽  
Lei Li ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mouse Model ◽  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
High Efficiency ◽  
Muscle Fibers ◽  
Muscle Cells

Abstract Background Mutations in the DMD gene encoding dystrophin—a critical structural element in muscle cells—cause Duchenne muscular dystrophy (DMD), which is the most common fatal genetic disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene editing is a promising strategy for permanently curing DMD. Methods In this study, we developed a novel strategy for reframing DMD mutations via CRISPR-mediated large-scale excision of exons 46–54. We compared this approach with other DMD rescue strategies by using DMD patient-derived primary muscle-derived stem cells (DMD-MDSCs). Furthermore, a patient-derived xenograft (PDX) DMD mouse model was established by transplanting DMD-MDSCs into immunodeficient mice. CRISPR gene editing components were intramuscularly delivered into the mouse model by adeno-associated virus vectors. Results Results demonstrated that the large-scale excision of mutant DMD exons showed high efficiency in restoring dystrophin protein expression. We also confirmed that CRISPR from Prevotella and Francisella 1(Cas12a)-mediated genome editing could correct DMD mutation with the same efficiency as CRISPR-associated protein 9 (Cas9). In addition, more than 10% human DMD muscle fibers expressed dystrophin in the PDX DMD mouse model after treated by the large-scale excision strategies. The restored dystrophin in vivo was functional as demonstrated by the expression of the dystrophin glycoprotein complex member β-dystroglycan. Conclusions We demonstrated that the clinically relevant CRISPR/Cas9 could restore dystrophin in human muscle cells in vivo in the PDX DMD mouse model. This study demonstrated an approach for the application of gene therapy to other genetic diseases.

scholarly journals Role of Sigma Factors in Controlling Global Gene Expression in Light/Dark Transitions in the Cyanobacterium Synechocystis sp. Strain PCC 6803

2007 ◽  
Vol 189 (21) ◽  
pp. 7829-7840 ◽  
Author(s):  
Tina C. Summerfield ◽  
Louis A. Sherman
Keyword(s):  
Gene Expression ◽  
Global Gene Expression ◽  
Growth Conditions ◽  
Day Length ◽  
Regulation Of Transcription ◽  
Wild Type ◽  
Protein Levels ◽  
In The Wild ◽  
Altered Gene ◽  
Pcc 6803

ABSTRACT We report on differential gene expression in the cyanobacterium Synechocystis sp. strain PCC 6803 after light-dark transitions in wild-type, ΔsigB, and ΔsigD strains. We also studied the effect of day length in the presence of glucose on a ΔsigB ΔsigE mutant. Our results indicated that the absence of SigB or SigD predominately altered gene expression in the dark or in the light, respectively. In the light, approximately 350 genes displayed transcript levels in the ΔsigD strain that were different from those of the wild type, with over 200 of these up-regulated in the mutant. In the dark, removal of SigB altered more than 150 genes, and the levels of 136 of these were increased in the mutant compared to those in the wild type. The removal of both SigB and SigE had a major impact on gene expression under mixotrophic growth conditions and resulted in the inability of cells to grow in the presence of glucose with 8-h light and 16-h dark cycles. Our results indicated the importance of group II σ factors in the global regulation of transcription in this organism and are best explained by using the σ cycle paradigm with the stochastic release model described previously (R. A. Mooney, S. A. Darst, and R. Landick, Mol. Cell 20:335-345, 2005). We combined our results with the total protein levels of the σ factors in the light and dark as calculated previously (S. Imamura, S. Yoshihara, S. Nakano, N. Shiozaki, A. Yamada, K. Tanaka, H. Takahashi, M. Asayama, and M. Shirai, J. Mol. Biol. 325:857-872, 2003; S. Imamura, M. Asayama, H. Takahashi, K. Tanaka, H. Takahashi, and M. Shirai, FEBS Lett. 554:357-362, 2003). Thus, we concluded that the control of global transcription is based on the amount of the various σ factors present and able to bind RNA polymerase.

