Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.692559
Title: A gene therapy approach for treating muscle degeneration in neuromuscular disorders using IGF-I splice variants
Author: Ates, K.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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Abstract:
Neuromuscular disorders, which associate with muscle degeneration, are not rare and affect millions of people worldwide. The impacts of such disorders vary from gradual loss of mobility and independence to severe disability and death, and therefore millions of patients suffer from them at every stage of their life. Because, there is currently no treatment of any form of such disorders, this study was aimed to develop a novel treatment for such disorders. For a long time it has been known that Insulin-like Growth Factor (IGF-I) influences several cellular processes, including proliferation, differentiation, repair and maintenance. Like many genes, the IGF-I gene can be spliced to produce several isoforms, and in human muscle, it expresses at least two main isoforms which are a liver type, systemic form (IGF-I Ea) and an autocrine / paracrine form (IGF-I Ec). This second isoform has been named the Mechano Growth Factor (MGF) because of its mechanosensitivity. The in vitro and in vivo effects of these two splice variants of the IGF-I gene were investigated in this study. In vitro roles of two splice variants of the gene were studied by the proliferation / differentiation effects of two alternative splice isoforms of the gene in animal muscle cell lines (mouse C2C12 and rat L6 E9). Proliferation / differentiation assays were carried out using human primary cell cultures from biopsied muscles from congenital muscular dystrophy (CMD), fascioscapulohumeral muscular dystrophy (FSHD) and amyotrophic lateral sclerosis (ALS) patients as well as from healthy volunteers. Human primary muscle cells were treated with IGF-I Ea (long r IGF-I) and MGF E domain peptides, and immunocytochemistry techniques with Desmin, DAPI and FITC markers were used to detect proliferation state of myogenic commitment. The CPK and BCA protein assays were also used to determine the differentiation state following such peptide treatments. The results showed that MGF significantly increased muscle stem (satellite) cell proliferation in both animal and human muscles, both in healthy and in severe muscle wasting disorders. E domain of MGF dramatically increased proliferation in progenitor cell in CMD (68%), FSHD (74%) and ALS (49%) primary cultures. The results also confirmed that the MGF had no effect on myotube formation but that it increases myoblast progenitor cell proliferation, whilst systemic IGF-I peptide (IGF-I Ea) increased cell differentiation and facilitated myotube formation. The effects of two IGF-I splice variants in muscle fibre growth were also studied in relation to Duchenne Muscular Dystrophy (DMD) by in vivo gene transfer method using the mdx mouse model. Such effects were investigated in both young and old mdx mice TA muscles by intramuscular injection of cDNAs in plasmid vectors pcDNA3.1NT/GFP. Maximum muscle tetanic contractile force was measured to determine the changes of muscle strength at 21 days after gene injection. The results showed that cDNA of MGF dramatically increased muscle fibre strength in young mdx mice (37 %) in only 3 weeks time. The MGF also increased muscle strength and mass in the older mdx mice but to a moderate level (11%). In mdx mice, the changes in gene expressions of satellite cell markers (MyoD and myogenin) were determined by quantitative real-time reverse transcriptase PCR, 21 days after injection of the gene constructs into TA muscle comparing with TA muscle of untreated leg. The results showed that MGF had an effect in satellite cell activation, and it activated quiescent satellite cells in mdx mice. This study showed that MGF has a significant effect in both in vitro and in vivo models, which were used in relation to treatment of muscle degeneration in DMD, CMD, FSHD and ALS. The study also showed that MGF has considerable potential to use as a therapeutic agent to treat muscle degeneration in such neuromuscular disorders.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.692559  DOI: Not available
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