Exon Skipping: Methods and Protocols PDF
Preview Exon Skipping: Methods and Protocols
M M B ™ ETHODS IN OLECULAR IOLOGY Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Exon Skipping Methods and Protocols Edited by Annemieke Aartsma-Rus Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Editor Annemieke Aartsma-Rus Department of Human Genetics Leiden University Medical Center Leiden, The Netherlands ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-766-8 e-ISBN 978-1-61779-767-5 DOI 10.1007/978-1-61779-767-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012933585 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or d issimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com) Preface It was discovered back in 1977 that for most genes the genetic code is dispersed over the gene. Before the messenger RNA can be translated into protein noncoding intron frag- ments have to be removed from RNA transcripts during a process called splicing. This complex process is coordinated by the splicing machinery, which consists of hundreds of proteins, and sequence motifs in introns and exons are important for recognition by splic- ing factors and proper processing of pre-mRNA into mRNA. Some exons are not always included in the mRNA depending, e.g., on the developmental state of an organism or the type of tissue (alternative splicing). Soon after the discovery of splicing, it became apparent that genetic mutations affecting splicing motifs or introducing “false” splicing motifs can disrupt splicing and underlie many genetic diseases. In addition, the disruption of alterna- tive splicing can give rise to or exacerbate genetic and acquired disease processes. Due to their larger size, introns are generally not included in standard diagnostic pro- tocols. Nevertheless, for multiple diseases it has been shown that deep intronic mutations can activate false splice sites, leading to the aberrant inclusion of a piece of intron into the mRNA. Furthermore, previously silent mutations (or substitutions) within an exon were often thought to be polymorphic. Now, it is recognized that these mutations can also cause an exon to be no longer recognized by the splicing machinery, leading to exon skipping. Even though the splicing process has not been elucidated completely, it is possible to intentionally manipulate it. This can be achieved by preventing binding of splicing factors to their respective motifs, e.g., using chemical compounds, modifi ed pieces of RNA or DNA (antisense oligonucleotides) or through expression of a small nuclear ribonucleopro- tein in which the natural antisense part is replaced with an antisense sequence targeting the splicing motif. These tools all induce “skipping” of the targeted exon and can be used to prevent the inclusion of an aberrant exon, to modify levels of alternatively spliced exons or to decrease protein expression levels by skipping an exon. I felt that a Methods book on exon skipping was timely for two reasons: (1) Now that “next generation” sequencing techniques allow a more detailed analysis of exons and introns in multiple genes at the same time, many alterations will be identifi ed in the near future for which the impact on splicing is uncertain. Methodology on how to assess this will be crucial to discriminate “real polymorphisms” from mutations that affect splicing. (2) Antisense-mediated exon skipping is currently tested in phase 3 clinical trials for Duchenne muscular dystrophy and the encouraging results in this fi eld have incited many groups to apply the antisense-mediated exon skipping approach to their own favorite gene(s). Methodology on how to go about this should facilitate obtaining proof of concept for new exon skipping applications and prevent duplication of errors that have been made already by others (after all as Einstein pointed out: “An expert is someone who has made all pos- sible mistakes in a limited fi eld.”). v vi Preface While for mutations that potentially lead to exon skipping a lot can be achieved with a limited set of protocols (Chaps. 1 – 5 ), for the intentional induction of exon skipping differ- ent tools and target genes and the translational path from in vitro splicing to in vivo tests in animal models are involved, requiring a more extensive set of protocols (Chaps. 