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Title: Antisense-mediated myostatin downregulation by destructive exon skipping using 2'O-methyl RNA and morpholino oligomers in skeletal muscle cells and animal models
Author: Kang, Jagjeet Kaur
Awarding Body: Royal Holloway, University of London
Current Institution: Royal Holloway, University of London
Date of Award: 2012
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Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knock down the expression of the myostatin gene as a means to bring about improvements in muscle wasting conditions, including Duchenne muscular dystrophy (DMD). Improved muscle regeneration in the absence of myostatin has been demonstrated in mdx mice by-crossing them with myostatin null mice. Increased muscle strength and improved dystrophic pathophysiology has been found in the mdx mice treated with myostatin antibodies, purified myostatin propeptide (natural binding partner of myostatin) with a mouse fusion protein and AA V -mediated transfer of propeptide. Virus-based strategies have the major drawbacks of uncontrolled insertion into the target genome and undesirable immune response; whereas, injecting binding partners to the target-tissue might have low sustainability over a longer period of time. In this study the design and use of anti sense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing and knockdown myostatin has been described. AOs of both 2'O-methyl RNA (2'OMePS-with a phosphorothioate backbone) and phosphorodiarnidate morpholino (PMO) chemistries were designed using different bioinformatics algorithms. Efficiency of destructive myostatin exon skipping was then demonstrated and evaluated comparatively by oligomer transfection of cultured muscle cells, and RT-PCR and bioactivity analyses. Sustained and high levels of destructive exon skipping of the myostatin mRNA, and increases in skeletal muscle mass and fibre sizes was observed up toi months following a single injection of Vivo-PMO (PMO conjugated to a cell-penetrating octa-guanidine moiety) into the tibialis anterior muscle in normal mice. The efficiency of Vivo-PMO was also compared to a PMO conjugated to a cell penetrating peptide moiety (B-PMO) by single intra -muscular treatment of wild type mice. Weekly intravenous injections of Vivo- PMO in normal mice also lead to myostatin exon skipping in selected skeletal muscles, and associated increases in tissue mass, cross-sectional area, and average fibre diameter. Dual exon skipping of myostatin and dystrophin was also carried out successfully in mdx mice. These studies indicate that (i) anti sense-mediated destructive exon skipping can be induced in the myostatin RNA, (ii) anti sense AO treatment reduces myostatin bioactivity and enhances muscle mass in vivo, and (iii) AO-induced myostatin exon- skipping may be a potential therapeutic strategy to counter muscular dystrophy, muscular atrophy, cachexia and sarcopenia.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available