M E T H O D S I N M O L E C U L A R B I O L O G Y

Series EditorJohn M. Walker

School of Life SciencesUniversity of Hertfordshire

Hat fi eld, Hertfordshire, AL10 9AB, UK

For further volumes: http://www.springer.com/series/7651

RNA Interference

Challenges and Therapeutic Opportunities

Edited by

Mouldy SioudInstitute for Cancer Research, Oslo University Hospital, Oslo, Norway

ISSN 1064-3745 ISSN 1940-6029 (electronic)ISBN 978-1-4939-1537-8 ISBN 978-1-4939-1538-5 (eBook) DOI 10.1007/978-1-4939-1538-5 Springer New York Heidelberg Dordrecht London

Library of Congress Control Number: 2014949774

© Springer Science+Business Media New York 2015 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. Exempted from this legal reservation are brief excerpts in connection with reviews or scholarly analysis or material supplied specifi cally for the purpose of being entered and executed on a computer system, for exclusive use by the purchaser of the work. Duplication of this publication or parts thereof is permitted only under the provisions of the Copyright Law of the Publisher’s location, in its current version, and permission for use must always be obtained from Springer. Permissions for use may be obtained through RightsLink at the Copyright Clearance Center. Violations are liable to prosecution under the respective Copyright Law. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specifi c statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. While the advice and information in this book are believed to be true and accurate at the date of publication, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

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Editor Mouldy Sioud Institute for Cancer ResearchOslo University Hospital Oslo , Norway

v

Subsequent to the discovery that small interfering RNAs (siRNAs) mimicking the Dicer cleavage products can silence mammalian genes, RNA interference (RNAi) has become the experimental tool of choice to suppress gene expression in a wide variety of cells and organisms. The catalytic potency of siRNAs lies in the key discovery that endogenous RNAi gene silencing machinery can be hijacked to artifi cially knock down genes of inter-est. Today, RNAi has also become a method of choice for key steps in the development of therapeutic agents, from target discovery and validation to the analysis of the mechanisms of action of small molecules. To date, several strategies have been devised to trigger the RNAi pathway, each of which is adapted and optimized for different cell types. Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of therapeutic siRNAs devoted to off-target effects is still a challenging task. Also strategic success of therapeutic siRNA or micro (mi)RNAs will depend on the development of versatile delivery systems due to the poor cellular uptake of naked RNA molecules. The purpose of this book is to provide readers with the recent advances in siRNA design, delivery, targeting, and methods to minimize siRNA unwanted effects. Preclinical and clinical use of synthetic siRNAs, the roles of miR-NAs in cancer, and the promise of extracellular miRNAs for diagnosis are also covered in this issue along with novel methods for identifying endogenous siRNAs and annotation of small RNA transcriptomes.

To design an effective siRNA sequence, one must consider the base composition of the chosen target site and whether it will be accessible. Delivery methods should be also con-sidered when dealing with primary cells, preclinical, and clinical studies. Chapter 1 critically reviews several parameters such as design, delivery, and chemical modifi cations that are important for successful siRNA applications. Nanostructured RNAs capable of inducing RNAi and new shRNA constructs are described in Chapters 2 and 3 , respectively. With respect to delivery, new siRNA formulations, including polymers, PGLA microspheres, magnetic nanoparticles, microwell-based transfection as well as bacteria-based approaches, are adequately described in Chapters 4 – 8 .

The effi cacy and safety of siRNA drugs heavily depends on their delivery to the intended target. Selectively targeting siRNAs to diseased cells or tissues increases their accumulation at the site of interest, thus increasing the silencing potency and limiting toxicity to normal tissues. While Chapter 9 deals with direct injection of siRNAs into tumors, Chapter 10 describes a strategy for siRNA delivery to hepatocytes for the treat-ment of chronic hepatitis B virus infection. Interestingly, the addition of a hepatocyte-targeted endosome-releasing agent enhances gene silencing. Local delivery of siRNAs avoids systemic exposure and reduces the likelihood of unexpected harmful effects else-where in the body as results of the unwanted siRNA effects. Chapter 11 deals with the development of aptamers, which are in vitro-evolved RNA or DNA oligomers that bind to target ligands with a high degree of specifi city. Chapter 12 critically reviews the currently used targeting strategies for directing siRNA molecules to desired cells, including peptides, antibodies, and CpG oligonucleotides.

