Pharmaceutical Aspects of Oligonucleotides (Taylor & Francis Series in Pharmaceutical Sciences)

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Pharmaceutical Aspects of Oligonucleotides

New and Forthcoming Titles in the Pharmaceutical Sciences Nuclear Medicine in Pharmaceutical Research Perkins and Frier (Eds) 1999 0 7484 0688 3 Hbk Electrically Assisted Transdermal and Topical Drug Delivery Ajay K.Banga 1998 0 7484 0687 5 Hbk Physiological Pharmaceutics Barriers to Drug Absorption 2nd Edition Washington & Wilson 1999 0 7484 0562 3 Hbk 0 7484 0610 7 Pbk Intelligent Software for Product Formulation Rowe & Roberts 1998 0 7484 0732 4 Hbk Flow Injection Analysis of Pharmaceuticals Automation in the Laboratory Martínez Calatayud 1996 0 7484 0445 7 Hbk Photostability of Drugs and Drug Formulations Tønneson (Ed) 1996 0 7484 0449 X Hbk Microbial Quality Assurance in Cosmetics, Toiletries and Non-Sterile Pharmaceuticals 2nd Edition Baird with Bloomfield (Eds) 1996 0 7484 0437 6 Hbk Immunoassay A Practical Guide Law (Ed) 1996 0 7484 0560 7 Hbk Cytochromes P450 Structure, Function and Mechanism Lewis 1996 0 7484 0443 0 Hbk Autonomic Pharmacology Broadley 1996 0 7484 0556 9 Hbk Pharmaceutical Experimental Design and Interpretation 2nd Edition Armstrong & James 1996 0 7484 0436 8 Hbk Pharmaceutical Production Facilities 2nd Edition Cole 1998 0 7484 0438 4 Hbk full pharmaceutical science catalogue available or visit our new website on: http://www.tandf.co.uk 325 Chestnut Street, 8th Floor, Philadelphia, PA 19106, USA Tel: 215–625–8900 Fax: 215–625–2940 11 New Fetter Lane London, EC4P 4EE Tel: +44 (0)20–7583 9855

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Pharmaceutical Aspects of Oligonucleotides Edited by

PATRICK COUVREUR University of Paris-Sud CNRS Unit ‘Physico-chimie, Pharmacotechnie, Biopharmacie’, Chatenay-Malabry, France

CLAUDE MALVY CNRS, Institut G.Roussy, Villejuif, France

First published 2000 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc., 325 Chestnut Street, 8th Floor, Philadelphia, PA 19106 Taylor & Francis is an imprint of the Taylor & Francis Group This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” © 2000 Taylor & Francis Limited All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data Pharmaceutical aspects of oligonucleotides/edited by Patrick Couvreur, Claude Malvy. p. cm.—(Taylor & Francis series in pharmaceutical sciences) Includes bibliographical references and index. 1. Oligonucleotides—Therapeutic use. I. Couvreur, Patrick. II. Malvy, Claude. III. Series. [DNLM: 1. Oligonucleotides—therapeutic use. 2. Oligonucleotides— adverse effects. QU 57 P536 2000] RM666.N87P47 2000 615′.31—dc21 99–34210 ISBN 0-203-30566-3 Master e-book ISBN

ISBN 0-203-34211-9 (Adobe eReader Format) ISBN 0-748-40841-X (Print Edition)

Contents

PART ONE

Contributors

xii

Preface

xiv

General Features 1

1

Mechanisms of Action of Antisense Oligonucleotides D.M.Tidd and R.V.Giles

2

1.1

Introduction

2

1.2

Cell Proliferation Arrest through Release of Deoxynucleosides

3

1.3

Extracellular Aptameric Effects of Phosphorothioate Oligodeoxynucleotide Analogues

5

1.4

Aptameric Effects of G-quartet Oligonucleotides and Analogues

6

1.5

Immune Stimulation by Oligodeoxynucleotides Containing CpG Motifs

10

1.6

Other Activities of Oligodeoxynucleotides Containing CpG Motifs

11

1.7

Antisense Inhibition of Gene Expression by Steric Block

11

1.8

Antisense Inhibition of Gene Expression through Ribonuclease H-mediated Destruction of Target mRNA

13

1.9

Oligonucleotides in Vivo

20

Conclusions

21

Acknowledgements

21

References

22

1.10

vii

PART TWO

Chemical Aspects 2

32

Chemistry of Oligonucleotides J.W.Engels and E.Uhlmann

33

2.1

Introduction

33

2.2

Design of Oligonucleotides

37

2.3

Oligonucleotide Modifications: Synthesis and Properties

40

2.4

Analysis of Oligonucleotides

59

2.5

Conclusion

68

Acknowledgement

68

References

68

3

The Oligonucleotide Prodrug Approach: The 80 Pro-oligonucleotides F.Morvan, J.-J.Vasseur, E.Vivès, B.Rayner and J.-L.Imbach

3.1

Introduction

80

3.2

What Kind of Enzymolabile Group?

81

3.3

First Pro-oligonucleotide Models

84

3.4

Pro-oligonucleotides of the Second Generation

87

3.5

Conclusion

94

Acknowledgements

96

References

96

4

Peptide Nucleic Acids P.E.Nielsen

100

4.1

Introduction

100

4.2

Antisense

101

4.3

Antimicrobials

103

4.4

Anti-telomerase

103

4.5

Antigene

103

4.6

Anti-HIV

105

4.7

Pharmacology

105

4.8

Further Developments

106

viii

Acknowledgement

106

References

107

PART THREE Delivery 5

110

Peptide-mediated Delivery of Oligonucleotides E.Vivès and B.Lebleu

111

5.1

Delivery Vehicles for the Improved Uptake of Nucleic Acids: a Survey

111

5.2

The Potential of Peptides for Nucleic Acids Delivery

113

5.3

Strategies for the Coupling of Peptides to Oligonucleotides

114

5.4

Poly (L-lysine)-based Delivery Systems

116

5.5

Conjugation to Fusogenic Peptides Allowing Membrane Fusion or Membrane Translocation

117

5.6

Conclusions

122

Acknowledgements

124

References

124

Polymeric Nanoparticles and Microparticles as Carriers for Antisense Oligonucleotides E.Fattal and P.Couvreur

129

6.1

Introduction

129

6.2

Nanoparticles

130

6.3

In Vitro Stability of ONs Adsorbed onto Nanoparticles

138

6.4

Cell Interactions with ON Loaded Nanoparticles

139

6.5

In Vitro Pharmacological Activity of Oligonucleotideloaded Nanoparticles

140

6.6

In Vivo Studies with Oligonucleotide Nanoparticles

140

6.7

Microparticles

141

6.8

Conclusion

142

References

142

Liposomes for the Delivery of Oligonucleotides P.Couvreur, C.Malvy, C.Dubernet and E.Fattal

148

Introduction

148

6

7 7.1

ix

7.2

Anionic Liposomes

149

7.3

Cationic Liposomes

152

7.4

pH-sensitive Liposomes

159

7.5

Immunoliposomes and Other Molecularly Targeted Liposomes

163

7.6

Fusogenic Liposomes and Proteoliposomes

165

7.7

Conclusions

167

References

168

Comb-type Polycation Copolymer for Antigene Strategy and DNA Carrier A.Maruyama

176

8.1

Introduction

176

8.2

Comb-type Polycations as a Stabilizer for DNA Duplex and Triplex

176

8.3

Comb-type Polycation Copolymers with Cell-specific Polysaccharide Side-chains as cell-specific DNA Carrier

189

Acknowledgements

196

References

196

8

PART FOUR Biopharmaceutics 9

203

Delivery of Antisense Oligonucleotides in Vitro: Experimental Points G.B.Takle and C.A.Stein

204

9.1

Introduction

204

9.2

Oligonucleotide-Binding Proteins on the Cell Surface

206

9.3

Intracellular Compartmentalization

208

9.4

Oligonucleotide Delivery Reagents—Practical Considerations

210

9.5

Experimental Antisense: Points to Consider

211

References

214

Mechanisms of Transmembrane Transport of Oligonucleotides

217

10

x

R.L.Juliano 10.1

Overview of Cellular Uptake of Antisense Oligonucleotides

217

10.2

Permeation of Oligonucleotides across Membranes

219

10.3

Mechanisms of Enhancement of Oligonucleotide Permeation across Membranes

222

10.4

Summary

225

References

225

Pharmacokinetics of Oligodeoxynucleotides A.Gouyette

230

11.1

Introduction

230

11.2

Pharmacokinetics

231

11.3

Chemistry of Oligonucleotides and Formulations

233

11.4

Cellular Pharmacokinetics

233

11.5

Preclinical Pharmacokinetics

235

11.6

Clinical Pharmacokinetics

239

11.7

Conclusions

240

References

241

11

PART FIVE

Pharmacological Activity 12

246

Antisense as a Novel Therapy for Cancer B.P.Monia

247

12.1

Novel Approaches for Anticancer Therapy

247

12.2

Antisense Approaches for Cancer

249

12.3

Antisense as a Novel Anticancer Approach against ras

255

12.4

Conclusions and Future Prospects

263

Acknowledgements

264

References

264

Modulation of Inflammatory Processes with Antisense Oligonucleotides C.F.Bennett

268

Introduction

268

13

13.1

xi

13.2

ICAM-1, a Case Study

269

13.3

Other Examples

281

13.4

Regulation of Immune Response by Non-antisense Mechanisms

285

13.5

Conclusions

286

References

286

Oligonucleotides as Antiparasite Compounds J.-J.Toulmé

293

14.1

Introduction

293

14.2

Design of Antisense Oligonucleotides for Antiparasite Use

294

14.3

Antiparasite Effects of Antisense Oligonucleotides

297

14.4

RNA Structures are Valid Targets for Regulatory Oligonucleotides

305

14.5

Conclusion

310

Acknowledgements

311

References

311

Index

318

14

Contributors

C.F.BENNETT, ISIS Pharmaceutical, 2292 Faraday Avenue, Carlsbad, CA 92008, USA. P.COUVREUR, Laboratoire de Physico-Chimie-PharmacotechnieBiopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. C.DUBERNET, Laboratoire de Physico-Chimie-PharmacotechnieBiopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. J.W.ENGELS, University of Frankfurt, Institut for Organische Chemir, Marie Curie Str 11, 60439 Frankfurt am Main, Germany. R.V.GILES, School of Biological Sciences, The University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. E.FATTAL, Laboratoire de Physico-chimie-Pharmacotechnie-Biopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. A.GOUYETTE, Institut Gustave Roussy, CNRS, 140 rue Camille Desmoulins, 94805 Villejuif Cedex, France. J.-L.IMBACH, Laboratoire de Chimie Bio-Organique, UMR CNRS-UMII 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. R.L.JULIANO, Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA. B.LEBLEU, Institut de Génétique Moléculaire, CNRS-UMR 5535, 1919, Route de Mende, 34293 Montpellier Cedex 5, France. C.MALVY, Laboratoire de Régulation Artificielle de l’Expression Génétique, UMR CNRS 8532, Institut Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif, France. A.MARUYAMA, Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda-Cho, Midori-Ku, Yokohama 226–8501, Japan.

xiii

B.P.MONIA, Department of Molecular Pharmacology, ISIS Pharmaceuticals, 2280 Faraday Avenue, Carlsbad, CA 92008, USA. F.MORVAN, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. P.E.NIELSEN, Center for Biomolecular Recognition, Department of Medical Biochemistry and Genetics, Biochemical Laboratory B, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark. B.RAYNER, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. C.A.STEIN, Departments of Medicine and Pharmacology, Columbia University, 630 W 168th Street, New York, NY 10032, USA. G.B.TAKLE, Innovir Labs Inc., 510 E 73rd Street, New York, NY 10021, USA. D.M.TIDD, School of Biological Sciences, The University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. J.-J.TOULMÉ, INSERM U 386, IFR Pathologies Infectieuses, Université Victor Segalen, 146 rue Léo-Saignat, 33076 Bordeaux Cedex, France. E.UHLMANN, Hoehst Marion Rounel Deutschland GmbH, Chemical Research G838 D-65 926, Frankfurt am Main, Germany. J.-J.VASSEUR, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. E.VIVÈS, Institut de Génétique Moléculaire, CNRS-UMR 5535, 1919, Route de Mende, 34293 Montpellier Cedex 5, France.

Preface

The dream of modern drug research is to discover a biologically active molecule or a class of biologically active molecules, 100% specific, able to act efficiently only on the function responsible for the disease. If this dream became a reality, treatment of fatal illnesses (AIDS, cancer, chronic immune disorders, etc.) would be possible without the severe side-effects and toxicities that are the main limitations of these treatments. Many drugs work by interfering with critical proteins which have been identified as responsible for dysfunction of cells or tissues. However, conventional therapeutic agents now on the market which tend to act on proteins in the body also often bind to non-target proteins, or exert an effect through unknown interactions. Fortunately, progress in genetics and genomics has enabled definitive studies on some of the fundamental molecular mechanisms that regulate the expression of genes, as well as the dysfunction of these mechanisms. From this mass of knowledge, the idea has emerged of drugs that may turn off genes by targeting the RNA that codes for the protein instead of the protein product. This strategy displays several advantages: (i) an active antisense oligonucleotide can be identified in a short time, (ii) the subcellular location of the protein is not important, (iii) first results do not show any antigenicity of oligonucleotides. One antisense oligonucleotide was approved by the FDA as a drug in 1998. The ‘Antisense’ concept is based on the ability of cellular RNA macromolecules to bind to complementary (or antisense) sequences of oligonucleotides. Since the antisense oligonucleotides interact with their targets by Watson-Crick base pairing, this should lead, in theory, to the perfect specificity and affinity as dreamed of by the drug researcher. However, despite their exciting potential for selectively modulating the expression of an individual gene, oligonucleotides are still far from becoming drugs. They suffer from numerous limitations: they are rapidly degraded in vivo by nucleases, they diffuse poorly through the biological barriers (including cell membranes), and they fail to reach the right intracellular compartments. There are also questions about reproducibility of biological efficacy of oligonucleotides, and about whether the pharmacological effects are definitely the result of an antisense activity.

xv

This book is intended as a condensed work on oligonucleotides from the perspective of their pharmaceutical application, trying to answer the following questions: what technological bolts are hindering the development of oligonucleotides as pharmaceuticals, and how can these be overcome? Contributors were asked to emphasize their own experience with oligonucleotides, and to deal with aspects not normally covered in specialized reviews. The objective was to provide a book which, although written by specialists, could be easily understood by people in different fields of life science, and especially scientists interested in drug discovery and pharmaceutical development. The book starts with a general account of how oligonucleotides work at the molecular level, and goes on to discuss the challenge of improving the administration of these compounds. Therefore, particular emphasis is placed on the development of the chemistry (peptidic nucleic acids) as well as of the formulation and the controlled delivery of oligonucleotides with the aid of molecular (peptides, polymers) or particular/supramolecular systems (liposomes, nanoparticles). Background information concerning intracellular trafficking and tissue distribution/ pharmacokinetics is reviewed and discussed, because these areas should also give new ideas for improving oligonucleotide delivery. The last part of the book is devoted to therapeutical aspects (treatment of cancer and of inflammatory and parasitic diseases). Clearly, the potential provided by oligonucleotides as shown in this book will be realized only through a multidisciplinary and innovative research effort. Patrick Couvreur and Claude Malvy (Eds) Université Paris-Sud. CNRS. France

