MEDICAL INTELLIGENCE UNIT

Peptide Nucleic Acids, and Related Antisense Biomolecules

Christopher G. Janson, M.D. Departments of Neurosurgery, Neurology and Molecular Genetics 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 NUCLEIC ACIDS, MORPHOLINOS AND RELATED ANTISENSE BIOMOLECULES

Medical Intelligence Unit

Landes Bioscience / Eurekah.com Kluwer Academic / Plenum Publishers

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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 . 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 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 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 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 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 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 Activation of Human CREB Gene Promoter Using Bis-PNA (Peptide Nucleic Acid) 265 Christopher G. Janson, Matthew J. During, Yelena Shifinan and Paola Leone Materials and Methods 266 Results 267 Future Directions for Trans-Activation in the Brain 268

Index 271 EDITORS

Christopher G. Janson Departments of Neurosurgery, Neurology and Molecular Genetics Cell and Gene Therapy Center UMDNJ-Robert Wood Johnson Medical School Camden, New Jersey, U.S.A. Email: [email protected] Chapter 18

Matthew J. During Department of Molecular Medicine and Pathology University of Auckland Auckland, New Zealand Email: [email protected] Chapter 18 ^=^= CONTRIBUTORS =^^

David O. Azorsa Natasha J. Caplen Translational Genomics Research Medical Genetics Branch Institute (TGen) National Human Genome Research Gaithersburg, Maryland, U.S.A. Institute Chapter 17 National Institutes of Health Bethesda, Maryland, U.S.A. Lionel Bastide Email: [email protected] Institute de Genetique Moleculaire Chapter 17 de Montpellier Centre National de la Recherche David R. Corey Scientifique Departments of Pharmacology Universite Montpellier 2 and Biochemistry UMR5124,IGMM University of Texas Southwestern Montpellier, France Medical Center Chapter 2 Dallas, Texas, U.S.A. Email: [email protected] Ruben J. Boado Chapter 15 Department of Medicine UCLA Los Angeles, California, U.S.A. Email: [email protected] Chapter 4 Jiin Feigner Leaf Huang Gene Therapy Systems Inc. Center for Pharmacogenetics San Diego, California, U.S.A. Department of Pharmaceutical Sciences Chapter 12 School of Pharmacy University of Pittsburgh Philip Feigner Pittsburgh, Pennsylvania, U.S.A. University of California, Irvine Email: [email protected] Center for Virus Research Chapter 3 Irvine, California, U.S.A. Email: [email protected] Troels Koch Chapter 12 Santaris Pharma A/S Horsholm, Denmark Marco Folini Email: [email protected] Dipartimento di Oncologia Sperimentale Chapter 7 Istituto Nazionale per lo Studio e la Cura dei Tumori Lars Kongsbak Milan, Italy Exiqon A/S Chapter 10 Vedbaek, Denmark Email: [email protected] Miriam Frieden Chapter 13 Santaris Pharma A/S Horsholm, Denmark Bernard Lebleu Email: [email protected] Institute de Gdn^tique Mol^culaire Chapter 7 de Montpellier Centre National de la Recherche Carlo Gambacorti-Passerini Scientifique Oncogenic Fusion Proteins Unit Universite Montpellier 2 Department of Experimental Oncology UMR5124,IGMM National Cancer Institute Montpellier, France Milan, Italy Email: [email protected] Chapter 11 Chapter 2