Pref ace

vi

Being RNA, siRNAs are prone to nuclease-mediated degradation in biological fl uids, which has a negative impact on their use in patients. Chapters 13 and 14 describe the devel-opment of nuclease-resistant siRNAs with the potential to progress into a new class of therapeutic drugs against virus-related diseases and cancers.

Notably, the enthusiasm for siRNA-based therapies is refl ected by the large number of pharmaceutical companies pursuing this strategy. siRNA-targeted therapies could also be used to enhance or to prevent resistance to standard chemotherapeutic agents or other biological agents. Chapter 15 describes the design of a new vaccine formulation (dendritic cells loaded with tumor antigens that have been transfected with an interleukin-10 siRNA) capable of killing leukemic cells in a rat model of acute myeloid leukemia. Chapters 16 and 17 describe the use of siRNA in cancer patients. Reprogramming DC vaccines with siRNA targeting immunosuppressive factors enhanced DC function and clinical responses in patients with ovarian cancer. Moreover, targeting bcr-abl transcripts in a patient with chronic myeloid leukemia with siRNA resulted in inhibition of BCR-ABL, which led to chronic myeloid leukemia cell apoptosis without any associated adverse effects that could be ascribed to the siRNA drug. These clinical studies should facilitate the progression of synthetic siRNA-based drugs to clinical trials.

Although siRNAs are known to induce specifi c degradation of the target RNA in a sequence-dependent manner, some concern has been raised about the specifi city of gene silencing. To overcome this challenge, multiple statistical and computational models have been proposed in recent years to design functional siRNAs. Regardless of the method used, in my opinion unwanted effects mediated by both the sense and antisense siRNA strands cannot be eliminated. Chapters 18 and 19 provide researchers with reliable means to mini-mize off- targeting concerns associated with RNAi experiments.

Small noncoding RNA molecules such as miRNAs, are abundantly expressed in all cell types and are involved in the regulation of key cellular process such as metabolism, prolif-eration, DNA repair, apoptosis, and differentiation. Dysregulation of certain miRNAs expression in the cell was consistently observed during certain pathologies including can-cers. Chapter 20 critically reviews the miRNA fi eld and describes a method for gene silenc-ing using intronic miRNAs. Chapters 21 – 23 describe new strategies for in silico identifi cation for novel endogenous siRNAs, annotation of small RNA transcriptomes, and a miRNA capture affi nity method, respectively. Chapters 24 and 25 critically review the involvement of miRNAs in cancers and critically discuss the rationale and the strategies for the therapeu-tic targeting of miRNAs.

Recent studies have shown that, in addition to being expressed endogenously, miRNAs can be secreted from cells. Most of these extracellular miRNAs are associated with lipid car-riers known as exosomes, which are membrane-derived vesicles. A signifi cant amount of miRNAs were detected in all biological fl uids including blood plasma, urine, and cerebro-spinal fl uids. The diagnostic potential of extracellular miRNAs for liver injury, cardiovascu-lar, neurological, autoimmune, infl ammatory, and metabolic diseases has been documented by several studies. Chapter 26 describes the recent advances in using urinary miRNAs as a new class of noninvasive biomarkers in oncology, nephrology, and cardiology.

Circulating exosome-containing small RNAs have been demonstrated in vitro to be taken up by recipient cells and to alter gene expression through RNAi. Chapter 27 describes an immunomagnetic method for the isolation of exosomes from human cell lines. This improved isolation method should have utility in exosome characterization and clinical applications. Previous work has shown that small RNA such as siRNAs and exosome-derived miRNAs can bind to Toll-like receptors (TLR)-7/8 resulting in the activation of innate

Preface

vii

immunity. Chapter 28 describes the design of 2’-modifi ed small RNAs capable of blocking TLR-7/8 signaling and thus functioning as antagonists. Given the involvement of TLRs in infl ammatory diseases, the development of TLR antagonists might have clinical applications.