PART ONE General Features

1 Mechanisms of Action of Antisense Oligonucleotides D.M.TIDD and R.V.GILES

1.1 Introduction As well as inhibiting gene expression through steric blockade of pre-mRNA splicing or initiation of translation, or through ribonuclease H-mediated ablation of mRNA, antisense oligonucleotides have been found to induce a variety of biological effects by mechanisms other than, or in addition to, those which might result from hybridization to the targeted mRNA. Such additional mechanisms of action include: 1 release of pharmacologically active concentrations of deoxyribonucleosides, through nucleolytic degradation 2 aptameric binding to proteins 3 immune stimulation by oligodeoxynucleotides containing CpG motifs 4 other activities of oligodeoxynucleotides containing CpG motifs. The interpretation of results of experiments with antisense oligonucleotides requires that account be taken of all potential mechanisms that might have been entrained, as well as the possibility that the expression of non-targeted genes may have been suppressed by antisense mechanisms, through substantial partial complementarity between their mRNAs and the antisense effector. Antisense oligonucleotides were initially hailed as agents for achieving highly specific inhibition of pathologic gene expression, whether that be of a virus (Cohen, 199la; Degols et al., 1992), a cancer cell (Calabretta, 1991; Dolnick, 1991; Carter and Lemoine, 1993), a neuropathology (Eng, 1993; Wahlestedt, 1994), or indeed of almost any disease of humans imaginable (Miller and Ts’o, 1987; Cohen, 1991b; Crooke, 1992; Gura, 1995). In reality, these molecules have turned out to induce a variety of biological effects, many of which may be ascribed to mechanisms other than Watson-Crick hybridization to the miscreant mRNA (Stein, 1995; Neckers and Iyer, 1998). Furthermore, the recognition that oligonucleotides may bind to proteins and subvert their function has led to a

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 3

growing following for the pursuit of such aptamers, through their evolution in repetitive selection—amplification procedures from libraries of random base sequence (Bock et al., 1992). Apart from such non-antisense mechanisms, there is increasing awareness also that a specific base sequence in an oligonucleotide might exert its antisense effects on several of the mRNAs from the as yet mostly undiscovered 60 000 to 80 000 genes thought to be contained in the human genome. Despite a lack of complete complementarity between the oligonucleotide and the non-targeted mRNAs, the activity at these sites could be as potent as, or more potent than, that achieved against the intended target, if the former and not the latter were devoid of secondary and tertiary structure, and therefore more readily accessible (Giles et al., 1998). In this chapter we will discuss some of the mechanisms by which antisense oligonucleotides may achieve their biological effects. 1.2 Cell Proliferation Arrest through Release of Deoxynucleosides Consideration of the intracellular trafficking (see also Chapters 9 and 10) of any drug is fundamental to an understanding of its mechanism of action upon the cell. Antisense oligonucleotides simply added to the bathing medium do not gain access to the cytoplasm and hence their target mRNAs in cells in culture. Small amounts of oligonucleotide are taken up through processes of endocytosis and/or fluid phase pinocytosis, but they remain in endosomes or vacuoles, excluded from the nucleus and still topologically exterior to the cell, being separated by a membrane from the cytoplasm (Spiller and Tidd, 1992; Giles et al., 1993; Crooke and Lebleu, 1993; Wagner et al., 1993). Experiments in which the media were replaced by oligonucleotide-free solution have demonstrated that the bulk of such molecules are subsequently quite rapidly expelled from cells by exocytosis (Tonkinson and Stein, 1994). There is no evidence to support the suggestion that oligonucleotides are even delivered to lysosomes to any significant extent, although Tonkinson and Stein (1994) have demonstrated that phosphorothioate oligodeoxynucleotide analogues penetrate into a deeper, acidic vesicular compartment within cells, as well as the shallow compartment attained predominantly by normal phosphodiester oligodeoxynucleotides. In contrast, when fluorescently tagged oligonucleotides are specifically delivered into the cytoplasm by direct microinjection, they have been observed to accumulate rapidly in the nuclei, presumably as a result of their affinity for binding to nuclear structures (Chin et al., 1990; Leonetti et al., 1991; Fisher et al., 1993; Wagner et al., 1993). In fact, nuclear accumulation of oligonucleotides is indicative that intracytoplasmic delivery has been achieved by other means, such as electroporation (Bergan et al., 1996; Spiller et al., 1998a), reversible plasma membrane permeabilization with streptolysin O (Spiller and Tidd, 1995; Giles et al., 1997), or lipofection with cationic liposomes (see also Chapter 7) (Bennett et

4 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

al., 1992; Gewirtz et al., 1996; Lewis et al., 1996; Marcusson et al., 1998). However, such observations require the concomitant exclusion of propidium iodide by the cells in order to establish that the cells are still living, since dead cells readily accumulate oligonucleotide in their nuclei by passive diffusion of the molecules through the holes in their ruptured plasma membranes. It was originally reported that primary cultures of keratinocytes did not require any special manipulation for intracytoplasmic delivery of oligonucleotides from the media (Nestle et al., 1994), but it has now been shown that all such cells exhibiting nuclear localization of oligonucleotide were also stained by propidium iodide or merocyanine 540, indicating that they were either already dead or undergoing apoptosis, respectively (Wingens et al., 1998; Giachetti and Chin, 1996). Also, it is important that observations of the intracellular localization of oligonucleotides be made on living and not fixed cells, since vesicular-contained oligonucleotide in living cells has been seen to translocate instantaneously to their nuclei upon fixation with ethanol (Tidd, 1998). It is evident from the foregoing considerations that early reports of inhibition of cell proliferation by a variety of oncogene antisense oligodeoxynucleotides added to the culture media were unlikely to have been achieved by an antisense mechanism against the targeted mRNA. In addition, such unprotected normal phosphodiester oligodeoxynucleotides are rapidly degraded to mononucleotides by endonuclease and 3′-phosphodiesterase activities present in the serum component of the culture medium, where the latter is the most important activity (Tidd and Warenius, 1989). Although monodeoxynucleotides also do not gain access to the cytoplasm of cells when presented exogenously, phosphatases present on the cell surface and possibly released into the media readily catalyse their hydrolysis to deoxynucleosides (unpublished observations), which are then rapidly transported into the cells and rephosphorylated by intracellular deoxynucleoside kinases (Henderson and Paterson, 1973). It is likely that at least some of the reported inhibitions of cell proliferation observed with oncogene antisense oligodeoxynucleotides were the result of cell uptake of their deoxynucleoside breakdown products. The consequent induction of gross imbalances in intracellular deoxynucleoside triphosphate pools would have prevented passage of the cells through the S-phase of the cell cycle. Limited exposure to excess thymidine is a long-established method for synchronizing cells for their entry into S-phase (Doida and Okada, 1967). Progressive replacement of internucleoside phosphodiester linkages by nuclease-resistant methylphosphonates (Miller and Ts’o, 1987; Miller, 1998) from each end of a cmyc antisense oligodeoxynucleotide resulted in proportionately less inhibition of proliferation of the cells by the same base sequence (Tidd, 1993). The terminal methylphosphonates protected the oligodeoxynucleotide from exonuclease attack and reduced its susceptibility to endonuclease-mediated hydrolysis. The predominant 3′-exonucleolytic degradation of oligodeoxynucleotides also permits an apparent sequence specificity for inhibition of cell proliferation. Vaerman et al. (1997) have demonstrated that oligodeoxynucleotides terminating


at the 3′-end in the bases adenine, guanine or thymine were inhibitory, while those ending in cytosine at either of the two most terminal 3′-positions were not. They determined the toxicity of deoxynucleoside 5′-monophosphates to haematologic cells and demonstrated that only deoxycytidine 5′-monophosphate was non-toxic, and that this mononucleotide neutralized the cytotoxicity of deoxyadenosine, deoxyguanosine and thymidine monophosphates. Added to this, there is the possibility of observing spurious, apparent inhibition of proliferation when 3H-thymidine or 5-bromodeoxyuridine incorporation assays are used (Matson and Krieg, 1992). Thymidine released from degraded oligodeoxynucleotides competes with these labelled deoxynucleosides, reducing their incorporation into DNA. This effect is greatest when the thymidine nucleotides are present at the 3′-end of an oligodeoxynucleotide. 1.3 Extracellular Aptameric Effects of Phosphorothioate Oligodeoxynucleotide Analogues Recognition of the poor biological stability of phosphodiester oligodeoxynucleotides led to a search for analogues of the structure which would be resistant to nucleases while retaining the hybridization properties of the natural molecule. A variety of structures have been developed, but phosphorothioate oligodeoxynucleotides, in which a non-bridging oxygen of the internucleoside linkage is replaced by sulphur, have become the most popular analogues. These are comparatively, though not completely, nuclease-resistant, while their hybridization potential with complementary nucleic acids is only moderately reduced relative to the corresponding phosphodiester molecules (Stein et al., 1988; Hoke et al., 1991). There has been a plethora of reports of putative antisense effects on gene expression induced through application of phosphorothioate oligodeoxynucleotides, where no special measures were taken to ensure intracytoplasmic delivery of the molecules. However, more careful examination of some of these claims has demonstrated that the biological effects observed were unrelated to inhibition of expression of the target gene. Barton and Lemoine (1995, 1997) have demonstrated that the established antiproliferative effects of phosphorothioate p53 antisense oligodeoxynucleotides applied exogenously to cells were unrelated to any effects on p53 expression. Growth of breast and lung cancer cell lines was inhibited by the c-myc (see also Chapter 12) antisense, phosphorothioate oligodeoxynucleotide via a sequencespecific, non-antisense mechanism which was correlated with inhibition of cell adhesion (Watson et al., 1992; Saijo et al., 1997). Similarly, growth inhibition of chronic myelogenous leukaemia cells, induced by phosphorothioate bcr-abl antisense oligodeoxynucleotides added to the culture medium, represented non-antisense, base sequence-dependent but not base sequence-specific toxicity of the oligomers, where the lower toxicity of the sense sequence controls was fortuitous (O’Brien et al., 1994; Smetsers et al., 1995).


Coulson et al. (1996) have also reported that a phosphorothioate oligodeoxynucleotide antisense to the epidermal growth factor receptor inhibited the proliferation and altered the morphology of A431 cells by a sequenceselective, but non-antisense, mechanism apparently affecting receptor tyrosine kinase activity. Phosphorothioate oligodeoxynucleotide analogues bind promiscuously to proteins (Stein and Cheng, 1993; Stein and Krieg, 1994; Stein, 1995) such as protein kinase C isoforms (Khaled et al., 1995), HIV gp120 (Stein et al., 1993) and heparin binding proteins such as members of the fibroblast growth factor family (Guvakova et al., 1995), integrin and fibronectin (Khaled et al., 1996) and soluble recombinant CD4 (Yakubov et al., 1993). Furthermore, since intracytoplasmic access would not have been achieved by the oligomers in the experiments described in the preceding paragraph, it is likely that the observed biological activities were the result of aptameric effects of their competitively binding essential growth factors (Fennewald and Rando, 1995; Guvakova et al., 1995), proteins in the plasma membrane or membrane proteins within endosomes. Antiviral effects of phosphorothioate antisense oligodeoxynucleotides may for the most part be accounted for by their binding to viral envelope proteins and/or cell surface receptors and thereby inhibiting cell entry, rather than through antisense inhibition of targeted viral gene expression (Stein, 1995). Likewise, sequence-specific inhibition of cell adhesion may be the result of direct interactions with proteins at the cell surface and substratum, rather than the consequence of altered gene expression (Neckers and Iyer, 1998). 1.4 Aptameric Effects of G-quartet Oligonucleotides and Analogues Guanine-rich oligonucleotides and polynucleotides can form intermolecular or intramolecular structures whereby four guanines are arranged in a planar array, known as a G-tetrad or G-quartet. In general, intermolecular G-quartets require four, or more, consecutive guanine residues and present structures in which all four sugar-phosphate backbones are in a parallel arrangement. Intramolecular Gquartets fold into a structure with a minimum of two adjacent G-tetrads and with the backbone oriented so as to present two parallel and two antiparallel strands. These arrays are stabilized by hydrogen bonding in which each of the guanines acts as an acceptor and a donor for two bonds (discussed in Williamson, 1994). ) In solution, G-quartets are stabilized by alkali metal ions ( which occupy a position in the core between two square arrays of guanine, and it would appear that potassium ions are preferred due to their possessing an optimal radius (Williamson, 1994). Residues outside the G-quartet region can stabilize the structure, particularly with intramolecular G-tetrads, by T–T or T–A hydrogen bonding (Schultze et al., 1994; Murchie and Lilley, 1994; Keniry et al., 1997). Intermolecular and intramolecular G-quartets may be identified and


distinguished by X-ray crystallography, circular dichroism (CD) spectroscopy (Williamson, 1994), nuclear magnetic resonance (Feigon et al., 1995) and thermal denaturation with UV spectroscopy (Mergny et al., 1998). Inhibition of smooth muscle cell hyperplasia by phosphorothioate oligodeoxynucleotides antisense to c-myb in vitro (see also Chapter 12) (Simons and Rosenburg, 1992) and in vivo (Simons et al., 1992) and to c-myc in vitro and in vivo (Shi et al., 1994; M.R.Bennett et al., 1994), but not by control oligonucleotide analogues, was presented as a potential antisense approach to treatment of restenosis following balloon angioplasty. However, these effects were subsequently shown to occur by a non-antisense mechanism (Villa et al., 1995; Burgess et al., 1995; Castier et al., 1998). The presence of four contiguous guanines in the c-myb (Villa et al., 1995; Burgess et al., 1995; Castier et al., 1998) and the c-myc (Burgess et al., 1995; Saijo et al., 1997) antisense oligodeoxynucleotides, and in otherwise random oligodeoxynucleotides (Castier et al., 1998; Saijo et al., 1997) was responsible for their antiproliferative activity. Such runs of G residues permit the formation of intermolecular G-quartets which stabilize oligodeoxynucleotide quadruplexes, and it was intimated that these phosphorothioate quadruplex structures were responsible for at least some of the aptameric activities observed. On the other hand, it has been suggested that the c-myc and c-myb antisense phosphorothioate oligodeoxynucleotides are incapable of forming intermolecular G-tetrad stabilized quadruplexes under physiological ionic and temperature conditions (Basu and Wickstrom, 1997). However, other studies have indicated that pre-formed phosphorothioate intermolecular quadruplexes are quite stable under such conditions (50–80% quadruplex remaining at six days; Wyatt et al., 1994). To complicate the picture further, it appears that non-G-quartet forming phosphorothioate oligonucleotides (e.g. S-dC28) may also potently inhibit smooth muscle cell proliferation in vitro and in vivo (Wang et al., 1996). The mechanism responsible for the biological effects of a number of ‘antisense’ phosphodiester and phosphorothioate oligomers, which contain motifs that admit the potential to form intermolecular quadruplexes, has been investigated. Phosphodiester oligodeoxynucleotides antisense to the vpr gene of HIV1 were shown to be potent inhibitors of HIV replication and presented CD spectra consistent with the formation of intermolecular quadruplexes. Heat denaturation prior to use or mutation of the G4 run, so as to inhibit G-quartet formation, was found to abolish activity (Tondelli et al., 1996). Antisense NB1 phosphorothioate oligodeoxynucleotides which contained G4 runs, but not sense or antisense analogues lacking G4 tracts, were found to alter the morphology and inhibit proliferation of normal breast epithelial cells without concom itant reduction in NB1 mRNA or protein expression. Further control compounds were synthesized and activity was found to correlate directly with the presence of four contiguous G residues. Examination of phosphodiester or alternating phosphodiester/phosphorothioate congeners revealed that activity also correlated with the degree of phosphorothioate substitution (Yaswen et al., 1993). It is not