Peter M. Glazer Paola Leone Departments of Therapeutic Radiology Cell and Gene Therapy Center and Genetics UMDNJ Yale University School of Medicine Robert Wood Johnson Medical School New Haven, Connecticut, U.S.A. Camden, New Jersey, U.S.A. Email: [email protected] Chapter 18 Chapter 14 Kenneth W. Liang Center for Pharmacogenetics Department of Pharmaceutical Sciences School of Pharmacy University of Pittsburgh Pittsburgh, Pennsylvania, U.S.A. Chapter 3 Xiaowu Liang Henrik 0rum Gene Therapy Systems Inc. Santaris Pharma A/S San Diego, California, U.S.A. Horshom, Denmark Chapter 12 Email: [email protected] Chapter 13 Robert N. Lightowlers Department of Neurology William M. Pardridge University of Newcastle upon Tyne Department of Medicine Medical School UCLA Framlington Place Los Angeles, California, U.S.A. Newcastle upon Tyne, U.K. Email: [email protected] Email: [email protected] Chapter 4 Chapter 16 Michael Petersen Feng Liu Nucleic Acid Center Center for Pharmacogenetics Department of Chemistry Department of Pharmaceutical Sciences University of Southern Denmark School of Pharmacy Odense, Denmark University of Pittsburgh Email: [email protected] Pittsburgh, Pennsylvania, U.S.A. Chapter 7 Chapter 3 Ian Robbins Luca Mologni Institute de Genetique Moleculaire Department of Clinical Medicine de Montpellier University of Milano-Bicocca Centre National de la Recherche Monza, Italy Scientifique and Montpellier, France Department of Experimental Oncology Email: [email protected] National Cancer Institute Chapter 2 Milan, Italy Email: [email protected] Gunther F. Ross Chapter 11 Mitochondrial Research Group School of Neurology, Neurobiology Spyro Mousses and Psychiatry Translational Genomics Research University of Newcastle upon Tyne Institute (TGen) Framlington Place Gaithersburg, Maryland, U.S.A. Newcasde upon Tyne, U.K. Chapter 17 Chapter 16

Peter E. Nielsen John J. Rossi Center for Biomolecular Recognition Division of Molecular Biology Department of Medical Biochemistry Beckman Research Institute and Genetics of the City of Hope The Panum Institute Duarte, California, U.S.A. Copenhagen, Denmark Email: [email protected] Email: [email protected] Chapter 8 Chapter 1 Edward R. Sauter Robert W.Taylor Department of Surgery Mitochondrial Research Group Ellis Fischel Cancer Center School of Neurology, Neurobiology University of Missouri and Psychiatry Columbia, Missouri, U.S.A. University of Newcastle upon Tyne Chapter 5 Framlington Place Newcasde upon Tyne, U.K. Lisa Scherer Chapter 16 Division of Molecular Biology Beckman Research Institute Mathew L. Thakur of the City of Hope Department of Radiology Duarte, California, U.S.A. Kimmel Cancer Center Email: [email protected] Jefferson Medical College Chapter 8 Thomas Jefferson University Philadelphia, Pennsylvania, U.S.A. Shizuko Sei Chapter 5 Laboratory of Antiviral Drug Mechanisms Douglass M. TurnbuU Screening Technologies Branch Mitochondrial Research Group Developmental Therapeutics Program School of Neurology, Neurobiology SAIC-Frederick, NCI-Frederick and Psychiatry Frederick, Maryland, U.S.A. University of Newcastle upon Tyne Email: [email protected] Framlington Place Chapter 9 Newcasde upon Tyne, U.K. Chapter 16 Yelena Shifman Cell and Gene Therapy Center Raffaella Villa UMDNJ Dipartimento di Oncologia Sperimentale Robert Wood Johnson Medical School Istituto Nazionale per lo Studio Camden, New Jersey, U.S.A. e la Cura dei Tumori Chapter 18 Milan, Italy Chapter 10 Paul M. Smith Mitochondrial Research Group Can Wang School of Neurology, Neurobiology Department of Cell Biology and Psychiatry and Neuroscience University of Newcastle upon Tyne University of South Alabama Framlington Place Mobile, Alabama Newcastle upon Tyne, U.K. Chapter 14 Chapter 16 Xiaodong Wang James E. Summerton Gene Therapy Systems Inc. Gene Tools, LLC San Diego, California, U.S.A. Philomath, Oregon, U.S.A. Chapter 12 Email: [email protected] Chapter 6 Yan Wang Andreas Wolter Gene Therapy Systems Inc. Proligo GmbH San Diego, California, U.S.A. Hamburg, Germany Chapter 12 Email: [email protected] Chapter 13 Theresa M. Wardell Mitochondrial Research Group Nadia Zaffaroni School of Neurology, Neurobiology Dipartimento di Oncologia Sperimentale and Psychiatry Istituto Nazionale per lo Studio University of Newcastle upon Tyne e la Cura dei Tumori Framlington Place Milan, Italy Newcastle upon Tyne, U.K. Email: [email protected] Chapter 16 Chapter 10