Topics covered in this volume will be of interest to researchers, clinicians, teachers, and biotech companies interested in RNA-based therapies. I would like to thank the authors for their contributions, the series editor John Walker, and all those involved at Springer for the production of the book. It is my hope that you will fi nd the book informative and a valuable addition to your textbook and laboratory bookshelf.

Oslo, Norway Mouldy Sioud

Preface

ix

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii

1 RNA Interference: Mechanisms, Technical Challenges, and Therapeutic Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Mouldy Sioud

2 Nanostructured RNAs for RNA Interference. . . . . . . . . . . . . . . . . . . . . . . . . . 17 Yuko Nakashima, Naoko Abe, Yoshihiro Ito, and Hiroshi Abe

3 One Long Oligonucleotide or Two Short Oligonucleotides Based shRNA Construction and Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Xue-jun Wang and Sheng-qi Wang

4 PLGA Microspheres Encapsulating siRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Giuseppe De Rosa and Giuseppina Salzano

5 Magnetic Nanoparticle and Magnetic Field Assisted siRNA Delivery In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Olga Mykhaylyk, Yolanda Sanchez-Antequera, Dialechti Vlaskou, Maria Belen Cerda, Mehrdad Bokharaei, Edelburga Hammerschmid, Martina Anton, and Christian Plank

6 Cytoplasmic Delivery of siRNAs to Monocytes and Dendritic Cells via Electroporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Mouldy Sioud

7 Delivery of siRNAs to Cancer Cells via Bacteria. . . . . . . . . . . . . . . . . . . . . . . . 117 Omar Ahmed, Andrea Krühn, and Hermann Lage

8 Microwell Array-Mediated Delivery of Lipoplexes Containing Nucleic Acids for Enhanced Therapeutic Efficacy . . . . . . . . . . . . . . . . . . . . . . 131 Yun Wu, Daniel Gallego-Perez, and L. James Lee

9 Modulating the Tumor Microenvironment with RNA Interference as a Cancer Treatment Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Karin Zins, Mouldy Sioud, Seyedhossein Aharinejad, Trevor Lucas, and Dietmar Abraham

10 Targeted In Vivo Delivery of siRNA and an Endosome- Releasing Agent to Hepatocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Magdolna G. Sebestyén, So C. Wong, Vladimir Trubetskoy, David L. Lewis, and Christine I. Wooddell

11 Cell-Internalization SELEX: Method for Identifying Cell- Internalizing RNA Aptamers for Delivering siRNAs to Target Cells . . . . . . . . . . . . . . . . . . . 187 William H. Thiel, Kristina W. Thiel, Katie S. Flenker, Tom Bair, Adam J. Dupuy, James O. McNamara II, Francis J. Miller, and Paloma H. Giangrande

x

12 Strategies for siRNA Navigation to Desired Cells. . . . . . . . . . . . . . . . . . . . . . . 201 Mouldy Sioud

13 Use of Guanidinopropyl-Modified siRNAs to Silence Gene Expression . . . . . . 217 Maximilian C.R. Buff, Stefan Bernhardt, Musa D. Marimani, Abdullah Ely, Joachim W. Engels, and Patrick Arbuthnot

14 Therapy of Respiratory Viral Infections with Intranasal siRNAs . . . . . . . . . . . . 251 Sailen Barik and Patrick Lu

15 Engineering Therapeutic Cancer Vaccines That Activate Antitumor Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 Per Ole Iversen and Mouldy Sioud

16 Immunosuppressive Factor Blockade in Dendritic Cells via siRNAs Results in Objective Clinical Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 Mouldy Sioud, Anne Mobergslien, and Stein Sæbøe-Larssen

17 Targeting bcr-abl Transcripts with siRNAs in an Imatinib- Resistant Chronic Myeloid Leukemia Patient: Challenges and Future Directions . . . . . . 277 Michael Koldehoff

18 A Facile Method for Interfering with Off-Target Silencing Mediated by the Sense Strand. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Anne Mobergslien and Mouldy Sioud

19 Overcoming the Challenges of siRNA Activation of Innate Immunity: Design Better Therapeutic siRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301 Mouldy Sioud