clear whether these results reflected the relative nuclease stability of the phosphorothioate internucleoside linkage or indicated a requirement for the phosphorothioate backbone, i.e. a ‘phosphorothioate’ effect overlaying the Gquartet effect, in this system. Specific inhibition of human type II phospholipase A2 was shown to require both the phosphorothioate backbone and the G-quartet forming sequence. Nuclease-resistant, 2′-modified congeners with a phosphodiester backbone were inactive and heat denaturation of the phosphorothioate oligonucleotide stock, prior to use, abolished activity (C.F.Bennett et al., 1994). Phosphorothioate oligomers antisense to the mRNA of the RelA subunit of NF-κB have been shown to inhibit adhesion of a range of tumor cell lines to substrata, at the same time as down-regulating relA mRNA, and NF-κB and Sp 1 DNA binding activities by non-antisense mechanisms. For the most part, single base mismatches which destroyed the G4 tract in the antisense effector abolished both inhibition of adhesion and down-regulation of expression, whereas sequential 3′-end deletions, which retained the G4 run, were found to inhibit adhesion but not to reduce NF-κB DNA binding activity (Maltese et al., 1995). Further investigations were carried out using oligonucleotides with long GN tracts (N = 7 or 9), or mutations which interrupted the G4 run (Khaled et al., 1996), or 7-deazaG substitutions (which inhibit G quadruplex formation but not normal base-pairing; Benimetskaya et al., 1997). These confirmed that a significant proportion of the biological activity of the relA antisense effector resulted from its potential to form G-quartet structures that interacted with cell surface or extracellular proteins. An in vitro selected phosphorothioate octamer, S-T2G4T2, has been isolated by its ability to inhibit HIV1 replication efficiently (Wyatt et al., 1994). This compound was shown to form stable intermolecular quadruplex structures under physiological conditions. Nuclear magnetic resonance (NMR) of a similar compound (TG4T) demonstrated that the structure in solution comprised an allparallel arrangement of the phosphodiester backbones (Aboul-ela et al., 1994). Heat denaturation of S-T2G4T2 prior to use abolished anti-HIV l activity, confirming that the active species was a quadruplex. In addition, replacement of the phosphorothioate backbone with phosphodiester internucleoside linkages also abolished activity. Clearly, as for the phospholipase A2 system described above, the phosphorothioate internucleoside structure was required for activity since nuclease-resistant, quadruplex forming, α-phosphodiester congeners were equally inactive (Wyatt et al., 1994). It was shown that the mode of action of ST2G4T2 involved binding to the v3 loop of HIV1 gp120, thereby inhibiting adsorption of the virus onto CD4+ cells. Another oligonucleotide containing only T and G residues, but with the sequence GTG2TG3TG3TG3T and a phosphodiester backbone, has been described as a potent inhibitor of HIV1 integrase but an inefficient inhibitor of the gp120-CD4 interaction (Ojwang et al., 1994, 1995). The folded intramolecular quadruplex structure adopted by this molecule has been assessed by nondenaturing gel electrophoresis and NMR, and shown to possess two adjacent G-


quartets with the backbones aligned two parallel and two antiparallel (Rando et al., 1995). The partially phosphorothioate-protected analogue of this structure (a single phosphorothioate residue between the terminal and penultimate base at both the 5′ and the 3′ end) has been most extensively investigated. Comparison to a 10–15 fold less active compound, G2T2G2TGTG2T2G2 (thrombin binding aptamer; Bock et al., 1992), and intermediates between that and the parent oligodeoxynucleotide, which adopt similar three-dimensional structures (Macaya et al., 1993; Wang et al., 1993; Schultze et al., 1994), revealed that the precise sequence in the loops surrounding the G-quartets was important for interaction with HIV1 integrase (Mazumder et al., 1996). Detailed analysis of the mode of action indicated that the GTG2TG3TG3TG3T oligonucleotide bound directly to a zinc finger domain of the integrase (Mazumder et al., 1996) and that this inhibited oligomerization of the enzyme into the active complex (Cherepanov et al., 1997). However, it was also noted that a HIV1 isolate which was insensitive to this aptamer, in cell culture replication assays, did not possess a mutated integrase gene. In fact, the gp120 gene was mutant (Cherepanov et al., 1997), and thus it would appear that the major effect of this compound in cell culture is inhibition of viral adsorption to the cell surface. In addition to the oligodeoxynucleotide G-quartet aptamers (see also Chapter 14) that have been evolved using in vitro selection protocols (Bock et al., 1992; Wyatt et al., 1994), a number of RNA (or 2′-NH2 modified RNA) intramolecular quadruplex compounds have been selected which interact with specific proteins. Hamm (1996) demonstrated that a G-quartet RNA bound at, or very close to, the active site of an anti-ferritin antibody (H107) in vitro. Normal prion protein (PrPc) from hamster, mouse and cow, and recombinant prion protein (rPrP), but not PrPSc from scrapie-infected mice, was recognized by an RNA aptamer. The aptamer was shown to interact specifically with the N terminus of rPrP and the intramolecular quadruplex (composed of three adjacent G-quartets) was shown to be essential for the interaction (Weiss et al., 1997). A 2′-NH2 nuclease protected RNA aptamer was evolved (Wiegand et al., 1996) which bound to human immunoglobulin E (IgE) with a Kd of ~30 nM. The dissociation constants for mouse and rat IgE or human IgG were found to be at least 300 and 600 fold higher, respectively. Binding of the RNA aptamer to human IgE inhibited the interaction between the antibody and its receptor, FcεRI on RBL SX-38 cells and inhibition of the antibody-receptor binding on sensitized RBL SX-38 cells inhibited serotonin release in cell culture. G-quartet forming oligodeoxynucleotides and oligoribonucleotides are capable of forming intermolecular and intramolecular quadruplexes. The quadruplexes facilitate binding to proteins, and protein binding may result in substantial biological effects. The specific protein bound by a given quadruplex structure appears to depend in part on the backbone structure of the oligonucleotide and in part on the sequence context of the quadruplex. It may be that the quadruplex defines a rigid structure that permits reproducible presentation of the aptamer to its protein binding site and that the specificity of interaction depends on other


aspects of the chemistry of the compound. However, there appears to be no way to predict, a priori, what the protein binding specificity of a given quadruplex structure will be. 1.5 Immune Stimulation by Oligodeoxynucleotides Containing CpG Motifs Krieg (1998) has reviewed the original reports of unexpected, base sequencespecific, immune stimulation, B-cell proliferation, and immunoglobulin secretion induced by antisense and control sense phosphorothioate oligodeoxynucleotides. Intrigued by the profound magnitude of the immune stimulation, he synthesized and tested several hundred oligodeoxynucleotides to identify the base sequences responsible. These results established that all the previously observed effects could be accounted for by the presence in the oligodeoxynucleotides of a simple motif based on a cytosine phosphate guanine (CpG) dinucleotide in the optimal consensus sequence context, ‘R1R2CGY1Y2, where R1 is a purine (mild preference for G), R2 is a purine or T (preference for A; T can be substituted with minimal loss of activity if the rest of the motif is intact), and Y1 and Y2 are pyrimidines (preference for T)’ (Krieg et al., 1995; Krieg, 1998). The effects of the motif were enhanced when preceded on the 5′side by T, whereas a C on the 5′-side of the CpG greatly reduced activity. The optimal minimal sequence that would stimulate B-cells was TCAACGTT. Krieg (1998) has re-evaluated his own previous, apparently successful antisense experiments, where immune stimulation was the expected result, as well as those of others, in terms of the possible non-antisense, involvement of active CpG motifs. In addition, although phosphorothioate oligodeoxynucleotides containing CpG motifs were reported to exhibit immune stimulatory activity while the corresponding phosphodiester oligomers were inactive, the differences may have been more apparent than real, and related to the greater biological stability of the former. Phosphodiester oligodeoxynucleotides with CpG motifs mediated B-cell activation in media containing foetal calf serum which had been heat inactivated at 65°C for 30 minutes to reduce nucleases. Oligodeoxynucleotide-induced B-cell activation was achieved without any specific intervention to secure intracytoplasmic delivery of the oligomers, and consequently it is almost certainly the result of interactions at the cell surface or within endosomes. However, Krieg (1998) has reported that active oligodeoxynucleotides failed to trigger B-cell proliferation when immobilized on surfaces or on beads. Apart from the significance of the above findings in terms of their impact on antisense strategies, they have broader biological implications. CpG dinucleotides are far less prevalent in vertebrate genomes as compared to bacteria, and where they occur they are often methylated on the 5 position of the cytosine. Krieg et al. (1995) have advanced the hypothesis that the established immunostimulatory activity of bacterial DNA is mediated through detection of


unmethylated CpG motifs by B lymphocytes, and that the lymphoproliferative response may represent a first line, innate immune defence against infection when such DNA is detected in host tissues. In support of their hypothesis they demonstrated that methylation of bacterial DNA abolished its mitogenicity. Likewise, replacement of cytosine by 5-methylcytosine in CpG motifs of B-cell mitogenic oligodeoxynucleotides led to loss of their activity, whereas molecules in which other cytosines were methylated retained their stimulatory properties. 1.6 Other Activities of Oligodeoxynucleotides Containing CpG Motifs In addition to the immune stimulatory properties of CpG containing oligodeoxynucleotides, Krieg et al. (1997) have identified a sequence motif (CGT [C]GA) in phosphorothioate modified oligodeoxynucleotides that specifically inhibited the enzymatic activity of several recombinant or immunoprecipitated protein tyrosine kinases in vitro, without affecting the enzyme activity of a serine/threonine protein kinase. It is conceivable that antisense oligodeoxynucleotides containing these sequence motifs might have profound effects when introduced into cells, which would be unrelated to any suppression of the targeted gene. In our own work, we have observed that suitably endprotected phosphodiester oligodeoxynucleotides containing CpG motifs, rather than stimulating proliferation, rapidly induced fulminating apoptosis, when delivered into the cytoplasm of human leukaemia cells by reversible plasma membrane permeabilization with streptolysin O (unpublished observations). The minimum length of an oligodeoxynucleotide required to elicit this effect was just five bases, and inversion of the sequence from 5′–3′ to 3′–5′ completely abolished activity, as did mutation of the CpG dinucleotide. However, oligodeoxynucleotides in which the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine retained the ability to induce apoptosis. 1.7 Antisense Inhibition of Gene Expression by Steric Block Early investigations of the potential for inhibiting gene expression with antisense oligonucleotides were founded on the premise that their hybridization to the complementary site in the target mRNA might present a block to the passage of the translating ribosome complex. Such a mechanism, which was referred to as hybrid arrest of translation, was invoked to account for apparent antisense effects of oligonucleotides and cDNA in early work on cell-free systems and microinjected Xenopus oocytes (Paterson et al., 1977; Kawasaki, 1985). However, other experimental results demonstrated that the fully assembled ribosomal complex is able to locally destabilize secondary structures as it moves along the message such that DNA antisense to the translated region of the mRNA was


without effect, and that hybridization to part of the 5′-untranslated region extending from the cap site to the initiation codon was required for inhibition of translation (Liebhaber et al., 1984; Shakin and Liebhaber, 1986). These conflicting results in cell-free protein synthesizing systems were resolved by the observations that where inhibition of translation was achieved with antisense DNA targeting the coding region of mRNA, the latter was found to have been degraded during the course of the incubations (Haeuptle et al., 1986; Minshull and Hunt, 1986). The enzyme present in the cell extracts responsible for elimination of intact mRNA was shown to be ribonuclease H (RNase H) (Haeuptle et al., 1986; Minshull and Hunt, 1986; Cazenave et al., 1987; Walder and Walder, 1988), a ubiquitous activity that catalyses the hydrolysis of RNA in RNA—DNA heteroduplexes (Hausen and Stein, 1970). Degradation of mRNA by RNase H was also shown to contribute to inhibition of cell-free protein synthesis achieved by antisense oligodeoxynucleotides targeting 5′-untranslated and translation initiation codon regions (Walder and Walder, 1988; Cazenave et al., 1987). However, oligonucleotide analogues that do not form substrate hybrids for RNase H, such as a-anomeric (Bertrand et al., 1989; Boiziau et al., 1991) or morpholino (Partridge et al., 1996; Summerton et al., 1997) structures, may block translation in cell-free and cellular systems through hybridization to 5′-untranslated and initiation codon sites. Such activity apparently results from their preventing formation of the translation initiation complex, and they are generally inactive when targeted further downstream. The only exception to the rule would appear to be for RNase H-inactive, peptide nucleic acid oligomers (see also Chapter 4), oligonucleotide analogues in which a polyamide structure replaces the phosphodiester backbone. When these are synthesized as triplex forming homopyrimidine base sequences, they have sufficient affinity for RNA that they are able to withstand the unwinding activity of the fully assembled ribosomal complex, and are thus able to inhibit peptide chain elongation through a non-RNase H mechanism (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Another approach to inhibition of gene expression with antisense oligonucleotides which may exploit a steric blocking mechanism is through targeting splice sites in pre-mRNA. There have been several reports of the application of splice site antisense oligonudeotides, but in the absence of definitive evidence, it is not possible to conclude that their observed biological effects on living cells were necessarily induced through inhibition of maturation of the targeted pre-mRNA. However, Kole (1998) has provided convincing evidence for inhibition of splicing by RNase H-inactive 2′-O-methyloligoribonucleotides in nuclear splicing extracts from HeLa cells, and by phosphorothioate 2′-O-methyl-oligoribonucleotides delivered into HeLa cells by lipofection with Lipofectamine. In this case the strategy was somewhat different from that of the usual antisense approach, since the oligonudeotides were targeted at cryptic splice sites activated by mutation in introns of the β-globin gene, which are responsible for a majority of β-thalassemia cases worldwide.


Oligonucleotide-induced restitution of normal pre-mRNA splicing and normal βglobin synthesis in HeLa cells transfected with mutant constructs confirmed that steric blocks to splicing at the cryptic sites were established within living cells. We have observed strong evidence for the inhibition of gene expression with a splice site targeted antisense oligonucleotide in living cells. A 28-mer RNase Hinactive, morpholino oligonucleotide analogue, antisense to human c-myc premRNA across the intron 1/exon 2 boundary and initiation codon, induced the accumulation of high molecular weight, myc probe hybridizing RNA species, when introduced into a number of human leukaemia cell lines by reversible plasma membrane permeabilization with streptolysin O (Giles et al., 1999). The p64 Myc protein had completely disappeared from the cells by four hours and a Myc epitope reactive protein of approximate molecular weight 47 kDa was readily apparent on Western blots by 24 hours. However, mature c-myc mRNA seen on Northern blots was apparently unaffected throughout, if not somewhat increased in amount. Kole (1998) has suggested that accumulation of unspliced RNA is unlikely to occur even if splice sites are blocked by oligonudeotides that do not support RNase H, since the likely outcome of the oligonucleotide’s action would be skipping of the exon or removal of a portion of the exon due to activation of a cryptic splice site (Mayeda et al., 1990). Subsequent reverse transcription—polymerase chain reaction (RT-PCR) amplification of c-myc mRNA followed by DNA sequencing demonstrated that the morpholino oligonucleotide had indeed forced the splicing machinery to skip the normal acceptor site at exon 2 in favour of a cryptic site downstream of the initiation codon within the exon. The aberrant, c-myc epitope bearing, 47 kDa protein was probably the product of a fortuitously in-frame initiation of translation at an AUG codon within exon 2 of the truncated c-myc mRNA. However, the inhibition of splicing at the normal site was not complete, and therefore, the total loss of p64 Myc protein was probably the result of the combined effects of steric blocks to both splicing of pre-mRNA and initiation of translation from normally spliced mRNA. 1.8 Antisense Inhibition of Gene Expression through Ribonuclease H-mediated Destruction of Target mRNA Most antisense oligonucleotide analogues are unable to direct RNase H, and have generally proved less effective inhibitors of translation in cell-free systems and microinjected cells than RNase H-active oligomers that induce irreversible destruction of the message, even when targeting the cap, 5′-untranslated, and initiation codon sites on the mRNA (Bonham et al., 1995). At low concentrations, phosphorothioate oligodeoxynucleotide analogues do form hybrids with RNA which are still recognized as substrates by RNase H, but at higher concentrations the analogue oligomers apparently bind non-specifically to the enzyme and inhibit its activity (Gao et al., 1992). The protein-binding avidity


