Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout (KO) mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the eGFP gene in the tibialis anterior muscle of the Dmd KO mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out-of-frame to in-frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9 has great potential for the treatment of DMD and other neuromuscular diseases

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases. Koo et al. demonstrate that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. This study provides the therapeutic utility of CjCas9 for the treatment of Duchenne muscular dystrophy and other neuromuscular diseases

Inhibition of Myostatin Reduces Collagen Deposition in a Mouse Model of Oculopharyngeal Muscular Dystrophy (OPMD) With Established Disease

Copyright © 2020 Harish, Forrest, Herath, Dickson, Malerba and Popplewell. Background: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease presented by ptosis, dysphagia, and limb weakness. Affected muscles display increased fibrosis and atrophy, with characteristic inclusion bodies in the nucleus. Myostatin is a negative regulator of muscle mass, and inhibition of myostatin has been demonstrated to improve symptoms in models of muscular dystrophy. Methods: We systemically administered a monoclonal antibody to block myostatin in the A17 mouse model of OPMD at 42 weeks of age. The mice were administered a weekly dose of 10 mg/kg RK35 intraperitonially for 10 weeks, following which serum and histological analyses were performed on muscle samples. Results: The administration of the antibody resulted in a significant decrease in serum myostatin and collagen deposition in muscles. However, minimal effects on body mass, muscle mass and myofiber diameter, or the density of intranuclear inclusions (INIs) (a hallmark of disease progression of OPMD) were observed. Conclusion: This study demonstrates that inhibition of myostatin does not revert muscle atrophy in a mouse model with established OPMD disease, but is effective at reducing observed histological markers of fibrosis in the treated muscles

Comprehensive characterization of molecular interactions based on nanomechanics

Molecular interaction is a key concept in our understanding of the biological mechanisms of life. Two physical properties change when one molecular partner binds to another. Firstly, the masses combine and secondly, the structure of at least one binding partner is altered, mechanically transducing the binding into subsequent biological reactions. Here we present a nanomechanical micro-array technique for bio-medical research, which not only monitors the binding of effector molecules to their target but also the subsequent effect on a biological system in vitro. This label-free and real-time method directly and simultaneously tracks mass and nanomechanical changes at the sensor interface using micro-cantilever technology. To prove the concept we measured lipid vesicle (approximately 748*10(6) Da) adsorption on the sensor interface followed by subsequent binding of the bee venom peptide melittin (2840 Da) to the vesicles. The results show the high dynamic range of the instrument and that measuring the mass and structural changes simultaneously allow a comprehensive discussion of molecular interactions

Elara: A Phase 2 Trial Investigating The Efficacy And Safety Of Tisagenlecleucel In Adult Patients With Refractory/Relapsed Follicular Lymphoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/149492/1/hon6_2632.pd