20 Gene Silencing In Vitro and In Vivo Using Intronic MicroRNAs . . . . . . . . . . 321 Jia Han Deng, Peter Deng, Shi-Lung Lin, and Shao-Yao Ying

21 In Silico Identification of Novel Endo-siRNAs . . . . . . . . . . . . . . . . . . . . . . . . 341 Andrew Schuster, Grant W. Hennig, Nicole Ortogero, Dickson Luong, and Wei Yan

22 Computer-Assisted Annotation of Small RNA Transcriptomes. . . . . . . . . . . . . 353 Nicole Ortogero, Grant W. Hennig, Dickson Luong, and Wei Yan

23 miR-CATCH: MicroRNA Capture Affinity Technology . . . . . . . . . . . . . . . . . 365 Sebastian Vencken, Tidi Hassan, Noel G. McElvaney, Stephen G.J. Smith, and Catherine M. Greene

24 Roles of MicroRNAs in Cancers and Development . . . . . . . . . . . . . . . . . . . . . 375 Shigeru Takasaki

25 Targeting MicroRNAs to Withstand Cancer Metastasis . . . . . . . . . . . . . . . . . . 415 Valentina Profumo, Valentina Doldi, Paolo Gandellini, and Nadia Zaffaroni

26 Urinary MicroRNAs as a New Class of Noninvasive Biomarkers in Oncology, Nephrology, and Cardiology . . . . . . . . . . . . . . . . . . . . . . . . . . . 439 Hana Mlcochova, Renata Hezova, Albano C. Meli, and Ondrej Slaby

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xi

27 Magnetic Bead-Based Isolation of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . 465 Morten P. Oksvold, Axl Neurauter, and Ketil W. Pedersen

28 Modified 2′-Ribose Small RNAs Function as Toll-Like Receptor-7/8 Antagonists. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483 Mouldy Sioud

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

Contents

xiii

HIROSHI ABE • Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, Japan; Nano Medical Engineering Laboratory , RIKEN, Saitama , Japan ; PRESTO , Japan Science and Technology Agency , Saitama , Japan

NAOKO ABE • Nano Medical Engineering Laboratory , RIKEN, Saitama , Japan DIETMAR ABRAHAM • Laboratory for Cardiovascular Research, Center for Anatomy

and Cell Biology , Medical University of Vienna , Vienna , Austria SEYEDHOSSEIN AHARINEJAD • Laboratory for Cardiovascular Research, Center for Anatomy

and Cell Biology , Medical University of Vienna , Vienna , Austria OMAR AHMED • Charité Campus Mitte , Institute of Pathology , Berlin , Germany MARTINA ANTON • Institute of Experimental Oncology , Klinikum rechts der Isar der

Technischen Universität München , Munich , Germany PATRICK ARBUTHNOT • Health Sciences Faculty, Antiviral Gene Therapy Research Unit

and African Network for Drugs and Diagnostics Innovation (ANDI) Centre of Excellence, School of Pathology , University of the Witwatersrand , Johannesburg , South Africa

TOM BAIR • Department of Internal Medicine , University of Iowa , Iowa City , IA , USA SAILEN BARIK • Department of Biological, Geological and Environmental Sciences,

Center for Gene Regulation in Health and Disease , Cleveland State University , Cleveland , OH , USA

STEFAN BERNHARDT • Institute of Organic Chemistry and Chemical Biology , Goethe- University , Frankfurt am Main , Germany

MEHRDAD BOKHARAEI • Faculty of Pharmaceutical Sciences , The University of British Columbia , Vancouver , BC , Canada

MAXIMILIAN C.R. BUFF • Institute of Organic Chemistry and Chemical Biology , Goethe-University , Frankfurt am Main , Germany

MARIA BELEN CERDA • National Atomic Energy Commission , Buenos Aires , Argentina JIA HAN DENG • Department of Cell and Neurobiology, Keck School of Medicine ,

University of Southern California , Los Angeles , CA , USA PETER DENG • Department of Cell and Neurobiology, Keck School of Medicine ,

University of Southern California , Los Angeles , CA , USA VALENTINA DOLDI • Molecular Pharmacology Unit, Department of Experimental

Oncology and Molecular Medicine , Fondazione IRCCS Istituto Nazionale dei Tumori , Milan , Italy

