MEDICAL INTELLIGENCE UNIT Peptide Nucleic Acids, Morpholinos and Related Antisense Biomolecules Christopher G. Janson, M.D. Departments of Neurosurgery, Neurology and Molecular Genetics Cell and Gene Therapy Center UMDNJ-Robert Wood Johnson Medical School Camden, New Jersey, U.S.A. Matthew J. During, M.D., ScD. Department of Molecular Medicine and Pathology University of Auckland Auckland, New Zealand LANDES BIOSCIENCE / EUREKAH.COM KLUWER ACADEMIC / PLENUM PUBLISHERS GEORGETOWN, TEXAS NEW YORK, NEW YORK USA U.SA PEPTIDE NUCLEIC ACIDS, MORPHOLINOS AND RELATED ANTISENSE BIOMOLECULES Medical Intelligence Unit Landes Bioscience / Eurekah.com Kluwer Academic / Plenum Publishers Copyright ©2006 Eurekah.com and Kluwer Academic / Plenum Publishers All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher, vdth the exception of any material supplied specifically for the purpose of being entered and executed on a computer system; for exclusive use by the Purchaser of the work. Printed in the U.S.A. Kluwer Academic / Plenum PubHshers, 233 Spring Street, New York, New York, U.S.A. 10013 http ://www.wkap .nl/ Please address all inquiries to the Publishers: Landes Bioscience / Eurekah.com, 810 South Church Street, Georgetown, Texas, U.S.A. 78626 Phone: 512/ 863 7762; FAX: 512/ 863 0081 http ://v^^ww.eurekah. com http://www.landesbioscience.com Peptide Nucleic Acids, Morpholinos and Related Antisense BiomoleculeSy edited by Christopher G. Janson and Matthew J. During, Landes / Kluwer dual imprint / Landes series: Medical Intelligence Unit ISBN: 0-306-48230-4 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein. Library of Congress Cataloging-in-Publication Data Peptide nucleic acids, morpholinos, and related antisense biomolecules / [edited by] Christopher G. Janson, Matthew J. During. p. ; cm. ~ (Medical intelligence unit) ISBN 0-306-48230-4 1. Antisense nucleic acids. 2. Antisense peptides. I. Janson, Christopher G. II. During, Matthew J. III. Series: Medical intelligence unit (Unnumbered : 2003) [DNLM: 1. Peptide Nucleic Acids. 2. Antisense Elements (Genetics) QU 58 P42394 2006] QP624.5.A57P47 2006 612'.015756-dc22 2005016425 This volume is dedicated to Dr. Linda Bartoshuk, who facilitated its planning by assisting one of the editors (Dr. Janson) during his leave from Yale University; and to Dr. Stanley Miller, who first proposed the role of PNA in prebiotic chemical evolution and the ancient history of life on Earth, thereby raising the question if the recent discovery of PNA was in fact a fortuitous rediscovery of our common pre-DNA, pre-RNA origins. CONTENTS Preface xvii Parti Research Applications 1. The Many Faces of PNA 3 Peter E. Nielsen Introduction to PNA 3 PNA Chemistry 3 Cellular Uptake of PNA 4 Antisense Applications 5 Antigene Properties 5 Antimicrobial PNAs 9 Genetic Information Carrier 11 PNA in Diagnostics 12 Prospects 13 2. Modulation of Nucleic Acid Information Processing by PNAs: Potential Use in Anti-Viral Therapeutics 18 Lionel Bastide, Bernard Lebleu and Ian Robbins PNAs as Gene-Modulator Agents 21 Virus Specific Nucleic Acid Processing 25 PNA-Tuning 26 3. Targeted Gene Delivery: The Role of Peptide Nucleic Acid 30 Kenneth W. Liangs Feng Liu and Leaf Huang Targeted Gene Delivery through Polycation/DNA Complex 31 Targeted Delivery of Naked DNA 33 4. Imaging Gene Expression in the Brain with Peptide Nucleic Acid (PNA) Antisense Radiopharmaceuticals and Drug Targeting Technology 38 Ruben]. Boado and William M. Pardridge Mechanism of Action of Antisense Drugs 40 Medical Diagnostic and Therapeutic Applications 41 Functional Genomics 45 Overview of Antisense Molecules and Effective Delivery 45 Brain Drug Targeting Systems A7 Physiological Brain Efflux of IgG 52 Imaging of Brain Gene Expression 52 5. Receptor-Specific Targeting with Complementary Peptide Nucleic Acids Conjugated to Peptide Analogs and Radionuclides 61 Eric Wickstrom, Mathew L Thakur and Edward R, Sauter Synthesis of Probes 68 AppHcations in Cells 71 Applications in Animals 7G Applications in Patients 80 6. Morpholinos and PNAs Compared 89 James E. Summerton Classification of Antisense Structural Types 90 Preparation of Morpholinos and PNAs 91 Properties of Morpholinos and PNAs 93 Applications 106 7. Chemistry of Locked Nucleic Acids (LNA): Design, Synthesis and Bio-Physical Properties 114 ]esper WengeU Michael Petersen, Miriam Frieden and Troels Koch Synthesis of LNA