of phosphorothioate oligodeoxynucleotides seemed once again to counter their ability to induce sequence-specific antisense suppression of gene expression, by obstructing the very enzyme that would mediate it. Following the discovery of the involvement of RNase H in the antisense oligodeoxynucleotide-induced inhibition of protein synthesis in cell-free systems, it became generally assumed that the enzyme activity was also responsible for at least some of the biological effects of oligodeoxynucleotides on living cells. However, tell-tale fragments of the targeted mRNA which should have been produced by RNase H-mediated cleavage could not be detected in RNA isolates from treated cells. This led to a general consensus that such fragments would be extremely unstable in the biological milieu and would be degraded by ribonucleases as rapidly as they were produced. In fact, failure to detect mRNA cleavage products probably indicated that the observed biological effects of the antisense oligodeoxynucleotides were not the result of their hybridization to the complementary regions in mRNAs within the cells. RNase H-generated fragments of mRNA are actually readily detectable on Northern blots of RNA isolated at early times following the introduction of antisense oligodeoxynucleotides into cells by lipofection with cationic lipids, electroporation or reversible plasma membrane permeabilization with streptolysin O (Dean et al., 1998; Marcusson et al., 1998; Giles et al., 1995a, 1995c; Spiller et al., 1998b). RNase H cleaves RNA to produce 3′-fragments bearing phosphate groups at their 5′-ends (Eder et al., 1993). The identity of such fragments may be positively confirmed by DNA sequencing of their reverse ligation-mediated polymerase chain reaction (RLPCR) products, derived without an intermediate polynucleotide kinase phosphorylation step prior to ligation to the RNA linker (Bertrand et al., 1993). RNA was isolated from human leukaemia cells previously permeabilized with streptolysin O in the presence of antisense oligodeoxynucleotides, and samples were subjected to ligation reactions with an oligoribonucleotide of known sequence, such that any RNase Hgenerated 3′-fragments present would be tagged with the linker at their 5′-ends. The RNA was then reverse transcribed from primers specific for the antisense targeted genes and the cDNA products amplified by polymerase chain reactions using nested, gene-specific 3′-primers and linker sequence 5′-primer. Subsequent DNA sequencing of the amplified products confirmed not only that endogenous RNase H of the leukaemia cells had cleaved the targeted mRNAs, but also that cleavage had occurred in vivo at the site of complementarity to each of the antisense oligodeoxynucleotides (Giles et al., 1995a, 1995c). It has generally been the case that early RNase H-mediated antisense effects of phosphorothioate oligodeoxynucleotides, delivered into cells by streptolysin O permeabilization, have been less dramatic, in terms of the extent of ablation of the target mRNA, than those of the corresponding molecules with normal phosphodiester internucleoside linkages (Giles et al., 1995a, 1995b, 1995c; Spiller et al., 1998a). Indeed, in a comparison of mixed structure, antisense oligonucleotides, the extent of loss of efficacy correlated with the degree of


phosphorothioate substitution in the molecules (Giles et al., 1998). This is more likely due to sequestration of phosphorothioates through binding to cellular proteins, with less being freely available to interact with target mRNA, than to non-specific inhibition of RNase H. Of course, it is not only the immediate extent to which the target mRNA is ablated that is important if phenotypic effects of antisense inhibition of gene expression are to ensue. It is also essential that antisense activity be maintained for a sufficient duration to enable the pre-existing protein product of the gene to decay by natural turnover (Spiller et al., 1998a). Normal phosphodiester oligodeoxynucleotides, even when 3′-end protected against exonuclease with a 3hydroxypropyl phosphate group, are rapidly degraded by nucleases in living cells and their potent, RNase H-mediated antisense effects on mRNA are short-lived. At the same time, nuclease-resistant oligodeoxynucleotide analogues other than phosphorothioates are unable to recruit RNase H activity to cleave the target mRNA. Our solution to this problem has been to replace several internucleoside linkages at both ends of a phosphodiester oligodeoxynucleotide by nucleaseresistant, non-ionic methylphosphonate groups, while retaining a central, RNase H-active, phosphodiester section (Tidd and Warenius, 1989). The methylphosphonate modification (Miller and Ts’o, 1987; Miller, 1998) served to protect the oligodeoxynucleotides against exonuclease attack when incorporated into such chimeric molecules, while at the same time reducing their susceptibility to endonuclease-initiated degradation (Giles et al., 1995b). The methylphosphonate sections of the oligodeoxynucleotides are unable to direct RNase H activity against the target mRNA (Maher and Dolnick, 1987; Furdon et al., 1989; Quartin et al., 1989), and therefore serve to focus enzyme activity to the central section which may contribute towards achieving single base mismatch specificity against point mutations (Giles et al., 1995a). The concept of using antisense chimeric structures of non-activating analogue and enzyme directing phosphodiester oligodeoxynucleotide to achieve site-directed RNase H cleavage of RNA was originally developed by Ohtsuka’s group (Inoue et al., 1987b; Shibahara et al., 1987). They utilized the 2′-O-methyl analogue of RNA (Inoue et al., 1987a) in chimeras with normal phosphodiester oligodeoxynucleotide and demonstrated RNase H-mediated cleavage in vitro at a single internucleoside linkage in target RNA when the phosphodiester section was sufficiently short. However, the 2′-O-methyl-oligoribonucleotide structure was apparently of insufficient resistance to nucleases for effective application against living cells (Shibahara et al., 1989). In a recent comparison of the activities of c-myc antisense, methylphosphonodiester/phosphodiester oligodeoxynucleotides and chimeric oligonucleotides with the more nuclease-resistant 2′-Oalkyloligoribonucleotide wings, having 2′-methoxyethoxy or 2′methoxytriethoxy substituents, and either phosphodiester or phosphorothioate internucleoside linkages, the former proved to be greatly superior in promoting RNase H-mediated ablation of c-myc mRNA in living human leukaemia cells (Giles et al., 1998). It may well be that the 2′-O-alkyl substituents distort the


helix such that hybrids between RNA and chimeric oligonucleotides with these structures are less efficient substrates for RNase H (Crooke et al., 1995), than duplexes formed between RNA and methylphosphonodiester/phosphodiester oligodeoxynucleotides. In addition, the 2′-O-alkyl modifications are helixstabilizing (Cummins et al., 1995; Monia et al., 1996), which tends to counter the activity of RNase H. Measurements of the hybridization potential of methylphosphonate oligodeoxynucleotide analogues indicated that, while replacement of phosphodiester internucleoside linkages by methylphosphonate was moderately helix-destabilizing for complexes with a complementary phosphodiester oligodeoxynucleotide as the second strand, the modification induced profound reductions in stability of hybrids with RNA oligomers (Tidd, 1990; Giles and Tidd, 1992a). Hybrids of methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides with sense oligoribonucleotides exhibited melting temperatures (Tm) for half dissociation of the hybrids that were intermediate between those of their allphosphodiester and all-methylphosphonate counterparts, and were directly related to the degree of methylphosphonate substitution. However, the reduced hybridization potential of such chimeric oligodeoxynucleotides correlated inversely with increases rather than decreases in the initial rates of hydrolysis of the complementary oligoribonucleotide in the presence of Escherichia coli RNase H (Tidd, 1990; Giles and Tidd, 1992a). Possibly their lower affinity enhanced turnover rates for the enzyme by facilitating dissociation of enzyme—product complexes. Whatever the mechanism, it was evident that avid binding to target mRNA need not necessarily be of prime concern for optimizing antisense oligonucleotide activity, when this is mediated by RNase H. The last point was brought home by experiments in cell-free systems with in vitro transcribed, high molecular weight RNAs. Phosphodiester and phosphorothioate antisense oligodeoxynucleotides induced fragmentation of the RNAs by E.coli RNase H and RNase H activity in human leukaemia crude cell extracts, not only at the target site of full base complementarity, but also at nontargeted sites of partial complementarity within the same and different RNA molecules (Giles and Tidd, 1992b; Giles et al., 1993, 1995a, 1995b). Similar observations by others caused general concern about the ability to achieve specific antisense effects on gene expression with oligodeoxynucleotides under constant physiological conditions in living cells. Herschlag (1991) discussed helix destabilization as a means to enhance the specificity of ribozymes and antisense oligonucleotides, and Woolf et al. (1992) suggested the need ‘to devise chemically modified oligomers that hybridize less strongly’ on the basis of their demonstration of RNase H cleavage of RNAs at imperfectly matched target sites in Xenopus oocytes microinjected with antisense oligodeoxynucleotides. In fact, the helix-destabilizing property of the methylphosphonate substitution in chimeric, methylphosphonodiester/ phosphodiester, antisense oligodeoxynucleotides goes some way towards achieving this function, and


improved specificity for cleavage of RNA at the target site was observed with such antisense effectors in assays with E.coli, human and wheat germ RNase H (Giles and Tidd, 1992b; Giles et al., 1993, 1995a, 1995b; Larrouy et al., 1992). That the gain in specificity was due to increased stringency of hybridization was demonstrated by the observation that corresponding 2′-O-alkyloligoribonucleotide/phosphodiester oligodeoxynucleotide chimeras antisense to α-globin, with enhanced rather than reduced hybridization potential, directed non-targeted cleavage of β-globin mRNA by RNase H, and did not elicit similar selective inhibition of α-globin synthesis in the same in vitro protein synthesizing system (Larrouy et al., 1995). However, in using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides to achieve single base mismatch discrimination in targeting point mutations in p53 mRNA (Giles et al., 1995a; Ruddell et al., 1996), as well as stringency of hybridization achieved through the methylphosphonate modifications, there would appear to be a contribution from the mismatch at the central phosphodiester section in reducing RNase H activity. Corresponding mutant and wild type, chimeric, antisense oligomers with 2′-Omethyl oligoribonucleotide modifications exhibited a degree of discrimination between perfect and partial complementarity in the extent of RNase H-mediated ablation of the p53 mRNAs. Essentially, complete discrimination between wildtype and Harlow point mutant sequences for cleavage of p53 mRNA was achieved in living MOLT-4, human leukaemia cells following streptolysin Omediated delivery of 15-mer chimeric oligodeoxynucleotides with three methylphosphonate linkages at each end and antisense base sequences centred on codon 273. However, it would seem that secondary and tertiary structure in this region of the mRNA contributed to the achievement of this degree of specificity by effectively competing out binding by the mismatched oligomer. In targeting a point mutation in an apparently more readily accessible region of p53 mRNA at codon 248, in KYO1 leukaemia cells, further methylphosphonate substitutions were required in order to destabilize the interaction between the chimeric antisense oligodeoxynucleotides and the mRNA, and thereby to maximize the difference in RNase H cleavage activity at this site induced by mutant and wild type sequences. The degree of accessibility of the chosen target site within an mRNA can have a profound effect on the level of activity of an antisense oligodeoxynucleotide. The b3a2 junction in bcr-abl mRNA of human chronic myeloid leukaemia would appear to be less readily accessible than the b2a2 junction, and antisense oligodeoxynucleotides of equivalent structure consistently produced greater reductions in b2a2 mRNA than b3a2 mRNA, when introduced into leukaemic cells (Giles et al., 1995b). Also, the popular translation initiation region for targeting c-myc mRNA is in fact a poor antisense target site. Profound reductions in intracellular levels of c-myc mRNA and protein were achieved shortly after reversible plasma membrane permeabilization of human leukaemia cells with streptolysin O in the presence of comparatively high concentrations of a chimeric


15-mer oligodeoxynucleotide with three methylphosphonate internucleoside linkages at each end and base sequence complementary to codons 1–5 (Giles et al., 1997; Spiller et al., 1998a). However, the effects were short-lived, the mRNA and protein began to recover by four hours as degradation of the antisense effector proceeded by endonucleolytic attack, and the proliferation of the cells was essentially unaffected. In addition, replacement of just one more phosphodiester linkage by a helix-destabilizing methylphosphonate effectively abolished antisense activity of the chimeric oligodeoxynucleotide, suggesting that the target was involved in secondary and tertiary structure, which the oligomer no longer had the hybridization potential to invade (Spiller et al., 1998a). In contrast, a methylphosphonodiester/phosphodiester chimeric oligodeoxynucleotide with just six phosphodiester linkages exhibited high activity against the accessible p53 codon 248 site. However, it was evident that a site in the middle of c-myc mRNA (bases 1147–1166, ‘HSMYC1’, GenBank accession number V00568) that had previously shown exquisite sensitivity to non-targeted, RNase H cleavage, through partial complementarity to the initiation codon antisense sequence (Giles and Tidd, 1992b; Giles et al., 1993), must necessarily be quite accessible. Chimeric methylphosphonodiester/ phosphodiester, 20-mer oligodeoxynucleotides complementary to this site were shown to suppress c-myc mRNA and protein expression in human leukaemia cells for more than 24 hours, during which time cell proliferation ceased and cells started to die (Giles et al., 1998; Spiller et al., 1998a). Even a chimeric effector with just four phosphodiester linkages induced essentially undiminished RNase H cleavage activity (unpublished observations). Dose—response curves showed substantial antisense effects on c-myc mRNA and protein persisting for more than 24 hours when cells were initially permeabilized in the presence of concentrations of oligodeoxynucleotide as low as 0.2 µM (Giles et al., 1998). Evidently, very low concentrations of an antisense oligodeoxynucleotide are able to sustain high rates of RNase H-mediated cleavage of the target mRNA if the complementary region is located in an open loop which is freely available for hybridization. Therefore, sustained inhibition of gene expression may be achieved by targeting an accessible site, since the threshold level of intact chimeric oligodeoxynucleotide required for effective ablation of the mRNA will be low and will be exceeded for longer in the cell as the oligomer is degraded. These experiments also highlighted another reason why it is essential to locate open loops in mRNA if sequence-specific antisense effects resulting from inhibition of expression of the selected gene alone are to be achieved. The oligodeoxynucleotides targeted to c-myc, bases 1147–1166, were partially complementary to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (bases 965–975, 10 base pairs in total and including nine contiguous base pairs, ‘HSGAPDR’, GenBank accession number X01677), and permeabilization of human leukaemia cells with streptolysin O in the presence of 20 µM chimeric cmyc antisense effector resulted in total ablation, not only of c-myc mRNA, but also of GAPDH mRNA (Giles et al., 1998), and probably the mRNAs of several


other genes which were not assayed. However, the non-targeted cleavage of GAPDH mRNA was reduced to more acceptable levels by lowering the concentration of the oligodeoxynucleotide, without seriously affecting activity against c-myc. Therefore, the identification of good antisense sites in the mRNA of the gene of interest may permit a reduction in the concentration of antisense oligodeoxynucleotide required to induce the desired effect, which may at the same time minimize the extent of non-targeted, RNase H-mediated cleavage of other mRNAs through their partial complementarity to the antisense effector. Undesired side-effects may also be countered by reducing the central phosphodiester section of chimeric oligodeoxynucleotides to the minimum compatible with RNase H activity, although this did not appear to ameliorate the situation with regard to the c-myc antisense sequence and GAPDH mRNA (unpublished observations). Attempts to predict open loop regions in mRNA secondary structure using computer RNA folding routines have not been particularly successful (Giles et al., 1998; Spiller et al., 1998a). Stull et al. (1996) have described a gel shift assay for determining the efficiency of binding of a series of complementary radiolabelled oligodeoxynucleotides to in vitro transcribed RNA in order to identify accessible sites. A similar type of approach has been to measure binding of RNA to large oligodeoxynucleotide arrays as an empirical method for selecting effective antisense oligodeoxynucleotides (Milner et al., 1997; Southern et al., 1997). Accessible sites on in vitro transcribed RNA have also been identified in cell-free assays with RNase H and oligonucleotide libraries (Ho et al., 1996, 1998). However, there are doubts about the validity of using naked RNAs in in vitro assays as surrogates for intracellular RNAs, which exist in interaction with proteins (Branch, 1998). In the absence of a rational strategy for determining good antisense sites in mRNAs, an empirical approach using living cells would appear to be the optimum. Dean et al. (1998) have adopted this method by simply synthesizing a series of phosphorothioate oligodeoxynucleotide sequences designed to hybridize to multiple sites throughout the target mRNA, and evaluating them for their ability to reduce its expression. However, this approach should be treated with caution as it retains the potential to select sequences which induce potent down-regulation of the mRNA and protein studied by non-antisense aptameric effects, such as those obtained with RelA/NF-κB antisense oligonucleotides (see above). In our own work, we have synthesized selected sequences as normal phosphodiester oligodeoxynucleotides, end-protected against exonuclease degradation by 3′hydroxypropyl phosphate and 5′-fluorescein groups, where the latter also served as a reporter to monitor cell delivery. Sequences inducing substantial RNase Hmediated cleavage of the target mRNA, within 30 minutes after reversible membrane permeabilization of the cells with streptolysin O, were resynthesized for further evaluation as the more nuclease-resistant, methylphosphonodiester/ phosphodiester, chimeric antisense effectors.