ADAM J. DUPUY • Anatomy and Cell Biology , University of Iowa , Iowa City , IA , USA ABDULLAH ELY • Health Sciences Faculty, Antiviral Gene Therapy Research Unit

and African Network for Drugs and Diagnostics Innovation (ANDI) Centre of Excellence, School of Pathology , University of the Witwatersrand , Johannesburg , South Africa

JOACHIM W. ENGELS • Institute of Organic Chemistry and Chemical Biology , Goethe- University , Frankfurt am Main , Germany

Contributors

xiv

KATIE S. FLENKER • Department of Internal Medicine , University of Iowa , Iowa City , IA , USA

DANIEL GALLEGO-PEREZ • Nanoscale Science and Engineering Center for Affordable Nanoengineering , The Ohio State University , Columbus , OH , USA

PAOLO GANDELLINI • Molecular Pharmacology Unit, Department of Experimental Oncology and Molecular Medicine , Fondazione IRCCS Istituto Nazionale dei Tumori , Milan , Italy

PALOMA H. GIANGRANDE • Department of Internal Medicine , University of Iowa , Iowa City , IA , USA

CATHERINE M. GREENE • Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland , Beaumont Hospital , Dublin , Ireland

EDELBURGA HAMMERSCHMID • Institute of Experimental Oncology , Klinikum rechts der Isar der Technischen Universität München , Munich , Germany

TIDI HASSAN • Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland , Beaumont Hospital , Dublin , Ireland

GRANT W. HENNIG • Department of Physiology and Cell Biology , University of Nevada School of Medicine , Reno , NV , USA

RENATA HEZOVA • Central European Institute of Technology , Masaryk University , Brno , Czech Republic

YOSHIHIRO ITO • Nano Medical Engineering Laboratory , RIKEN, Saitama , Japan PER OLE IVERSEN • Department of Nutrition , University of Oslo , Oslo , Norway MICHAEL KOLDEHOFF • Department of Bone Marrow Transplantation, Faculty of Medicine,

West German Cancer Center , University of Duisburg-Essen , Essen , Germany ANDREA KRÜHN • Charité Campus Mitte , Institute of Pathology , Berlin , Germany HERMANN LAGE • Charité Campus Mitte , Institute of Pathology , Berlin , Germany L. JAMES LEE • Nanoscale Science and Engineering Center for Affordable

Nanoengineering , The Ohio State University , Columbus , OH , USA DAVID L. LEWIS • Arrowhead Research Corporation , Madison , WI , USA SHI-LUNG LIN • Division of Regenerative Medicine , WJWU & LYNN (W&L)

Institute for Stem Cell Research , Santa Fe Springs , CA , USA PATRICK LU • Sirnamoics Inc. , Gaithersburg , MD , USA TREVOR LUCAS • Laboratory for Cardiovascular Research, Center for Anatomy

and Cell Biology , Medical University of Vienna , Vienna , Austria DICKSON LUONG • Department of Physiology and Cell Biology , University of Nevada

School of Medicine , Reno , NV , USA MUSA D. MARIMANI • Health Sciences Faculty, Antiviral Gene Therapy Research Unit

and African Network for Drugs and Diagnostics Innovation (ANDI) Centre of Excellence, School of Pathology , University of the Witwatersrand , Johannesburg , South Africa

NOEL G. MCELVANEY • Respiratory Research Division, Department of Medicine, Royal College of Surgeons in Ireland , Beaumont Hospital , Dublin , Ireland

JAMES O. MCNAMARA II • Department of Internal Medicine , University of Iowa , Iowa City , IA , USA

ALBANO C. MELI • Department of Biology, Faculty of Medicine , Masaryk University , Brno , Czech Republic

FRANCIS J. MILLER • Department of Internal Medicine , University of Iowa , Iowa City , IA , USA

Contributors

xv

HANA MLCOCHOVA • Central European Institute of Technology , Masaryk University , Brno , Czech Republic

ANNE MOBERGSLIEN • Department of Immunology, Institute for Cancer Research , Radiumhospitalet-Rikshopitalet University Hospital , Oslo , Norway

