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A Cell-Based Gene Therapy Approach for Dysferlinopathy Using Sleeping Beauty Transposon

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A Cell-Based Gene Therapy Approach for Dysferlinopathy Using Sleeping Beauty Transposon A cell-based gene therapy approach for dysferlinopathy using Sleeping Beauty transposon Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by HELENA ESCOBAR FERNÁNDEZ from León, Spain August 2015 This work was prepared from October 2011 to July 2015 under the supervision of Dr. Zsuzsanna Izsvák (Max-Delbrück Center for Molecular Medicine, Berlin, Germany) and Prof. Simone Spuler (Experimental and Clinical Research Center, Berlin, Germany). All the experiments were conducted in the laboratories of Dr. Izsvák and Prof. Spuler. 1st reviewer: Prof. Dr. med. Simone Spuler Institute for Chemistry and Biochemistry Department of Biology, Chemistry and Pharmacy Freie Universität, Berlin and Department of Muscle Sciences and University Outpatient Clinic for Muscle Disorders Experimental and Clinical Research Center, Berlin 2nd reviewer: Prof. Dr. Sigmar Stricker Institute for Chemistry and Biochemistry Department of Biology, Chemistry and Pharmacy Freie Universität, Berlin and Development and Disease Group Max Planck Institute of Molecular Genetics, Berlin Date of Defense: November 18th, 2015 Preface The experiments with human muscle fiber fragments were done in collaboration with Dr. Andreas Marg, from the laboratory of Prof. Simone Spuler. Dr. Marg performed the isolation of human fiber fragments and the full characterization of the fiber fragment culture model. I performed the mouse irradiation and transplantation of human fiber fragments into mice. Dr. Marg also performed the mouse irradiation and the analysis of all grafted muscles. i ii Acknowledgements First of all, I want to thank Dr. Zsuzsanna Izsvák for supervising this work and giving me opportunity to carry out my PhD in her laboratory. I want to thank Dr. Luis García for agreeing to be my MyoGrad co-supervisor from Paris. I want to thank Prof. Simone Spuler for her supervision, support and encouragement, especially during the last two years of my PhD. I want to thank MyoGrad for the economic support not only to my doctoral studies but to a great number of local and international scientific events. I especially want to thank Susanne Wissler for her help with organizational issues. I also want to thank AFM-Téléthon for supporting me with a fourth year scholarship to complete my PhD. I want to thank Dr. Andreas Marg for being an excellent mentor, for his constructive criticism, for being always open to helpful discussions and for sharing his solid expertise with me. It made me a better scientist. I want to thank my colleagues in the lab of Zsuzsanna Izsvák for making work days fun. Especially, I want to thank Dr. Attila Szvetnik and Dr. Enikö Eva Nagy for many scientific and non-scientific late evening discussions, for being interested and always sincerely willing to help me and others. I want to thank Dr. Csaba Miskey, for being the best bench neighbor when I arrived at the lab. I want to thank Dr. Anantharam Devaraj for being a very friendly presence in my early days in the lab and for sharing his knowledge in transposons and molecular biology techniques. I want to thank Dr. Anna Osiak and Dr. Sanam Bashir and her family for their open minds and their friendship, advice and the fun times spent together. I want to thank Ana Jiménez Orgaz for being a great colleague and friend as well as the best companion to explore Berlin. I want to thank Angélica García Pérez and Julia Rugor for being the fellow PhD students and friends that prevailed with me until the end. For all the favors, the help, the discussions, the support, the fun, the trips, the trust. I especially want to thank Angélica for her incredible generosity and the huge amount of help she has given me. I want to thank Dr. Esther Grueso, Marta Holstein (to me, Pinito) and Alexander Holstein, for making me feel at home during my first months in Berlin. I want to thank Esther for singing in the cell culture in the evening and making it so easy to work until late, for being a great friend and my most thorough judge of scientific posters and applications. I really miss all of those who have left Berlin but I am thankful that they have given me such great memories of this time. From the lab of Simone Spuler, I want to thank Adrienne Rothe for her excellent technical support and for helping me with the mouse irradiation. I want to thank Kornelia Gräning for introducing me to the muscle histology techniques and for performing the histological stains for iii this work. I want to thank Stephanie Meyer-Liesener and Dr. Verena Schöwel for their help in the immunoblot analysis of some samples. I also want to thank Verena for her help with the applications for animal experiments. I want to thank Dr. Markus Kufeld and Diana Pasemann from the Cyberknife center of the Charité for their help with the mouse irradiation. I want to thank my friend Alena for being my family in Berlin, for her immense friendship and strong support and for sharing all good and bad moments here. Last, I want to thank my wonderful family, especially my parents Rosi and Félix, and my sister Laura. I want to thank them for encouraging me to look forward and always keep learning, for understanding my itinerant life style, and for taking part in it, for being always there. iv Table of contents PREFACE ........................................................................................................................................ I ACKNOWLEDGEMENTS .......................................................................................................... III TABLE OF CONTENTS ............................................................................................................... V SUMMARY .................................................................................................................................... IX ZUSAMMENFASSUNG ............................................................................................................... XI LIST OF FIGURES ..................................................................................................................... XIII LIST OF TABLES ........................................................................................................................ XV LIST OF ABBREVIATIONS .................................................................................................... XVII 1 INTRODUCTION .................................................................................................................. 1 1.1 DYSFERLINOPATHY .......................................................................................................... 1 1.1.1 Clinical and histological aspects of dysferlinopathy .................................................... 1 1.1.1.1 The distal myopathies – MM and DACM ......................................................................... 1 1.1.1.2 The limb girdle muscular dystrophies – LGMD2B .......................................................... 2 1.1.2 Genetic basis of dysferlinopathy................................................................................... 3 1.1.2.1 Discovery of the dysferlin gene ....................................................................................... 3 1.1.2.2 DYSF mutations and lack of genotype-phenotype correlation .......................................... 3 1.2 DYSFERLIN........................................................................................................................ 4 1.2.1 DYSF gene and dysferlin transcript variants ............................................................... 4 1.2.2 Dysferlin protein ........................................................................................................... 5 1.2.2.1 Dysferlin domain structure................................................................................................ 5 1.2.2.2 Dysferlin protein localization in skeletal muscle .............................................................. 6 1.2.2.3 Dysferlin function in membrane repair ............................................................................. 6 1.2.2.4 T-tubule associated functions .......................................................................................... 10 1.2.2.5 Dysferlin, immunity and inflammation ........................................................................... 11 1.2.2.6 Dysferlin function in muscle growth, regeneration and myoblast fusion........................ 12 1.2.2.7 Dysferlin function in the heart ........................................................................................ 13 1.2.2.8 Other functions of dysferlin ............................................................................................ 13 1.3 SKELETAL MUSCLE REGENERATION ............................................................................... 13 1.3.1 Satellite cells ............................................................................................................... 15 1.3.2 Regenerative potential of transplanted satellite cells ................................................. 17 1.3.3 Clinical trials of myoblast transplantation for muscular dystrophy .......................... 18 1.3.4 Contribution of other cell types to skeletal muscle regeneration ............................... 18 1.3.4.1 Bone marrow stem cells .................................................................................................. 18 1.3.4.2 Muscle side population (SP) cells ..................................................................................
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