Ablation of target mRNA occurs rapidly after introduction of antisense oligodeoxynucleotides into cells (Giles et al., 1997; Spiller et al., 1998a), but development of the phenotypic effects of blocking expression of a gene requires that its pre-existing protein product be degraded by the normal mechanisms of protein turnover within the cell. In the case of Myc protein, which has a very short half-life of around 10 minutes, the antisense oligodeoxynucleotide-induced reduction in its intracellular concentration closely followed the reduction in the level of c-myc mRNA (Giles et al., 1997; Spiller et al., 1998a). However, drastic reduction in mutant p53 mRNA in human chronic myeloid leukaemia KYO1 cells produced a much delayed and more modest decrease in p53 protein, as a result of the comparatively long half-life of the mutant protein of approximately eight hours (Spiller et al., 1998a). Endonuclease-mediated degradation of the chimeric, methylphosphonodiester/phosphodiester, p53 antisense oligodeoxynucleotide was such that levels of p53 mRNA were well on the way to recovering before all pre-existing p53 protein had time to decay. Methylphosphonodiester/ phosphorothiodiester, chimeric oligodeoxynucleotides exhibit greater nuclease resistance than their chimeric phosphodiester counterparts, but bcr-abl antisense oligomers with this structure had previously shown no activity against the relatively inaccessible b3a2 junction in mRNA in chronic myeloid leukaemia cells (Giles et al., 1995b). This was probably the result of partial sequestration of the molecules through protein binding via the phosphorothioate section, coupled with the combined helix-destabilizing effects of methylphosphonate and phosphorothioate substitutions reducing the hybridization potential of the oligomers below that required to invade the secondary structure at the b3a2 site. However, the mutant codon 248 site in p53 mRNA was apparently more readily accessible, and in this case a p53 antisense methylphosphonodiester/phosphorothiodiester oligodeoxynucleotide targeted to this site was able to maintain reduced p53 mRNA concentrations in KYO1 cells for sufficiently long that p53 protein decayed to low levels in the cells (Spiller et al., 1998a). On the other hand, experiments in which the general protein synthesis inhibitor cycloheximide was applied to KYO1 cells indicated that p210 Bcr-Abl protein had an apparent half-life in excess of 48 hours, and no effects whatsoever on the intracellular concentration of this protein were observed with any antisense oligodeoxynucleotide structure targeting bcrabl mRNA. 1.9 Oligonucleotides in Vivo There appears to be an increasing move towards using antisense oligonucleotides in animal models of disease, relegating cell culture work to the role of identifying compounds optimally active at suppressing the expression of the gene of interest. One of the ideas underlying this trend is that oligonucleotide uptake into cells in vivo may be completely different from that observed with cultured cells. Furthermore, targets may be identified where antisense suppression


actually alters the progression of a given disease in the organism, rather than targets which may be predicted to do so from in vitro work. Clearly, any compound found to produce a therapeutic advantage in such systems is of great interest, whatever the actual mechanism of action. However, it is vital to differentiate between real antisense effects and aptameric, and other nonantisense mechanisms so that rational progress may be maintained in targeting other genes in other disease states. One way that an antisense mechanism may be demonstrated to be at least part of the overall activity of an oligonucleotide in vivo is by RLPCR detection of mRNA 3′-fragments resulting from RNase H cleavage at the targeted site of RNA-oligomer hybridization, as described above for cell culture models. However, certain caveats need to be borne in mind. It is important that RLPCR is performed on RNA extracted from viable cells, as oligodeoxynucleotides rapidly redistribute to intracellular locations when the integrity of the cytoplasmic membrane is violated (Tidd, 1998), and RNase H activity is readily observed when cells are Iysed at room temperature under non-denaturing conditions in the presence of antisense compounds (Giles et al., 1995c). Therefore, care should be taken to address these concerns when samples are taken from a biopsy, and RNA should be extracted using a protocol which efficiently denatures proteins. DNA sequencing provides the means to identify positively any amplified fragments produced when the extracted RNA samples are subjected to the RLPCR procedure. 1.10 Conclusions The variety of potential mechanisms of action of antisense oligonucleotides makes it difficult to assign their biological effects to inhibition of expression of the targeted gene with any degree of certainty, even when the protein product of that gene is shown to have been suppressed. The question of the appropriate controls for antisense experiments is also still a matter of some debate (see Chapter 9 this volume) (Stein and Krieg, 1994; Wagner, 1995; Tidd, 1996, 1998), with certain participants arguing that each and every oligonucleotide is unique, and no ideal control exists (Neckers and Iyer, 1998). However, it would seem that inhibition of expression of a particular gene would be more strongly implicated in a biological response if the same phenotypic effects were induced by targeting different sites in its pre-mRNA/mRNA with both RNase H inactive, steric blocking, and RNase H active, antisense oligonucleotides. Acknowledgements The authors’ research is supported by the Leukaemia Research Fund of the UK and The Liposome Company, Inc., Princeton, NJ, USA.


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PART TWO Chemical Aspects

2 Chemistry of Oligonucleotides J.W.ENGELS AND E.UHLMANN

2.1 Introduction Oligonucleotides as drugs have attracted substantial interest in recent years. Sequencespecific interference with RNA function by complementary Oligonucleotides, as proposed by Belikowa et al. (1973) and experimentally investigated by Stephenson and Zamecnik (1978) and Zamecnik and Stephenson (1978), resulted in the ‘antisense approach’. Here the expression of a specific gene is blocked on the mRNA level by a complementary oligonucleotide called antisense oligonucleotide (Figure 2.1). Hybridization, according to Watson-Crick base pairing, should in theory provide high specificity and affinity. Thus Zamecnik and Stephenson (1978) could inhibit Rous sarcoma replication in a cellular assay. In these early days, the available chemistry and the inaccessibility of a large pool of synthetic Oligonucleotides, respectively, limited progress in this field. Some years later, the discovery of catalytically active RNA—so-called ribozymes—that can cleave in either cis or trans has created much interest. Their development as therapeutics has so far been limited to hammerhead and hairpin ribozymes. For practical purposes we shall limit our discussion to synthetic hammerhead ribozymes. The hammerhead ribozyme, originally found in the avocado sunblotch virus (Hutchins et al., 1986), owes its name to the two-dimensional description (Figure 2.2). Now, based on two X-ray structures (Pley et al., 1994; Scott et al., 1995), the two colinear helices, helices II and III, mean that it would be better described as ‘paperclip’. The hammerhead ribozyme catalyses a phosphodiester transesterification reaction at a specific NUH triplex (shown in italics in Figure 2.2) within a given RNA sequence. The final product is a 2′-3′-cyclic phosphate and a fragment with a 5′-hydroxyl group. Magnesium ions are essential for that cleavage (Uhlenbeck, 1987). Although conceptually appealing, antisense or ribozyme action does not play a major role in natural gene regulation. Here the regulation at the level

34 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.1 Schematic presentation of a cell with the antigene, antisense ribozyme and sense/aptamer approach

Figure 2.2 Hammerhead consensus sequence, conserved nucleotides in bold: N=A, C, G, U; R=A, G; H=A, C, U; numbering according to Hertel et al. (1992)

of transcription initiation is clearly favoured. A complex interaction of various transcription factors and the DNA itself guarantees the sequence-specific recognition. To date no simple polypeptide can recognize a particular DNA in a practical fashion. As early as 1957 (Felsenfeld and Miles, 1957) a triple-stranded nucleic acid structure was first reported.


Figure 2.3 Triple helices, Hoogsteen base pairing: above, pyrimidine motif (parallel); below, purine motif (antiparallel)

The basic recognition principle of the triplex is the Hoogsteen base pairing scheme (Figure 2.3). Here the major groove purine site is additionally involved in base pairing. Since there are several motifs, the TAT and C+GC as well as the GGC and AAT, the third strand can orient in either a parallel or an antiparallel fashion. With this concept in mind, it is obvious that so far only purine strands can be targeted. Furthermore, the thermodynamic stability of Hoogsteen base pairs is poorer than that of Watson-Crick base pairs. Therefore, triplex binders for physiological studies are either longer or have additional stabilizing features as compared to antisense agents. Sequence-specific recognition of DNA based on the pyrimidine motif was successfully shown by Moser and Dervan (1987). The recognition was detected by tethering an Fe(II)-EDTA chelate to the oligodeoxynucleotide (ODN) and observation of strand cleavage by footprinting techniques. Le Doan et al. (1987) shortly thereafter observed triplex formation using photocrosslinking techniques with psoralene. For the purine recognition motif, Cooney et al. (1988) demonstrated binding of a guanosine-rich ODN to a guanosine-rich promoter element of the human c-myc gene. An ODN with superior affinity to a complementary DNA strand can in theory replace one strand in a DNA duplex. Homopyrimidine peptide nucleic acid (PNA) (see also Chapter 4) binds under low salt condition to one strand of an AT


DNA-duplex forming a triple helix, thus leaving the second strand looped (Ploop). Table 2.1 Degree and type of modification: (A) 3′-end-capped; (B) 3′5′-end-capped; (C) minimally modified, which is 3′5′-end-capping plus protection at internal pyrimidines; (D) all-phosphorothioate; (E) all-phosphorothioate ‘gap-mer’ with 2′-Omethylribonucleoside wings and a seven deoxynucleotide window for activation of RNase H; (F) ‘mixed backbone’ with methylphosphonate wings and a phosphorothioate window A B

5′-N N N R Y R R Y R Y Y Y R YsNsNsN-3′ 5′-NsNsN R Y R R Y R Y Y Y R YsNsNsN-3′ 5′-NsNsN R YsR R YsR YsYsY R YsNsNsN-3′ 5′NsNsNsRsYsRsRsYsRsYsYsYsRsYsNsNsN3′ 5′NsNsNsRsYsRsRsYsRsYsYsYsRsYsNsNsN3′ 5′NmNmNmRmYsRsRsYsRsYsYsYmRmYmNm NmN-3′

C D

E

F

Abbreviations: N, any nucleotide; Y, pyrimidine; R, purine; s, phosphorothioate; m, methylphosphonate; italics, 2′-O-methylribonucleotide. Table 2.2 Clinical studies with antisense oligonucleotides Phase

Indication

Target protein

Name

Company

FDA approval August 1998

CMV-retinitis/ AIDS

CMV-protein

ISIS/Novartis

Phase II

Morbus Crohn, colitis ulcerosa, rheumatoid arthritis, psoriasis, kidney transplantation Solid tumors Solid tumors CMV-infection Non-Hodgkin lymphoma Solid tumors HIV-infection HIV-infection

ICAM-1

ISIS 2922 (Fomivirsen) Vitravene ISIS 2302

PKC α c-raf kinase CMV-protein bcl-2

ISIS 3521 ISIS 5132 GEM 132 G3139

ISIS/Novartis ISIS/Novartis Hybridon Genta

Ha-ras HIV-protein HIV-protein

ISIS 2503 ISIS 5320 Gps0193

ISIS ISIS/NCI Chugai

Phase I

ISIS/ Boehringer Ingelheim


Phase

Indication

Target protein

Name

Company

HIV-infection HIV-infection CML AML

HIV-protein HIV-protein − −

Arl77 Gem 92 − –

Aronex Hybridon Lynx Lynx

Abbrevations: CMV, cytomegalie virus; ICAM-1, intercellular adhesion molecule 1; PKC α, protein-kinase C α; c-raf kinase mitogen activated protein kinase; bcl-2, B cell leukemia/lymphoma 2; Ha-ras, Harvey-ras oncogene; HIV, human immuno deficiency virus; NCI, National Cancer Institute; CML, chronic myeloic leukaemia; AML, acute myeloic leukemia.

This is topologically equivalent to the D-loop structure formed under Rec A mediated recombination (Cherny et al., 1993). In gene transfer experiments a synthetic gene, a double-stranded piece of DNA, coding for a polypeptide, is introduced into a cell and expressed within it. This so-called gene therapy also relies in part on synthetic oligonucleotides, mostly unmodified ones so far. Synthetic oligonucleotides are defined as repetitive nucleotide units where the individual bases are properly spaced to interact specifically with the target nucleic acid. Originally, the oligonucleotide was derived from either DNA or RNA repetitive units. More recently, modifications have been undertaken in all the relevant subunits, i.e. sugar, base and phosphate backbone (Table 2.1 and Figure 2.4). Thus we shall discuss backbone-modified ODNs, notably phosphorothioates. These derivatives have so far met the criteria of antisense ODNs as the best drugs (Table 2.2). Repetitive nucleotides or analogues thereof can also be used as, for example, PNA units. Since Chapter 4 deals with PNA, only the analogues will be considered here when discussing chimeras, i.e. mixed backbone ODNs. A different way of dividing ODNs is the concept of their way of action. They can target either DNA or RNA. In the former case we deal with the so-called triplex or antigene approach, or even with strand invasion; in the latter with the antisense or ribozyme approach. 2.2 Design of Oligonucleotides Optimal design of ODNs for biological application demands good knowledge of the target to be addressed. The first decision is whether to target DNA or RNA. What is known about their accessibility, stability, and availability? Though several of the criteria for design are in parallel, we shall discuss the abovementioned approaches separately.


2.2.1 Design Criteria for Antisense, Ribozymes, Triplex Synthetic oligonucleotides designed to interact with specific stretches of RNA or DNA have to meet several criteria. For design purposes, three points are worth mentioning: the length, the type of modification and the mode of action. These in turn determine the affinity and specificity of the oligonucleotide utilized. In order to bind in a Watson-Crick fashion a given RNA in a human cell specifically, a minimum length of a 12-mer has been calculated; for triplex this may be closer to a 20-mer. Though it is difficult to define an upper limit in length, depending on the chemistry and base composition, 12–28mer oligonucleotides are most often used. By optimizing oligonucleotide length and concentration, high specificity of binding can be achieved in a cell. Second, the stability of synthetic oligonucleotides has to be ascertained. Natural oligonucleotides are susceptible to exonuclease and endonuclease degradation. Here a large variety of modified oligonucleotides will be discussed which circumvent this problem. Third, target selection plays an important role in finding the optimal binding site. Very often the region around the translation initiation site is targeted. It is well accepted now that the structure of RNA is of great importance for its accessibility and only a limited part of it can be reached. RNA folds into a complex secondary structure consisting of double-and singlestranded regions. This stem loop geometry is further folded into a tertiary structure of spatially arranged helices and single strands. It has been shown that therapeutic oligonucleotides bind well to only a limited number of sites. Site selection could in principle be based on secondary structure predictions (Zuker and Stiegler, 1981). Since our knowledge about folding and stability of RNA is rather limited, experimental approaches are advisable. These are based either on hybridization techniques (Milner et al., 1997) or on RNase H activity (DorisKeller, 1979). Though oligonucleotides directed to the translational initiation site often decrease the level of a particular mRNA, ODNs targeted to alternative regions frequently show superior downregulation of mRNA. Therefore screening of a set of overlapping ODN is necessary to find potent candidates. Recently a statistical analysis was presented of the correlation between in vitro accessibility and ODN intracellular activity (Matveeva et al., 1998). 2.2.2 Degree of Modification Uniformly phosphorothioate-modified oligonucleotides are still the best investigated and most broadly used type of antisense agents. Although numerous compounds of this first generation antisense oligonucleotide are in clinical trials, great effort is devoted to the development of second generation antisense


compounds in which the beneficial properties needed for antisense therapeutics are combined. Two basic aspects have to be discussed with respect to side-effects caused by phosphorothioates. First, uniformly phosphorothioate-modified oligonucleotides have a tendency for non-antisense effects which are mainly due to undesired binding to proteins (see also Chapter 9) (Milligan et al., 1993; Stein, 1995; Stein and Cheng, 1993; Uhlmann and Peyman, 1990). Second, the activation of RNase H by partial oligonucleotide/RNA duplices of only five to seven nucleotides in length can cause, under certain conditions, non-specific degradation of mRNA (Woolf et al., 1992). Both limitations may in principle be overcome by reducing the number of phosphorothioate linkages in oligonucleotides by modifications which do not lead to increased binding to proteins or which do not activate RNase H. This enzyme recognizes a duplex between DNA and RNA resulting in the cleavage of the RNA strand of this duplex. However, in view of the lability of unmodified oligonucleotides against nucleases and the weak antisense effects observed for most derivatives which do not activate RNase H, the development of mixed-backbone oligonucleotides or chimeric oligomers, in which the advantages of the individual structural elements are combined, appears to be the approach of choice at present. Although the metabolism of ODN involves cleavage by exonucleases and endonucleases, the major degrading activity in serum and cells is a 3′exonuclease (Shaw et al., 1991; Uhlmann and Peyman, 1990). Capping of the 3′end, or both the 3′- and 5′-ends, by phosphorothioate linkages was reported to protect the oligonucleotide significantly against 3′-exonuclease degradation (Daum et al., 1992; Gillardon et al., 1994; Gillardon et al., 1995; Hoke et al., 1991; Shaw et al., 1991; Stein et al., 1988). However, in in vivo experiments this protection strategy has proved to be only of limited success, since end-capped oligonucleotides are still subject to endonuclease degradation. Recently, a ‘minimal protection’ strategy has been suggested which is a combination of the end-capping technique and the protection at internal pyrimidine residues which are the major sites of endonuclease degradation (Peyman and Uhlmann, 1996; Uhlmann and Peyman, 1998). The endonuclease cleavage becomes especially serious if two or more pyrimidines are adjacent (Uhlmann et al., 1997). Minimally phosphorothioate modified oligonucleotides turned out to be particularly useful, since they are sufficiently stable to exonucleases and endonucleases, while at the same time undesirable non-antisense effects are strongly reduced (Peyman et al., 1997; Tanaka et al., 1996; Uhlmann and Peyman, 1998). Furthermore, the minimal protection scheme can be combined with secondary modifications, such as 2′-O-alkyl modification of ribose (Inoue et al., 1987; Sproat et al., 1991) or C5-alkynyl modification of the pyrimidine bases (Froehler et al., 1992; Uhlmann et al., 1997), in order to improve binding to the target mRNA. The activation of RNase H is not compromised by the C5alkynyl modification, whereas 2′-modified nucleotides do not stimulate RNase H. One possibility to limit the activation of RNase H to a certain region of the oligonucleotide is realized in the so-called gap-mers (see also Chapter 1)