6 – 27 ). The Methods chapters are written by experts who have hands on experience with the described protocols and who provide the readers with useful tips and tricks of the trade in the form of extensive Notes sections in each chapter, as is usual in the Methods in Molecular Biology series. The book also contains a number of overview chapters to provide the reader with a frame of reference for the exon skipping fi eld as well as some more background. This book is intended for researchers working or considering to start working in the exon skipping fi eld, and hopefully will also serve as a reference document to expert scientists in the exon skipping fi eld in academia or industry. While I am aware it is impossible to cover all spectra involved in exon skipping, I did my best to provide the reader with a manual as complete as possible and it is my sincerest hope that this book will prove useful to its readers. I would like to thank all authors for their excellent contributions and Prof. John Walker for his assistance during the editorial process. Leiden, The Netherlands Annemieke Aartsma-Rus Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I MUTATIONS 1 DNA Diagnostics and Exon Skipping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Umasuthan Srirangalingam and Shern L. Chew 2 Bioinformatics and Mutations Leading to Exon Skipping . . . . . . . . . . . . . . . . . . . . 17 F.O. Desmet and C. Béroud 3 Minigenes to Confirm Exon Skipping Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Lourdes R. Desviat, Belén Pérez, and Magdalena Ugarte 4 Analysis and Interpretation of RNA Splicing Alterations in Genes Involved in Genetic Disorders. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Maaike P.G. Vreeswijk and Heleen M. van der Klift 5 Exon Skipping Mutations in Neurofibromatosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Emanuele Buratti and Diana Baralle PART II TOOLS TO INDUCE EXON SKIPPING 6 Overview on Applications of Antisense-Mediated Exon Skipping. . . . . . . . . . . . . . . 79 Willeke M.C. van Roon-Mom and Annemieke Aartsma-Rus 7 Overview on DMD Exon Skipping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Annemieke Aartsma-Rus 8 Overview on AON Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Annemieke Aartsma-Rus 9 Optimizing RNA/ENA Chimeric Antisense Oligonucleotides Using In Vitro Splicing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Yasuhiro Takeshima, Mariko Yagi, and Masafumi Matsuo 10 Optimizing Antisense Oligonucleotides Using Phosphorodiamidate Morpholino Oligomers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Linda J. Popplewell, Alberto Malerba, and George Dickson 11 Optimizing Splice-Switching Oligomer Sequences Using 2¢-O-Methyl Phosphorothioate Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Carl Adkin, Sue Fletcher, and Steve D. Wilton 12 Exon Skipping Quantification by Real-Time PCR. . . . . . . . . . . . . . . . . . . . . . . . . . 189 Alessandra Ferlini and Paola Rimessi 13 Antisense-Mediated Exon Skipping to Shift Alternative Splicing to Treat Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 Jing Wan vii viii Contents 14 Antisense-Mediated Exon Skipping to Generate Soluble Receptors. . . . . . . . . . . . . 209 A. Seda Yilmaz-Elis and J. Sjef Verbeek 15 Antisense-Mediated Exon Skipping to Reframe Transcripts. . . . . . . . . . . . . . . . . . . 221 Sandrina Turczynski, Matthias Titeux, Nathalie Pironon, and Alain Hovnanian 16 U1 snRNA as an Effective Vector for Stable Expression of Antisense Molecules and for the Inhibition of the Splicing Reaction. . . . . . . . . . . . . . . . . . . . 239 Julie Martone, Fernanda Gabriella De Angelis, and Irene Bozzoni 17 Engineering U7snRNA Gene to Reframe Transcripts . . . . . . . . . . . . . . . . . . . . . . . 259 Aurélie Goyenvalle 18 Dynamic Fluorescent and Luminescent Reporters for Cell-Based Splicing Screens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273 Claude C. Warzecha, Ruben Hovhannisyan, and Russ P. Carstens 19 Antisense-Mediated Exon-Skipping to Induce Gene Knockdown . . . . . . . . . . . . . . 289 Petra Disterer and Bernard Khoo 20 Antisense-Mediated Exon Inclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 Yimin Hua and Adrian R. Krainer 21 Antisense Genes to Induce Exon Inclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325 Rachel Nlend Nlend and Daniel Schümperli 22 Using Mini-genes to Identify Factors That Modulate Alternative Splicing. . . . . . . . 349 Robert Morse, Adrian G. Todd, and Philip J. Young PART III DELIVERY 23 Overview of Alternative Oligonucleotide Chemistries for Exon Skipping. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 Amer F. Saleh, Andrey A. Arzumanov, and Michael J. Gait 24 Identification of Peptides for Tissue-Specific Delivery. . . . . . . . . . . . . . . . . . . . . . . 379 Hans Heemskerk 25 Systemic Delivery of Antisense Oligomer in Animal Models and Its Implications for Treating DMD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Qi Long Lu and Bo Wu 26 Cell-Penetrating Peptides Enhance Systemic Delivery of Antisense Morpholino Oligomers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407 Hong M. Moulton 27 Optimizing Tissue-Specific Antisense Oligonucleotide–Peptide Conjugates. . . . . . . 