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(A C L This Copy Has Been Deposited in the University of London Library, Senate □ House, Malet Street, London WC1E 7HU REFERENCE ONLY 2 8 0 9 4 4 2 6 1 3 UNIVERSITY OF LONDON THESIS D egree p ^ b Y ear Zoo~7 N am e of Author QvJ a f) COPYRIGHT This is a thesis accepted for a Higher Degree of the University of London. It is an unpublished typescript and the copyright is held by the author. All persons consulting the thesis must read and abide by the Copyright Declaration below. COPYRIGHT DECLARATION I recognise that the copyright of the above-described thesis rests with the author and that no quotation from it or information derived from it may be published without the prior written consent of the author. LOAN Theses may not be lent to individuals, but the University Library may lend a copy to approved libraries within the United Kingdom, for consultation solely on the premises of those libraries. Application should be made to: The Theses Section, University of London Library, Senate House, Malet Street, London WC1E 7HU. REPRODUCTION University of London theses may not be reproduced without explicit written permission from the University of London Library. Enquiries should be addressed to the Theses Section of the Library. Regulations concerning reproduction vary according to the date of acceptance of the thesis and are listed below as guidelines. A. Before 1962. Permission granted only upon the prior written consent of the author. (The University Library will provide addresses where possible). B. 1962 - 1974. In many cases the author has agreed to permit copying upon completion of a Copyright Declaration. C. 1975- 1988. Most theses may be copied upon completion of a Copyright Declaration. D. 1989 onwards. Most theses may be copied. This thesis comes within category D. □ This copy has been deposited in the Library of (a C L This copy has been deposited in the University of London Library, Senate □ House, Malet Street, London WC1E 7HU. Bound By Blissett Bookbinders 020 8992 3965 www.blissetts.com j OCX Department of Anatomy and Developmental Biology, and University Department of Surgery Royal Free and University College Medical School University College London A Gene Therapy Approach for Treating Muscle Degeneration in Neuromuscular Disorders Using IGF-I Splice Variants Kenan Ates, MD, MSc Supervised by Prof Geoffrey Goldspink, PhD Dr Mark P. Lewis, PhD Dr Paul Simons, PhD A thesis submitted for the degree of Doctor of Philosophy to the University of London - May 2007 UMI Number: U591290 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U591290 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 PhD Thesis Author: Kenan Ates Title: A Gene Therapy Approach for Treating Muscle Degeneration in Neuromuscular Disorders Using IGF-I Splice Variants Laboratory I Institution: Molecular Tissue Repair Unit, Department of Anatomy and Developmental Biology, University Department of Surgery, Royal Free and University College Medical School, University College London Submission to:University of London in May 2007 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 iv 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. V Acknowledgements I would like to thank my supervisors Prof Geoffrey Goldspink, Dr Mark P. Lewis and Dr Paul Simons for their help and advice. I must express my sincere gratitude to Prof G. Goldspink for that he kindly accepted me into the PhD programme, and he has always provided me great support and help thrawout the study. Dr M. Lewis was also very helpful and supportive, particularly in in-vitro cell culture part of the study. I would like to extend my special acknowledgement to Dr Shi-Yu Yang for his warm friendship, great scientific and technical advice and support in all stages of my study. I wish also to thank all research staff, both in Molecular Tissue Repair Unit in Department of Anatomy and Developmental Biology / University Department of Surgery in the Hampstead Campus of Royal Free and University College Medical School, and in Department of Materials and Tissue Engineering at Eastman Dental Institute. I specially thank Dr Andrea Sinanan of Eastman Dental Institute, and Drs Cristiana Velloso and Jeanny Weaker of Molecular Tissue Repair Unit, for their kindness, warmness, help and friendships. On the other hand, I thank my wife, Nuray Sancar for her support and patience. I also thank my dear friends for their great supports, encouragements and helps. I must particularly mention one of them, Taylan Sahbaz. I send my special acknowledgement to Medical Research Council for its financial support, which this study could not have been possible without it. vi Contents Pages C O V E R P A G E .................................................................................................................................................... I T IT L E P A G E ...................................................................................................................................................... II ABSTRACT ......................................................................................................................................................... Ill ACKNOWLEDGEMENTS ........................................................................................................................... VI CONTENTS .......................................................................................................................................................
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