scholarly journals Electroporation-mediated delivery of catalytic oligodeoxynucleotides for manipulation of vascular gene expression

2003 ◽  
Vol 285 (5) ◽  
pp. H2240-H2247 ◽  
Author(s):  
Elizabeth A. Nunamaker ◽  
Hai-Ying Zhang ◽  
Yuichi Shirasawa ◽  
Joseph N. Benoit ◽  
David A. Dean
Keyword(s):  
Gene Expression ◽  
Experimental Studies ◽  
Wild Type ◽  
Protein Levels ◽  
Dna Oligonucleotides ◽  
Pkc Isoforms ◽  
Cell Layers ◽  
Cultured Smooth Muscle Cells ◽  
Decrease Gene Expression

The development of inexpensive and effective approaches to transiently decrease gene expression in vivo would be useful for the study of physiological processes in living animals. DNAzymes are a novel class of DNA oligonucleotides that can catalytically cleave target mRNAs and thereby reduce protein production. However, current methods for their delivery in vivo are limited and inefficient. In this study, we show that electroporation can be used to deliver DNAzymes to the intact mesenteric vasculature of rats. With the use of PKC-ϵ as a target, a set of wild-type and mutant control DNAzymes was designed and shown to reduce both PKC-ϵ mRNA and protein levels in cultured smooth muscle cells in a specific manner. The wild-type DNAzyme reduced PKC-ϵ protein levels by 70% at 24 h in two different cell lines without decreasing the levels of the five other PKC isoforms tested. When delivered to the intact vasculature using electroporation, the DNAzyme reduced PKC-ϵ protein levels by >60% without affecting these other PKC isoforms. Electroporation was required for oligonucleotide transfer and was able to deliver the DNAzymes to multiple cell layers in the vessel wall. Protein levels were reduced maximally by 24 h postelectroporation and returned to normal by 48 h. These results suggest that electroporation can be used to deliver DNAzymes and other DNA oligonucleotides to the vasculature in vivo and can decrease gene expression for a window of time that can be used for experimental studies.

scholarly journals CRISPR-Generated Animal Models of Duchenne Muscular Dystrophy

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 342 ◽  
Author(s):  
Kenji Rowel Q. Lim ◽  
Quynh Nguyen ◽  
Kasia Dzierlega ◽  
Yiqing Huang ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Animal Models ◽  
Mechanical Stress ◽  
Muscle Cell ◽  
Neuromuscular Disorder ◽  
Reading Frame ◽  
Advantages And Disadvantages ◽  
Short Palindromic Repeat ◽  
Dmd Gene

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder most commonly caused by mutations disrupting the reading frame of the dystrophin (DMD) gene. DMD codes for dystrophin, which is critical for maintaining the integrity of muscle cell membranes. Without dystrophin, muscle cells receive heightened mechanical stress, becoming more susceptible to damage. An active body of research continues to explore therapeutic treatments for DMD as well as to further our understanding of the disease. These efforts rely on having reliable animal models that accurately recapitulate disease presentation in humans. While current animal models of DMD have served this purpose well to some extent, each has its own limitations. To help overcome this, clustered regularly interspaced short palindromic repeat (CRISPR)-based technology has been extremely useful in creating novel animal models for DMD. This review focuses on animal models developed for DMD that have been created using CRISPR, their advantages and disadvantages as well as their applications in the DMD field.

Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy

2007 ◽  
Vol 103 (2) ◽  
pp. 664-672 ◽  
Author(s):  
W. W. Dye ◽  
R. L. Gleason ◽  
E. Wilson ◽  
J. D. Humphrey
Keyword(s):  
Muscular Dystrophy ◽  
Axial Force ◽  
Carotid Arteries ◽  
Structural Integrity ◽  
Muscle Cells ◽  
Biomechanical Properties ◽  
Axial Stiffness ◽  
Wild Type ◽  
Functional Responses

Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-δ, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin ( mdx) or sarcoglycan-δ ( sgcd−/−) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd−/−, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd−/− arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd−/− arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd−/− arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.

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