415 Corinne A. Betts, Suzan M. Hammond, Hai-fang Yin, and Matthew J.A. Wood Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437 Contributors ANNEMIEKE AARTSMA-RUS (cid:129) Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands CARL ADKIN (cid:129) Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth , Australia FERNANDA GABRIELLA DE ANGELIS (cid:129) Department of Biology and Biotechnology “Charles Darwin”, “Sapienza” University of Rome, Rome , Italy ANDREY A. ARZUMANOV (cid:129) Laboratory of Molecular Biology , Medical Research Council, Cambridge, UK DIANA BARALLE (cid:129) Human Genetics Division, University of Southampton, Southampton General Hospital, Southampton, UK C. BÉROUD (cid:129) UMR_S 910, INSERM, Faculté de Médecine de la Timone, Marseille, France; UMR_S 910, Université de la Méditerranée, Faculté de Médecine de la Timone, Marseille, France; AP-HM, Département de Génétique Médicale, Hôpital d’enfants Timone, Marseille, France CORINNE A. BETTS (cid:129) Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford , UK IRENE BOZZONI (cid:129) Department of Biology and Biotechnology “Charles Darwin”, “Sapienza” University of Rome, Rome , Italy EMANUELE BURATTI (cid:129) Department of Molecular Pathology , ICGEB , Trieste , Italy RUSS P. CARSTENS (cid:129) Departments of Medicine and genetics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA , USA SHERN L. CHEW (cid:129) St Bartholomew’s Hospital, London , UK F.O. DESMET (cid:129) INSERM U1052 CNRS 5286, Lyon , France LOURDES R. DESVIAT (cid:129) Centro de Diagnóstico de Enfermedades Moleculares , Madrid , Spain; Centro de Biología Molecular Severo Ochoa , Madrid , Spain; UAM-CSIC, Universidad Autónoma de Madrid, Madrid , Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) , Madrid , Spain GEORGE DICKSON (cid:129) School of Biological Sciences, Royal Holloway, University of London, London , UK PETRA DISTERER (cid:129) Department of Endocrinology , UCL Medical School, London, UK ALESSANDRA FERLINI (cid:129) Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara , Italy SUE FLETCHER (cid:129) Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth , Australia MICHAEL J. GAIT (cid:129) Laboratory of Molecular Biology , Medical Research Council, Cambridge, UK ix x Contributors AURÉLIE GOYENVALLE (cid:129) MRC Functional Genomics Unit, Department of Physiology, Anatomy & Genetics, University of Oxford, Oxford , UK SUZAN M. HAMMOND (cid:129) Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford , UK HANS HEEMSKERK (cid:129) Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands RUBEN HOVHANNISYAN (cid:129) Department of Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA , USA ALAIN HOVNANIAN (cid:129) INSERM, U781, Paris , France; U niversity Paris V René Descartes, Paris , France; D epartments of Dermatology and genetics, Necker hospital for sick children, Paris , France YIMIN HUA (cid:129) Cold Spring Harbor Laboratory , Cold Spring Harbor, NY , USA BERNARD KHOO (cid:129) Department of Endocrinology , UCL Medical School, London, UK HELEEN M. VAN DER KLIFT (cid:129) Department of Clinical Genetics, Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands ADRIAN R. KRAINER (cid:129) Cold Spring Harbor Laboratory , Cold Spring Harbor, NY , USA QI LONG LU (cid:129) McColl-Lockwood Laboratory for Muscular Dystrophy Research, Neuromuscular/ALS Center, Carolinas Medical Center, Charlotte , NC , USA ALBERTO MALERBA (cid:129) School of Biological Sciences, Royal Holloway, University of London, London , UK JULIE MARTONE (cid:129) Department of Biology and Biotechnology “Charles Darwin”, “Sapienza” University of Rome, Rome , Italy MASAFUMI MATSUO (cid:129) Department of Pediatrics, Kobe University Graduate School of Medicine, Kobe , Japan ROBERT MORSE (cid:129) Clinical Neurobiology, Peninsula Medical School, University of Exeter, Exeter, UK HONG M. MOULTON (cid:129) Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis , OR , USA RACHEL NLEND NLEND (cid:129) Institute of Cell Biology, University of Bern, Bern , Switzerland BELÉN PÉREZ (cid:129) Centro de Diagnóstico de Enfermedades Moleculares , Madrid , Spain; Centro de Biología Molecular Severo Ochoa , Madrid , Spain; UAM-CSIC, Universidad Autónoma de Madrid, Madrid , Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid , Spain NATHALIE PIRONON (cid:129) INSERM, U781, Paris , France; U niversity Paris V René Descartes, Paris , France LINDA J. POPPLEWELL (cid:129) School of Biological Sciences, Royal Holloway, University of London, London , UK PAOLA RIMESSI (cid:129) Section of Medical Genetics, Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara , Italy WILLEKE M.C. VAN ROON-MOM (cid:129) Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands AMER F. SALEH (cid:129) Laboratory of Molecular Biology , Medical Research Council, Cambridge, UK DANIEL SCHÜMPERLI (cid:129) Institute of Cell Biology, University of Bern, Bern , Switzerland UMASUTHAN SRIRANGALINGAM (cid:129) St Bartholomew’s Hospital, London , UK
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