Jesper Wengel Olivier Zelphati Nucleic Acid Center University of California, Irvine Department of Chemistry Center for Virus Research University of Southern Denmark Irvine, California, U.S.A. Odense, Denmark Email: [email protected] Email: [email protected] Chapter 12 Chapter 7

Eric Wickstrom Department of Biochemistry and Molecular Biology Department of Microbiology and Immunology Kimmel Cancer Center Cardeza Foundation for Hematologic Research Jefferson Medical College Thomas Jefferson University Philadelphia, Pennsylvania, U.S.A. Email: [email protected] Chapter 5 -PREFACE --

When this book project was first contemplated, some of the molecules and applications discussed in this volume (such as mammalian siRNA) did not yet exist, which speaks to the relative progress in the antisense field and the likelihood that fiirther chemical modifications of existing classes of mol­ ecules will lead to even more enhanced and greater use of "gene tools" in the future. The original intention of the publisher was to devote an entire book to Peptide Nucleic Acid (PNA), which was an incipient but fast-growing field. Given the diversity of emerging antisense products, we felt that it would be more profitable to compare and contrast PNA with other available oligo­ homologues and to consider areas in which these biomolecules could be profitably applied to clinical and diagnostic applications. Because other books and research articles in the primary literature already provided specific protocols for use of PNA and related compounds, we preferred to take a broader review of the existing literature by some of the same innova­ tors who developed the molecules and associated techniques. There are currently a wide variety of research tools to choose from in the design of experiments utilizing gene knockdown and gene labeling, and the eight chapters in Part I address comparative strengths and weaknesses of various homologues: standard modified DNA oligonucleotides, peptide nucleic acid (PNA), (LNA), morpholinos, and small interfering RNA (siRNA). In terms of unique properties, PNA is espe­ cially useful in situations where DNA binding affinity and resistance to nu­ cleases is important such as gene-based diagnostics, or where another ligand is to be bound to DNA for site-specific mutagenesis, gene-specific drug de­ livery, or other demanding applications. Other currently popular molecules such as siRNA, LNA, and morpholinos are all efficient and versatile meth­ ods of knockdown for in vivo use, but each has distinct advantages and limitations. Some molecules are limited to acting on the RNA level (e.g., siRNA), while others work on the DNA or RNA level (e.g., LNA, PNA, mopholinos). After an overview of the basic characteristics of each "gene tool," the ten chapters in Part II address specific translational or clinical applications for PNA and related antisense biomolecules, such as anti-tu­ mor or anti-AIDS therapies, gene activation, and gene repair. The editors have aimed to present a balanced view of the methods available for gene targeting and modification, which will have broad appeal for either the research scientist or gene therapist. In the process we have omitted some techniques which originally appeared to have promise but which have subsequently been cast into serious doubt in terms of their speci­ ficity and effectiveness, such as DNA-RNA chimeraplasty. The molecules discussed in this volume are widely considered to be beyond reproach in terms of their potential utility in the research setting, despite the fact that they are still proving themselves in the laboratory and have yet to enter the clinic. Because the same "gene tools" may not be equally effective in research and in the clinic—indeed, it is quite possible that the opposite will be true— we have aimed to strike a balance between the bench and the bedside.

Christopher G. Janson Matthew J. During