(Crooke et al., 1995; Inoue et al., 1987), in which 2′-O-methylribonucleotide wings are introduced to enhance binding to mRNA, while a window of six to eight deoxynucleotides limits the stimulation of RNase H cleavage to this part of the duplex. Similarly, mixed-backbone oligonucleotides (Agrawal et al., 1997) consisting for example of methylphosphonate and phosphorothioate linkages, show improved properties: the methylphosphonate provides excellent nuclease resistance and non-ionic nature, while the phosphorothioate linkage is also sufficiently stable to nucleases and in addition allows activation of RNase H. Peptide nucleic acids (PNAs) are extremely stable during incubation in serum, but show poor solubility in aqueous medium and do not stimulate RNase H in a duplex with RNA (Uhlmann et al., 1998b). In contrast, 5′-DNA-PNA(pseudo-3′) chimeras (Uhlmann et al., 1996) with more than four deoxynucleotides at the 5′-part of the oligomer can induce RNase H and are completely resistant to 3′-exonucleases (Uhlmann, 1998). Additionally, these chimeras show improved binding affinity as compared to the natural counterparts. It has been found that phosphorothioate oligonucleotides with a CG sequence motif have immunostimulatory effects (Krieg et al., 1995). Substitution of 2′-Omethylribonucleosides for CG deoxynucleotides could minimize the undesired immunostimulatory effect in the 18-mer oligonucleotide 5′CCGCTCTTCCTCACTGGT-3′ (Agrawal and Zhao, 1998). Since the CG motif in this oligonucleotide was at the 5′-end, substitution of four deoxynucleotides at both the 5′- and the 3′-end resulted not only in reduced toxicity, but also in increased biological activity due to improved binding and enhanced stability against nuclease degradation. 2.3 Oligonucleotide Modifications: Synthesis and Properties In the following we will outline the structural chemistry and properties of antisense oligonucleotide derivatives, and discuss the different routes for synthesis of unmodified oligonucleotides and selected modified derivatives. Chemical variation of the natural oligonucleotide structure is necessary to render these compounds useful in biological systems, as follows. 1 They must be sufficiently stable against nucleases in serum and within cells. 2 They should enter the various organs of the body. After distribution to the desired tissue, they must be able to penetrate cellular membranes to reach their site of action. 3 They must form stable Watson-Crick or Hoogsteen complexes with complementary target sequences under physiological conditions. In recent years, a plethora of different oligonucleotide analogues have been described (Figure 2.4), in which the natural phosphodiester linkage, the sugar


part or the heterocyclic bases of the oligonucleotides were modified aiming at the improvement of properties (Beaucage, 1993; Beaucage and Iyer, 1993; Milligan et al., 1993; Uhlmann and Peyman, 1990). 2.3.1 Unmodified Oligonucleotides having 3′5′-Phosphodiester Linkages Although unmodified oligonucleotides are widely used as tools in molecular biology, they have serious limitations with respect to their use as gene expressioninhibiting therapeutics. In cellular and animal experiments one has to take account of the fact that oligonucleotides with a natural phosphodiester internucleoside linkage are degraded in serum within a few hours, mainly by the action of fast cleavaging 3′-exonucleases which are accompanied by slower cleavaging endonucleases. In certain tissues a significant 5′-exonuclease activity has been observed. In the following sections chemically modified oligonucleotides will be described. However, it should be noted that oligonucleotides can also be protected against nucleolytic cleavage to a certain extent by packaging into liposomes or nanoparticles which at the same time serve as carriers for in vivo application of these compounds (see Chapters 6 and 7). Unmodified oligonucleotides are taken up to a substantial extent by cultured living cells in a time- and energy-dependent endocytodic-like process (see also Chapters 9 and 10). However, a punctate intracellular distribution of oligonucleotides is observed using fluorescence microscopy. As the oligonucleotides are obviously trapped in endosomes or lysosomes, only a comparatively low concentration of ‘free’ hybridizable oligonucleotide in the cytoplasm or in the nucleus is obtained. This problem can be overcome by microinjection (Clarenc et al., 1993) of the antisense oligonucleotides into the cytosol from which they rapidly penetrate into the nucleus, or by administration of the oligonucleotides with cationic lipids, such as lipofectin™ (Bennett et al., 1993). The observed efficacy of unmodified oligonucleotides as antisense inhibitors can be explained by an antisense oligonucleotide-stimulated degradation of the target mRNA which is brought about by the cellular enzyme RNase H. In the following two sections, the basic possibilities for the synthesis of natural phosphodiester oligonucleotides are discussed briefly. We will see that both methods frequently also form the basis for the synthesis of modified oligonucleotides.


Figure 2.4 Different possibilities for the modification of oligonucleotides

2.3.1.1 Chemical synthesis The synthesis of unmodified oligonucleotides can be performed either in solution or on solid supports. At low or medium scale, oligonucleotide synthesis using polymeric supports and step-wise addition of monomeric building blocks is preferred. Although different chemistries, such as the phosphodiester, the phosphotriester and H-phosphonate methods have been used successfully in the past, the phosphoramidite chemistry clearly dominates oligonucleotide synthesis up to the multikilogram scale at present. The phosphoramidite method according to Matteucci and Caruthers (1981) involves the reaction of the 5′-hydroxy group of a nucleos(t)ide, which is bound to the solid-support via its 3′-hydroxy group, with a nucleoside-3′-β-cyanoethyl N, N-diisopropylphosphoramidite under catalysis using a weak acid (Figure 2.5). Although catalysis by 1H-tetrazole has been the method of choice for many years, more efficient catalysts, such as dicyanoimidazoles (Schell and Engels, 1998; Vargeese et al., 1998) are becoming of increasing interest. The phosphite triester resulting from the coupling is oxidized immediately with iodine/water to


Figure 2.5 Synthesis cycle for the preparation of oligonucleotides on a solid support according to the phosphoramidite chemistry

Figure 2.6 Synthesis of oligonudeotides by the H-phosphonate method

the corresponding phosphotriester. The coupling yields of the phosphoramidite method are usually >99%. As phosphate blocking group the β-cyanoethyl group (Sinha et al., 1983) is most popular; it can be cleaved with ammonia in a βelimination reaction, thus avoiding unfavourable cleavage of internucleotide linkages. For chain elongation the 5′-hydroxy group of the growing DNA chain is protected with 4, 4′-dimethoxytrityl (Dmt) group. It has the advantage that it can be introduced into the monomers quite regioselectively and renders the monomers easily soluble in organic solvents, such as methylenechloride and acetonitril. On deblocking with 3% trichloroacetic acid the resulting orange-red


Figure 2.7 Oligonucleotides with 2′5′-, 3′3′- and 5′5′-phosphodiester linkages

coloured Dmt cation allows easy monitoring (498 nm) of the efficiency of coupling steps. The exocylic amino functions of the bases are protected by acyl protecting groups, which are cleaved off by ammonia treatment at the end of the synthesis. Recently, the facile synthesis of oligonudeotides without the need for nucleobase protection has been reported (Hayakawa and Kataoka, 1998). The H-phosphonate method has the advantage that no phosphate protecting group is employed. This method involves the reaction of the 5′-hydroxy group of the growing DNA chain with a nucleoside-3′-H-phosphonate (Figure 2.6). Sterically hindered carbonyl chlorides, such as pivaloyl or adamantoyl chloride (Sinha and Cook, 1988), are used as coupling reagents. Only after the construction of the whole oligonucleotide chain has been completed, the Hphosphonate internucleoside linkages are oxidized in one step to the phosphotriester linkages. This is in contrast to the phosphoramidite method, in which the phosphite linkages are oxidized after each coupling step (Froehler et al., 1986). 2.3.1.2 Enzymatic synthesis and ligation The enzymatic synthesis of oligonudeotides, especially longer ones, containing modified building units is a valuable alternative to chemical synthesis. Enzymatic incorporation of natural or suitably modified nucleoside triphosphates by phage RNA polymerases (e.g. T7) has been successfully used. This approach is limited by the acceptance of the polymerase for sugar, backbone or base modification. For the backbone linkage, phosphorothioates are the most common derivatives. All four nucleoside thiotriphosphates are good substrates for RNA as well as DNA polymerases. Only the Sp-Isomer is actually accepted. Since the incorporation proceeds with inversion of configuration by an in-line mechanism, only the Rpphosphorothioate linkage is obtained (Eckstein, 1985). For the sugar modification, 2′-deoxynucleoside triphosphates (Milligan and Uhlenbeck, 1989), especially in the presence of Mn2+ instead of Mg2+ (Conrad et al., 1995), have


been successfully incorporated. In this case even 2′-O-methyl nucleoside triphosphates were incorporated. 2′-Aminonucleoside triphosphates are good substrates (Aurup et al., 1992) whereas 2′-fluoronucleoside triphosphates are less readily incorporated. Here Huang et al. (1997) introduced mutant T7 polymerase with improved efficiency. In a post-synthetic modification step, kinase easily introduces a 5′thiophosphoryl group (Igloi, 1988). Ligation of short oligonucleotides to synthesize long oligonucleotides is very attractive. This can be accomplished either by DNA, RNA-ligases or by chemical ligation. When DNA ligases are used, a 5′-phosphoryl donor group in a preformed duplex is ligated to a free 3′hydroxyl group acceptor (Maunders, 1993). In contrast, RNA ligases, particularly T4 RNA ligase, catalyse the formation of phosphodiester bonds between two single-stranded oligomers, whereby a 5′-phosphoryl donor and a 3′hydroxylacceptor are coupled. This enzyme accepts a variety of nucleoside analogues even at the ligation junction. By this method, longer RNA-sequences, which can be fluorescence labelled (Cosstick et al., 1984), in particular can be synthesized. Alternatively, T4 DNA ligase has been used to ligate two oligoribonucleotides with the aid of a short complementary oligodeoxynucleotide at the ligation junction (Moore and Sharp, 1992). 2.3.2 Oligonucleotides with 2′5'-Linkages and 3'3'-/ 5'5'Inversions A special type of minimal modification is oligonucleotide analogues which differ from their naturally occuring 3′, 5′-phosphodiester compounds only by changes in the linkage, but do not include new chemical entities. Oligonucleotides with 3′, 3′- or 5', 5'-end inversions (Seliger et al., 1991) (Figure 2.7) are very stable against exonucleases, and their half-life in human serum is several hours, as compared to minutes for completely 3′5′-linked oligonucleotides. The WatsonCrick base-pairing of the antiparallel portion of the oligonucleotide appears not to be disturbed by the inverted termini. In order to extend base recognition and to improve flexibility in triplex formation, the 3′3′- and 5′5′-inversions were also exploited. The 3′3′-inversions, also called 3′, 3′-switches, in which the polarity of the strand changes via a 3′, 3′linkage within the sequence, allow jumping from one purine tract of one strand to another purine tract on the complementary strand (McCurdy et al., 1991). Synthesis of these analogues is straightforward by using the corresponding ‘inverted’ nucleoside-5′-O-phosphoramidites or nucleoside-5′-O-succinylsupports combined with standard phosphoramidite chemistry (Seliger et al., 1991). The naturally occurring 2′, 5′-oligoadenylates (Figure 2.7) are involved in the stimulation of cellular RNase L as part of the cell’s defence system, e.g. after viral infection. RNase L cleaves single-stranded RNA adjacent to double-


stranded RNA in response to interferon treatment or viral infection. Interestingly, chimeric molecules having 5′-phosphorylated 2′, 5′-tri-adenylate conjugated to a normal 3′, 5′-linked antisense oligonucleotide have been successfully used to cleave a target RNA by activation of 2′, 5′-adenylatedependent RNase L (Torrence et al., 1993). Because 2′, 5′-adenylate-dependent RNase L is present in most mammalian cells, this strategy is of potential use when applied in combination with antisense oligonucleotide derivatives which do not stimulate RNase H cleavage, e.g. in combination with 2-Oalkylribonucleotide analogues or PNA. Synthesis of 2′5′-oligoribonucleotides can be achieved using protected nucleoside-2′-phosphoramidite building blocks, in which the 3′-hydroxy group is protected by the fluoride cleavable tert.-butyl dimethylsilyl protecting group (Ogilvie and Iwacha, 1973). 2.3.3 Modification on Phosphorus The nucleases achieve oligonucleotide degradation by nucleophilic attack at the phosphodiester linkage. Therefore, replacement of one non-bonding phosphate oxygen by other atoms is an easy way to make oligonucleotides resistant against nucleases. It also allows the alteration of other properties of oligonucleotides, such as binding affinity and cellular permeation. The most common types of modification are depicted in Figure 2.8. 2.3.3.1 Phosphorothioates Phosphorothioates, in which one non-bridging oxygen is replaced by a sulphur, are among the most obvious analogues of naturally occurring phosphodiesters (Eckstein, 1983). The phosphorothioate modification provides significant stabilization against degradation by nucleases. It must be mentioned, however, that nuclease resistance depends strongly on the configuration on the phosphorus. The Sp diastereomers are substrates of nucleases S1 or P1, while the Rp diastereomers are cleaved by snake venom phosphodiesterase. Cellular uptake of phosphorothioate oligonucleotides is similar to phosphodiester oligonucleotides. An important property of phosphorothioates is the ability to mediate RNase H degradation of the RNA after hybridization. One drawback of uniformly phosphorothioate-modified oligonucleotides is their propensity for non-specific effects, which result mainly from the interaction with cellular proteins (Stein, 1996). Phosphorothioate-containing oligonucleotides are easy to synthesize, as the same synthons as for phosphodiester oligonucleotide synthesis (phosphoramidite or H-phosphonate chemistry) can be used. The only difference is in the oxidation step, where sulphur is introduced instead of oxygen. The most widely used