Monomers 114 Solid Phase Synthesis of LNA Oligonucleotides 115 Hybridization Characteristics of LNA Oligonucleotides 118 Hybridization Kinetics of LNA Oligonucleotides 119 Structure of LNA Oligonucleotides 122 DNA and RNA Structure 122 LNA Structure 123 LNAiRNA Duplexes 123 LNArDNA Duplexes 125 LNA:LNA Duplexes 126 a-L-LNA:RNA Duplexes 126 a-L-LNA:DNA Duplexes 126 Implications for RNase H Activity of LNA:RNA and a-L-LNA:RNA Duplexes 127 Stability of LNA and a-L-LNA Modified Nucleic Acids 127 LNA Triplexes 128 8. Recent Applications of RNA Interference (RNAi) in Mammalian Systems 133 Lisa Scherer and John J. Rossi siRNA Design 133 Recent Applications of RNAi 137 Functional Genomics 142 RNAi versus Ribozymes 143 Part II Clinical Applications 9. Peptide Nucleic Acids as Epigenetic Inhibitors of HIV-1 151 Shizuko Set HIV-1 Life Cycle and Potential Molecular Targets 152 Genetic Strategies to Inhibit HIV-1 Replication 153 Peptide Nucleic Acids as Epigenetic HIV-1 Inhibitors 154 Potential Use of PNA Against Other Infectious Pathogens 161 Bio-Delivery of PNA 161 Future Perspectives 163 10. Therapeutic Uses of Peptide Nucleic Acids (PNA) in Oncology 171 Nadia Zaffaroniy Raffaella Villa and Marco Folini Potential of PNAs as Tools for Anticancer Therapeutic Interventions 172 Perspectives 178 11. PNAs as Novel Cancer Therapeutics 181 Luca Mologni and Carlo Gamhacorti-Passerini Biochemistry of Peptide Nucleic Acids 182 PNA as a Biomolecular Tool 183 Antisense and Anti-Gene Properties of PNA 184 Future Directions 189 12. Medicinal Chemistry of Plasmid DNA with Peptide Nucleic Acids: A New Strategy for Gene Therapy 195 Olivier Zelphati, Jiin Feigner^ Yan Wang, Xiaowu Liang, Xiaodong Wang and Philip Feigner Principle of PNA Dependent Gene Chemistry Technology 197 Labeling of Plasmid DNA to Study Gene Delivery Mechanism 198 Transition 199 Overcoming the Barriers to Improve Gene Delivery and Expression 200 PNA-Conjugates for Targeting DNA to Cell Surface Receptors 201 PNA-Peptide Conjugates to Overcome Cell Membrane Barriers 202 Other Potential Applications of PNA Conjugate for Gene Delivery 206 13. Locked Nucleic Acids (LNA) and Medical Applications 212 Henrik 0rumy Andreas Wolter and Lars Kongsbak Biochemistry of LNA 212 LNA in Diagnostics and Genomics Applications 215 LNA in Therapeutic Applications 218 Future Perspectives on LNA 221 14. Peptide Nucleic Acids as Agents to Modify Target Gene E3q)ression and Function 223 Gan Wang and Peter M. Glazer PNA Binding Affinity 224 PNA Binding Specificity 224 Detection of PNA Binding-Induced Transcription in Hela Nuclear Extract in Vitro Transcription System 224 Determination of the Initiation Sites of PNA Binding-Induced Transcription 226 PNA Binding-Generated D-Loops Lead to GFP Gene Expression in Mammalian Cells 228 PNA-Induced Endogenous y-Globin Gene Expression in Human Cells 228 The Correlation between PNA Binding-Generated D-Loops and the Natural Promoter of the Gene in Target Gene Transcription 229 The PNA Length Requirement for Inducing Transcription from the PNA Binding Sites 231 Transcription Components Involved in PNA Binding-Induced Transcription 232 The Limitation of PNA Binding-Induced Target Gene Expression 233 PNAs for Targeted Genome Modification 234 15. Peptide Nucleic Acids: Cellular Delivery and Recognition of DNA and RNA Targets 236 David K Corey Hybridization by PNA: Affinity Isn't Everything 236 Strand Invasion by PNAs 238 Intracellular Delivery of PNAs 239 Applications for PNAs Delivered within Cells 240 16. The Use of PNAs and Their Derivatives in Mitochondrial Gene Therapy 243 PaulM. Smith, Gunther F. Ross, Theresa M. Wardell, Robert W. Taylor, Douglass M. Turnhull and Robert N. Lightowlers The Antigenomic Hypothesis 244 PNA as the Choice of Antigenomic Agent 244 Cellular Uptake and the Problem of Mitochondrial Import 246 PNAs as Antigenomic Molecules - Do They Work? 247 Trouble-Shooting the Antigenomic Approach to mtDNA Disease .... 248 17. Gene Silencing through RNA Interference: Potential for Therapeutics and Functional Genomics 252 David O. Azorsa, Spyro Mousses and Natasha J. Caplen RNAi: An Historical Perspective 252 RNAi: Summary of Mechanism 253 Physiological Role of RNAi and Related Post-Transcriptional Gene Silencing Mechanisms 253 RNAi in Mammalian Cells 255 The Delivery of Triggers of RNAi 256 RNAi as a Functional Genomics Tool 257 RNAi as a Therapeutic Strategy 258 18. Transcriptional