OLGA MYKHAYLYK • Institute of Experimental Oncology , Klinikum rechts der Isar der Technischen Universität München , Munich , Germany

YUKO NAKASHIMA • Nano Medical Engineering Laboratory , RIKEN, Saitama , Japan AXL NEURAUTER • Life Technologies AS , Oslo , Norway MORTEN P. OKSVOLD • Department of Immunology, Institute for Cancer Research, Oslo

University Hospital, Oslo, Norway; Centre for Cancer Biomedicine, University of Oslo, Oslo, Norway

NICOLE ORTOGERO • Department of Physiology and Cell Biology , University of Nevada School of Medicine , Reno , NV , USA

KETIL W. PEDERSEN • Life Technologies AS , Oslo , Norway CHRISTIAN PLANK • Institute of Experimental Oncology, Klinikum rechts der Isar der

Technischen Universität München , Munich , Germany VALENTINA PROFUMO • Molecular Pharmacology Unit, Department of Experimental

Oncology and Molecular Medicine , Fondazione IRCCS Istituto Nazionale dei Tumori , Milan , Italy

GIUSEPPE DE ROSA • Department of Pharmacy , Università degli Studi di Napoli Federico II , Naples , Italy

STEIN SÆBØE-LARSSEN • Department of Cell Therapy, Institute for Cancer Research , Radiumhospitalet-Rikshopitalet University Hospital , Oslo , Norway

GIUSEPPINA SALZANO • Department of Pharmacy , Università degli Studi di Napoli Federico II , Naples , Italy

YOLANDA SANCHEZ-ANTEQUERA • Institute of Experimental Oncology , Klinikum rechts der Isar der Technischen Universität München , Munich , Germany

ANDREW SCHUSTER • Department of Physiology and Cell Biology , University of Nevada School of Medicine , Reno , NV , USA

MAGDOLNA G. SEBESTYÉN • Arrowhead Research Corporation , Madison , WI , USA MOULDY SIOUD • Department of Immunology, Institute for Cancer Research ,

Oslo University Hospital , Oslo , Norway ONDREJ SLABY • Central European Institute of Technology , Masaryk University , Brno ,

Czech Republic STEPHEN G.J. SMITH • Department of Clinical Microbiology, School of Medicine ,

Trinity College Dublin , Dublin , Ireland SHIGERU TAKASAKI • Toyo University , Ora-gun Gunma , Japan WILLIAM H. THIEL • Department of Internal Medicine , University of Iowa , Iowa City ,

IA , USA KRISTINA W. THIEL • Department of Internal Medicine , University of Iowa , Iowa City ,

IA , USA VLADIMIR TRUBETSKOY • Arrowhead Research Corporation , Madison , WI , USA SEBASTIAN VENCKEN • Respiratory Research Division, Department of Medicine,

Royal College of Surgeons in Ireland , Beaumont Hospital , Dublin , Ireland DIALECHTI VLASKOU • Institute of Experimental Oncology , Klinikum rechts der Isar der

Technischen Universität München , Munich , Germany SHENG-QI WANG • Department of Biotechnology , Beijing Institute of Radiation Medicine ,

Beijing , China

Contributors

xvi

XUE-JUN WANG • Department of Biotechnology , Beijing Institute of Radiation Medicine , Beijing , China

SO C. WONG • Arrowhead Research Corporation , Madison , WI , USA CHRISTINE I. WOODDELL • Arrowhead Research Corporation , Madison , WI , USA YUN WU • Department of Biomedical Engineering, University at Buffalo ,

State University of New York , Buffalo , NY , USA WEI YAN • Department of Physiology and Cell Biology , University of Nevada School of Medicine ,

Reno , NV , USA SHAO-YAO YING • Department of Cell and Neurobiology, Keck School of Medicine ,

University of Southern California , Los Angeles , CA , USA NADIA ZAFFARONI • Molecular Pharmacology Unit, Department of Experimental

Oncology and Molecular Medicine , Fondazione IRCCS Istituto Nazionale dei Tumori , Milan , Italy

KARIN ZINS • Laboratory for Cardiovascular Research, Center for Anatomy and Cell Biology , Medical University of Vienna , Vienna , Austria

Contributors