Figure 2.8 Examples of the modification on phosphorus

sulphurization agents are Beaucage’s reagent (Iyer et al., 1990) and tetraethylthiuram disulphide (Vu and Hirschbein, 1991). The phosphorothioate bridge also represents a centre of chirality. The hybridization properties of diastereoisomeric phosphorothioates are sufficiently good to use them under in vivo conditions, although there is an average loss of 0. 5 K/ phosphorothioate linkage in the melting temperature for the racemic mixture. Heterodimers formed between oligoribonucleotides and all-RPphosphorothioates showed a higher Tm as compared with the less stable heterodimers formed with all-Sp-phosphorothioates or the random mixture of diastereomers (Koziolkiewicz et al., 1995). The DNA—RNA complex containing the phosphorothioate of all-Rp configuration was found to be more susceptible to RNase H-dependent degradation than hybrids having either all-Sp counterparts or the random mixture of diastereomers. Interestingly, 3′exonucleases present in human plasma appear to degrade phosphorothioates of Rp configuration, but not of those of Sp configuration (Koziolkiewicz et al., 1997). 2.3.3.2 Alkylphosphonates and arylphosphonates After the phosphorothioates, the methylphosphonates are probably the second best investigated class of oligonucleotide derivatives with a modification on the phosphorus, and they were used very early for specific antisense inhibition of gene expression (Miller et al., 1985). In methylphosphonates the negatively charged phosphate oxygen is replaced by a neutral methyl group. The methylphosphonate linkage is highly stable against degradation by nucleases. However, a major problem is posed by the chirality of the methylphosphonate


bridge, which can have either the Rp or the Sp configuration. Therefore, methylphosphonate oligonucleotides as phosphorothioates resulting from standard synthesis usually consist of a mixture of 2n diastereoisomers, where n is the number of such linkages. It has been shown for short oligonucleotides with a methylphosphonate backbone that the all-Rp backbone shows a significantly higher melting point (Tm) than a mixture of diastereomers or even the corresponding phosphodiester oligonucleotide when hybridized to complementary nucleic acids. Uniformly methylphosphonate-modified oligonucleotides do not form duplexes with RNA that induce RNase H cleavage. This may be a limitation to their use as antisense oligonucleotides and could explain the relatively high concentration required for effective translation arrest in antisense experiments. As discussed in more detail in section 2.2, chimeric oligonucleotides containing both methylphosphonate and a window of at least five to seven phosphodiester linkages retain RNase H activity, and the poor solubility of uniformly modified compounds in biological systems can be overcome. Alkylphosphonates are available through automated oligonucleotide synthesis using methylphosphonamidites as synthons (Engels and Jaeger, 1982). Since the alkylphosphonate bridge is more base-labile than the natural phosphodiester linkage, much milder conditions are necessary for cleavage from the support and for deprotection. Similarly, phenylphosphonate—and phenylphosphonothioatecontaining oligonucleotides can be prepared from the corresponding nucleoside-3′-phenylphosphonamidites (Mag et al., 1997). Binding affinity of the arylphosphonate-containing oligomers depends strongly on the sequence, so that some duplexes are destabilized by −0.3 to −1.3 K, whereas others are stabilized by +0.2 to +0.5 K relative to their natural congeners. The synthesis of oligodeoxynucleotide pentadecamers containing two octylphosphonate linkages with stereoregular or stereorandom chirality has also been described (Mag et al., 1996). The difference in Tm was −3.4 K per modification for the stereorandom and about −2.3 to −4.0 K per modification for the stereoregular configurated oligonucleotides. 2.3.3.3 Other modifications on phosphorus The phosphoramidate linkage is also attractive, as introduction can easily be achieved using H-phosphonate chemistry with a final CCl4/amine oxidation step (Froehler, 1986). Alternatively, the oxidation of phosphite triesters by I2/amine allows the specific introduction of phosphoramidates in specific positions with moderate yields. However, the P–N bond is relatively easily cleaved by acid, resulting in phosphodiesters. Phosphoramidate oligonucleotides have a decreased binding affinity to complementary DNA (−1.5 K/modification). Phosphotriesters (Marcus-Sekura et al., 1987), like the alkylphosphonates, have been used as non-ionic analogues of oligonucleotides. However, because of


the lability of the phosphotriester function towards basic deblocking conditions (P-OCH3>P-OC2H5>P-OiC3H7) and their potential as alkylating reagents, they are only infrequently used as antisense agents at present. An interesting class of analogues are those with a ‘boronated’ internucleotide linkage (Sood et al., 1990), since they appear also to induce RNase H in a duplex with RNA. As phosphorothioates and methylphosphonates, they are also very resistant to degradation by nucleases. 2.3.4 Oligonucleotides Containing Dephospho Linkages Due to the stereochemical problems associated with methylphosphonates and phosphorothioates, there is a growing interest in oligonucleotide analogues containing achiral, uncharged and phosphate-free linkages (Uhlmann and Peyman, 1993). All investigated oligonucleotide analogues of this type are completely stable to nuclease degradation. Furthermore, many oligonucleotide analogues of this type form more stable complexes with complementary nucleic acids. Problems with these analogues include poor water-solubility and poor hydrolytic stability. Most of the methods reported so far for the synthesis of dephospho oligonucleotide analogues are difficult and not easily amenable to automated solid-phase synthesis. Oligonucleotide analogues have been described containing formacetal, 5′thioformacetal and 3′-thioformacetal linkages (Figure 2.8), which are uncharged, achiral isosteres of the normal phosphodiester linkage (Jones et al., 1993; Matteucci, 1991). In all cases dimers or trimers containing the modified linkages were synthesized in solution. After incorporation of these building blocks into oligonucleotides at appropriate sites, chimeric formacetal-phosphodiester oligonucleotides were obtained. Oligonucleotides containing formacetal linkages have favourable hybridization properties when hybridized to RNA. Thus, a tetradecamer containing four formacetal linkages showed a Tm value of 59°C when hybridized to single-stranded RNA, as compared to 60°C for the allphosphodiester-linked tetradecamer. In contrast, the binding affinity of formacetal analogues to single-stranded DNA turned out to be considerably lower (Tm=39°C) than that of the all-phosphodiester-linked oligomer (Tm=50°C). Oligonucleotides containing alternating 3′-thioformacetal-phosphodiester linkages showed improved binding affinity on hybridization with single-stranded RNA. In a DNase I footprinting assay, triple-helix formation equivalent to that of the control oligonucleotide was observed. Oligonucleotide analogues with dialkylsilyl internucleoside linkages were prepared by one-pot reactions starting from dialkyldichloro or chloro (dialkylamino)silane derivatives (Seliger and Feger, 1987). Phosphodiesterlinked oligonucleotides containing one or two diisopropylsiloxane linkages were synthesized using phosphoramidite derivatives of siloxane-linked dimers and trimers. The Tm values for undecamers containing one or two diisopropylsiloxane


Figure 2.9 Examples of the modification of the sugar moiety

links were 2–5 K less than that of the control of all-phosphodiester oligonucleotides. The diisopropylsiloxane link is again stable to 3′-exonuclease digestion (Cormier and Ogilvie, 1988). A plethora of other dephospho oligonucleotide analogues have been reported in the literature (Uhlmann and Peyman, 1993), including carbonate-, carboxymethyl-, acetamidate-, carbamate-, thioether-, sulphonate-, sulphonamide-, oxime, methyleneimino-, methylene methylimino (MMI)-, methylene dimethylhydrazo (MDH)-, methyleneoxy methylimino-, urea-, guanidine-, all-carbon-, riboacetal-, and amide-linked nucleosides. Among the most promising derivatives are the MMI-linked derivatives (Debart et al., 1992). 2.3.5 Modification of the Sugar Moiety The 2′-deoxy-β-D-ribofuranose unit of the DNA backbone is another suitable site for oligonucleotide modification (Figure 2.9). There are quite a number of sugar modifications that have been synthesized and tested for improvement of antisense oligonucleotide properties, such as improved binding affinity and enhanced stability against nucleases.


2.3.5.1 α-Anomeric oligonucleotides α-Anomeric oligonucleotides (Bloch et al., 1988; Morvan et al., 1991) show excellent binding properties. In some cases the melting temperature of a duplex with RNA is almost doubled compared to β-anomeric oligonucleotides. Furthermore, α-anomeric oligonucleotides are highly nuclease-resistant. Despite their excellent binding properties, they are not generally efficient in antisense experiments, since they do not activate RNase H, although good inhibition was obtained when the α-anomeric oligonucleotide was directed against the 5′-nontranslated mRNA region (Bertrand et al., 1989). The synthesis of α-anomeric oligonucleotides is very similar to the one used in β-deoxyribonucleotide synthesis, whereby α-anomeric monomeric phosphoramidite building blocks are employed (Debart et al., 1992). Similarly to their β-anomeric analogues, the α-oligodeoxynucleoside phosphorothioates can be obtained easily by oxidation with elemental sulphur or Beaucage’s reagent following the standard conditions. 2.3.5.2 2′-Modified oligonucleotides 2′-Modified oligonucleotides can be considered as analogues of oligoribonucleotides. They can be subdivided into 2′-O-alkyl RNA and other modifications at the 2′-position such as 2′-fluoro or 2′-amino-substituted oligonucleotides. The 2′-O-methyl ether of RNA is a naturally occurring modification which is found, for example, at certain positions in tRNAs, rRNAs and snRNAs. The stability of (RNA)•(2′-O-alkyl-RNA) heteroduplexes (Freier and Altmann, 1997) clearly depends on the nature of the 2′-O-alkyl group and decreases in the order 2′-methoxyethoxy>2′-O-methyl>2′-O-allyl>2′-OH>2′-O-butyl>2′-Odimethylallyl. As early as 1987 it was reported that 2′-O-methyl RNA forms a more stable duplex with a complementary RNA strand than unmodified DNA or even RNA (Inoue et al., 1987). The 2′-O-alkylribonucleotides prefer the C(3′)endo conformation in a duplex with RNA. This sugar pucker has been found as a key structural element in RNA•RNA duplexes which are generally more stable than DNA•DNA duplexes of the same sequence. It is worth mentioning that the 2′-O-methoxyethoxy modification not only results in enhanced binding affinity to RNA, but at the same time renders the oligomer more stable against nuclease degradation. Synthesis starts from the corresponding nucleosides containing 2′-O-alkyl groups combined with standard 2-cyanoethyl phosphoramidite chemistry (Shibahara et al., 1987). Replacement of the 2′-hydroxy groups in RNA by fluorine or the amino group can also be employed to obtain analogues with improved binding affinity or


enhanced stability towards nucleases, respectively. Unfortunately, the corresponding monomeric nucleosides are difficult to prepare and they also tend to be toxic. A uniformly modified 2′-deoxy-2′-fluoro oligonucleotide (Kawasaki et al., 1993) exhibits a considerably increased binding affinity for RNA as compared to the DNA oligonucleotide without compromising base pair specificity, whereas the 2′-amino modification (Aurup et al., 1994) destabilizes the duplex with RNA. However, additional modifications, such as phosphorothioate bridges, are necessary to render these oligonucleotide derivatives sufficiently stable to nucleases. As with 2′-O-alkyl RNA, introduction of uniform 2′-deoxy-2′-fluoro sugars lead to the loss of RNase H activation. 2.3.5.3 Sterically locked nucleic acid analogues Nucleic acid analogues with conformationally restricted sugar phosphate backbones based on (3′S, 5′R)-2′-deoxy-3′5′-ethano-β-D-ribofuranosyladenine and-thymine (bicyclo-DNA) were reported to form more stable duplexes than the corresponding natural congeners (Tarkov et al., 1993). The bicyclo DNA (A10) formed more stable triplexes with d(T10) of the pyrimidine-purine-pyrimidine motif than natural d(A10). Recently, a new DNA analogue, ‘bicyclo-[3.2.1]-DNA’, has been described (Epple and Leumann, 1998) which has a rigid phosphodiester backbone, emulating a B-DNA-type conformation, and to which the nucleobases are attached via a flexible open-chain linker. Although bicyclo-[3.2.1]-DNA forms less stable duplexes with complementary DNA than natural DNA, basemismatch discrimination is slightly enhanced compared to pure DNA duplexes. Importantly, bicyclo-[3.2.1]-DNA oligomers are resistant to 3′-exonuclease degradation. A novel modification with unusually high binding affinity is LNA (Locked Nucleic Acid), in which the 2′ oxygen is linked by a methylene bridge to the 4′ carbon forming a methylene-linked bicyclic ribofuranosyl nucleoside. The sugar conformation of this derivative is locked in the N-type (3′-endo) conformation (Koshkin et al., 1998). An unprecedented increase of 3 to 8 K per modification in the thermal stability of duplexes towards both DNA and RNA was reported when evaluating mixed sequences of partly or fully modified LNA. 2.3.5.4 Other sugar-modified analogues Oligonucleotide analogues consisting of 1′, 5′-anhydrohexitol nucleoside building blocks (hexitol nucleic acids, HNAs) (Hendrix et al., 1997) were shown to form stable duplexes with natural DNA and RNA. The stabilizing effect amounts to +1.3 K/base pair with DNA as complement and +3.0 K/base pair with RNA as complement. HNA is completely stable towards 3′-exonucleases.


Figure 2.10 Structure of peptide nucleic acids (PNAs) and DNA—PNA chimeras

A great number of other sugar modifications have been incorporated in antisense oligonucleotides, e.g. L-2′-deoxyribose (Garbesi et al., 1993; Urata et al., 1992), 2′-deoxy-β-D-xylofuranose (Rosemeyer et al., 1991), l-(Darabinofuranose) (Resmini and Pfleiderer, 1993), 4′-thio-β-D-ribose (Bellon et al., 1993), and carbocyclic (Szemzo et al., 1990) sugar analogues. 2.3.6 Peptide Nucleic Acids Peptide nucleic acids (PNAs) (see also Chapter 4), in which the entire sugarphosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure (Nielsen et al., 1991), bind with higher affinity to complementary nucleic acids than their natural counterparts following the Watson-Crick base-pairing rules (Egholm et al., 1993; Hyrup and Nielsen, 1996; Uhlmann et al., 1998b). The Nterminus of PNA corresponds to the 5′-end and the C-terminus to the 3′-end of DNA (Figure 2.10). In the case of the antiparallel PNA•DNA or PNA•RNA duplex, the melting temperature is increased by approximately 1 K/base or 1.5 K/base, respectively, as compared to the corresponding DNA•DNA or DNA•RNA duplex. A further advantage of PNA is that base-mismatches often give rise to a significantly larger reduction in the Tm value as compared to DNA. PNA is extremely stable to nucleases and peptidases. One limitation of PNA, however, is that it cannot stimulate RNase H cleavage. Depending on the targeted mRNA sequence, antisense oligonucleotides having the ability to stimulate RNase H (e.g. phosphodiester and phosphorothioate oligonucleotides) are often more effective antisense inhibitors than those without this ability. Consequently, it has been found that


Figure 2.11 Examples of the modification of the heterocyclic bases

PNAs are less effective antisense agents than phosphodiester (van der Laan et al., 1998) and phosphorothioate (Bonham et al., 1995) oligonucleotides. In contrast, DNA•PNA chimeras (Uhlmann, 1998) (Figure 2.10) with more than four nucleotides are able to stimulate the cleavage of RNA by RNase H on formation of a chimera•RNA duplex. RNA cleavage occurs at the ribonucleotides which base-pair with the DNA part of the chimera. PNA—DNA chimeras also obey the Watson-Crick rules on binding to complementary DNA and RNA (Uhlmann et al., 1996; van der Laan et al., 1997) whereby the Tm value strongly depends on the PNA/DNA ratio in the chimeras. The Tm value of 5′DNA–3′-PNA chimeras, in which the PNA and DNA parts are of equal length, lies roughly between those observed for the corresponding pure PNA and DNA. In contrast to pure PNA, 5′-DNA—PNA chimeras bind exclusively in the antiparallel orientation to DNA and RNA under physiological conditions. The chimeras show also much better cellular uptake than pure PNA. PHONAs are analogs of PNA in which the peptide bond has been replaced by a phosphonic acid ester bridge, rendering this negatively charged analogue more soluble in aqueous medium than PNA (Peyman et al., 1996, 1998). PNA can be synthesized by the Boc strategy (Egholm et al., 1992) or the Fmoc strategy (Breipohl et al., 1996; Thomson et al., 1995) in analogy to the established peptide solid-phase synthesis methods. PNA—DNA chimeras containing purine nucleotides cannot be synthesized by standard PNA synthesis methods, since strong acid is used for deprotection or cleavage of the PNA from the solid support. Therefore, a mild method for PNA synthesis has been introduced which is fully compatible with DNA and RNA synthesis (van der Laan et al., 1995; Will et al., 1995). This strategy makes use of the monomethoxytrityl group for temporary protection of the N-terminus and acyl groups for protection of the exocyclic amino groups of the nucleobases (Uhlmann et al., 1997). The monomethoxytrityl group can be cleaved under very


mild conditions (3% trichloroacetic acid in dichloromethane), thus avoiding depurination in the DNA part. The DNA part is synthesized according to standard methods. 2.3.7 Modification on Heterocyclic Bases Modified nucleobases (Figure 2.11) are mainly introduced into oligonucleotides to improve binding affinity and specificity, though they always influence other parameters, such as nuclease stability and induction of RNase H, as well. Thus, enhanced binding affinity is observed by incorporation of C5 propynyl (Froehler et al., 1992) and C5 hexynyl (Ojwang et al., 1997) pyrimidines into oligonucleotides which at the same time leads to enhanced specificity. In case of C5 substituted pyrimidines, significant stabilization to nucleolytic digestion was observed as compared to their unmodified controls (Uhlmann et al., 1997). Very recently, phenoxazine-substituted oligonucleotides were described as selfpermeable antisense therapeutics (Flanagan et al., 1999). Phenoxazine, a planar tricyclic cytosine analogue, was introduced to improve stacking interactions between the heterocycles of oligonucleotide•RNA hybrids while retaining the ability to activate RNase H. The substitution of phenoxazine for C5 propynynl cytosine in a heptamer antisense oligonucleotide resulted in a five-fold increase in the relative binding affinity. The ability to induce RNase H cleavage was retained, although the pattern of cleavage was altered relative to the propynyl analogue. However, the most unique feature of the phenoxazine analogue was its ability to confer cellular permeation to a short heptameric oligonucleotide. N4, N4-ethano-5-methyl-cytosine (Shaw et al., 1991) has been used for irreversible modification of target sequences in vitro. For triplex formation, cytosine must be protonated (C+) to allow binding to a G-C base-pair. Therefore, under physiological conditions cytosine is replaced by 5-methylcytosine to effect triplex formation. In addition, incorporation of pseudoisocytidine, e.g. as its 2′-Omethyl derivative, has been used successfully for triplex formation at neutral pH (Ono et al., 1992). To allow triplex formation on a homopurine stretch containing a pyrimidine inversion, N4-(3-acetamidopropyl)-cytosine was introduced opposite the inversion site (Huang et al., 1993). Substitution of guanine by 7-deazaguanine in GGGG-motifs was used to suppress non-antisense effects caused by these G-tetrads (Benimetskaya et al., 1997). This substitution retains Watson-Crick base pair hybridization but prevents Hoogsteen base-pair interactions. 2.3.7.1 Universal bases and abasic sites A universal base is defined as a base analogue that can substitute for all four natural bases without significant loss of duplex stability. This is mainly


accomplished by base stacking energy rather than hydrogen bond interaction, though the latter has been postulated for the imidazole-4-carboxamide (Sala et al., 1996). 3-Nitropyrrole (Nichols et al., 1994) and 5-nitroindole (Loakes and Brown, 1994) are among the best accepted base substitutions used for PCR so far. Deletion of a nucleoside base from the sugar unit gives rise to an abasic site. Any effect observed by this change is directly correlated to the loss of the heterocycle. Substitution of abasic ribonucleosides in the hammerhead ribozyme significantly impairs its catalytic activity (Peracchi et al., 1996). 2.3.8 Oligonucleotide conjugates By covalent attachment of non-nucleosidic molecules to either the 3′- or the 5′end of oligonucleotides, the properties of antisense oligonucleotides can also be modulated. This type of derivatization is chemically relatively simple and allows modulation of nuclease stability, cellular uptake and organ distribution of oligonucleotides. Besides the conjugation to the 5′- or the 3′-end of the oligonucleotide, the internucleoside linkages, the C5-position of pyrimidine bases and 2′-position of ribose are favoured. Conjugation to the 3′-end of oligonucleotides usually results in strong stabilization against 3′-exonucleases, which are the predominant nucleases in human serum. 2.3.8.1 5′-end conjugates Conjugation of molecules to the 5′-end of oligonucleotides is straightforward by coupling a phosphoramidite or H-phosphonate derivative of the desired molecule to the 5′-hydroxy group of the oligomer following chain elongation by solidphase synthesis. A broad range of phosphoramidite derivatives of ligands, such as fluorescein, biotin, cholesterol, dinitrophenyl, acridine and psoralen derivatives, are commercially available. Alternatively, the 5′-terminal hydroxy group of the oligonucleotide is reacted with an aminoalkyl linker phosphoramidite, which after deprotection results in a free aminoalkyl function. The amino function of the oligonucleotide can then be reacted post-synthesis in solution with suitably activated conjugate molecule derivatives, such as active esters, isothiocyanates, or iodo-acetamides. 2.3.8.2 3′-end conjugates The conjugation of molecules to the 3′-end of oligonucleotides is conveniently achieved by using correspondingly functionalized solid supports which in addition harbour a hydroxyl function from which the oligonucleotide chain is


extended during solid-phase synthesis. After oligonucleotide synthesis is complete, the oligonucleotide conjugate is cleaved from the solid support and deprotected by ammonia treatment. Using this method, relatively exotic derivatives, such as the anionophoric moiety of pamamycin (Uhlmann et al., 1998a) can be easily introduced. Similarly to the 5′-conjugation, appropriate 3′amino-modifier solid supports are commercially available which allow coupling of suitably activated derivatives of the molecule to be conjugated with the 3′amino alkyl group in a post-synthetic solution-phase step. 2.3.8.3 Conjugation at internucleoside linkages, nucleobases and ribose The attachment of conjugate molecules to the internucleoside linkages of oligonucleotides is possible via oxidative amination. On one hand, this can be accomplished by replacement of the normal iodine/water oxidation of the phosphite triester intermediate in phosphoramidite oligonucleotide synthesis by treatment with a solution of iodine and a derivative of the conjugate molecule bearing an amino function. On the other hand, replacement of the normal iodine/ water oxidation of the H-phosphonate intermediate in H-phosphonate oligonucleotide synthesis by treatment with a carbon tetrachloride solution of a derivative of the conjugate molecule bearing an amino function also leads to a modified phosphoramidate linkage. Although the oxidative amination method allows the conjugation of suitable molecules at any internucleoside linkage in an oligonucleotide, it has mainly been used for the synthesis of oligonucleotides with conjugate molecules attached to the 5′-and/or 3′-terminal internucleoside linkages. For conjugation of molecules to the heterocyclic bases, C5 trifluoroacetylprotected aminoalkenyl pyrimidine and C7-aminoalkynyl-7deazapurine nucleoside phosphoramidites can be used, which after deprotection can be further coupled with active esters and the like as described above. Similarly, suitably protected 2′-O-aminoalkylribonucleoside phosphoroamidites are useful for site-specific introduction of conjugate groups into the 2′-position of ribose of oligonucleotides. 2.3.8.4 Types of conjugate Many different oligonucleotide conjugates have been described in recent years aiming at the improvement of cellular uptake, exonuclease stability, binding affinity, or enhancement of biological activity due to cross-linking reactions or artificial nuclease cleavage. The relatively poor cellular uptake and unfavourable intracellular distribution of antisense oligonucleotides is still one of the major obstacles to their use as therapeutic agents. Much work has been devoted to the


conjugation of oligonucleotides with lipophilic molecules or with positively charged polypeptides (see also Chapter 5). Cholesterol groups (Krieg et al., 1993; Letsinger et al., 1989) were introduced by oxidative animation of H-phosphonate internucleoside linkages. Alternatively, H-phosphonate and phosphoramidite derivatives allowed the synthesis of 5′-cholesteryl oligonucleotides, whereas cholesterol derivatized controlled-pore glass solid supports are available for the synthesis of 3′-cholesteryl oligonucleotides. Interestingly, studies using fluorescently labelled or radiolabelled cholesteryl oligonucleotides showed that attachment of cholesterol to the oligonucleotide increases its cell association and uptake. In addition, cholesterol conjugation appears to influence the intracellular localization of the oligonucleotide in that the nuclear fraction is increased. However, the interaction of the cholesterol moiety with cellular membranes can also lead to complications in the interpretation of biological activity experiments. Vitamin E (Will and Brown, 1992) and polyalkyl chains (MacKellar et al., 1992) of various lengths have also been attached to oligonucleotides at both the 3′-and 5′-ends. In many cases higher cellular association and uptake are reported for these conjugates. Furthermore, 3′-and 5′-polylysine-modified oligonucleotides (Lemaitre et al., 1987) (see also Chapter 8 this volume) have revealed remarkable antisense activity in antiviral assay systems. However, the potential cytotoxicity of polylysine and some reports of sequence non-specific effects may limit the applicability of this approach. In order to enhance the duplex stability of oligonucleotides with its target sequence, intercalating agents were conjugated to both the 3′-and the 5′-end. Similarly, the stability of triple helices could be enhanced by attachment of intercalators to triple-helix-forming oligonucleotides. Acridine-derived moieties (Thuong et al., 1987) are commonly used as intercalating conjugates and a phosphoramidite of 9-amino-6-chloro-2-methoxy-acridine is commercially available for coupling to the 5′-end of oligonucleotides. Cross-linking agents were used for permanent inactivation of target nucleic acids (Knorre et al., 1985, 1989), which were based for example on aromatic (2-chloroethyl) amino (Vlasov et al., 1988) groups. Ideally, the alkylation reaction at the purine bases on the complementary strand should only occur on hybridization of the oligonucleotide to its target sequence. Chlorambucil, proflavine, azidoproflavine and pazidophenacyl cross-linkers have also been used for attachment to oligonucleotides. Conjugation of psoralen is frequently used, which reacts with thymine bases on exposure to UV light. As artificial endonucleases, which induce non-specific DNA cleavage, oligonucleotide conjugates with metal chelators were mainly employed, such as EDTAFe(II), o-phenanthroline-Cu(II), Bleomycin-Fe(II), bipyridinyl-Cu(II) and porphyrin chelators. Although oligonucleotide conjugates of this type have proved to be useful tools for probing oligonucleotide interactions in vitro, this approach cannot be applied to the in vivo situation. Of potential use for therapy are oligonucleotides with texaphyrins (ring expanded porphyrin analogues) (Magda et al., 1996, 1997) attached at the ends, which, depending on the


chelated lanthanide metal cation, can be used to cleave either single-stranded RNA (with europium) or DNA (with lutetium). 2.4 Analysis of Oligonucleotides The analysis of oligonucleotides (see also Chapter 11) and the analytical techniques are refined in the same way as synthesis methods are improved. Today a great diversity of analytical tools is available, ranging from classical electrophoresis via chromatographic techniques to nuclear magnetic resonance (NMR) and mass spectrometry (Schweitzer and Engels, 1997). For pharmaceutical application the purity and identity (base composition and sequence) of the oligonucleotides has to be proven. An incomplete coupling results in a contamination of the full length oligonucleotide with n–1 failure sequences. These can be monitored by chromatography or electrophoresis. Furthermore, the protecting groups applied in the synthesis have to be separated from the oligonucleotide product after cleavage. HPLC is a good method to verify the purity of the product. For physiological applications modification of the oligonucleotides is essential, since unmodified oligonucleotides are readily degraded in biological systems. The introduction of all kinds of modifications, such as phosphorothioates, methylphosphonates, O-methyl oligonucleotides or amino groups for the attachment of reporter groups has to be monitored. Mass spectrometry is a powerful tool for this purpose, and NMR allows the determination of the average amount of sulphurization. Radioactive or fluorescent-labelled molecules are used to monitor the course of the oligonucleotide in vivo and to determine organ distribution, half-life and degradation products. The stability of hybridization to the target sequence is an important criterion for antisense application. Here recording of UV melting curves allows the determination of the Tm value and the thermodynamic parameters of the transition. 2.4.1 UV Spectroscopy of Oligonucleotides UV Spectroscopy is an easy and sensitive technique, and among the first methods employed in the analysis of nucleic acids. A typical UV spectrum of an oligonucleotide shows the absorbance maximum at around 260 nm and the minimum at approximately 230 nm. Because of its sensitivity and nondestructive nature, UV spectroscopy is frequently utilized as a detection method for analytical procedures such as HPLC or CGE. UV spectra of nucleic acids show pronounced hypochromicity. The absorbance of a native DNA duplex is 20–30% lower than the absorbance of the


single strands in random coil conformation. This phenomenon is employed to measure the melting behaviour (Tm) of oligonucleotides. 2.4.1.1 Quantification of oligonucleotides UV-spectrophotometry is the main technique for quantification of nucleic acids, since the ionic character of these molecules results in an extensive hydration in the solid state. This and the possibility of salt contamination complicates the direct weighing of dry DNA samples. The determination of the absorbance or optical density (OD) is a good way to measure oligonucleotide amounts when the extinction coefficient (ε) of the molecule is known. The concentration (c) of the oligonucleotide solution is correlated with the absorbance reading (OD) according to Beer’s law: OD=εcl (l is the path length of the cuvette). The concentration of an oligonucleotide solution can be determined by hydrolysis of the oligonucleotide into mononucleotides, so minimizing stacking interactions between the bases. Provided that its base composition is known, this is the most accurate way to obtain the concentration but, especially for modified oligonucleotides, hydrolysis is not always possible. A method for the calculation of the extinction coefficient considering the nearest neighbour interactions between the bases is presented by Gray et al. (1989). An easier method that does not respect the sequence of the oligonucleotide is given by Brown and Brown (1991). In this formula the stacking interaction is estimated and considered by multiplying the sum of the individual extinction coefficients by a factor of 0.9. Both methods are valid only for single-stranded oligonucleotides. The extinction coefficient is almost independent of the nature of the phosphate group, so these calculations are applicable to backbone-modified oligonucleotides without corrections as well. Measurements are routinely performed in 1 cm cuvettes with a volume of 1 ml. The absorbance at 260 nm is then referred to as OD260. Calculate the extinction coefficient at 260 nm as follows:

a, t, g, c correspond to the number of the respective bases in the oligonucleotide sequence. 2.41.2 Melting curves of DNA In order to evaluate these effects and to determine the hybridization characteristics of an oligonucleotide, a melting curve has to be recorded (Figure 2.12). In UV melting experiments the change of absorbance with increasing temperature is measured (Puglisi and Tinoco, 1989). The measured hypochromicity is due to the stacking interaction between the chromophores of the nucleobases (sugar and phosphate backbones do not contribute to the UVabsorbance), resulting in a lower absorbance than expected for the sum of the


Figure 2.12 UV melting curve of a 20-mer ODN (Tm=62°C) and its derivative d (absorbance)/dT

extinction coefficients of the free nucleotides. The UV-absorbance increases as a duplex (with stacked bases) melts into two single strands with random coil conformation. The extent of hypochromicity is wavelength-dependent. For AT base pairs, maximum hypochromicity is observed at 260 nm while for CG base pairs the maximum is 280 nm. The optimum wavelength is between these two values, and depends on the base composition of the oligonucleotide investigated. The melting temperature Tm is commonly used as a measure for the binding affinity. It is the temperature at which 50% of the double strand has dissociated into its single strands. Once a curve is recorded, it has to be analysed to extract the Tm value and, if desired, the thermodynamic parameters of the transition. Several procedures for data analysis have been reported (Albergo et al., 1981; Marky and Breslauer, 1987; Puglisi and Tinoco, 1989): these are outside the scope of this chapter. A good way to obtain thermodynamic parameters of the transition is to plot 1/Tm versus In c. The resulting plot should be linear, the slope corresponds to R/∆H° and the intercept is ∆S°/∆H° (where H°, S° and R are enthalpy, entropy and the gas constant, respectively). Furthermore, the duplex to random coil transition is dependent on the ionic strength and the pH of the buffer. 2.4.2 Analysis of Oligonucleotides by High-Performance Liquid Chromatography High-performance liquid chromatography (HPLC) is the most widespread tool in the analysis of oligonucleotides (Oliver, 1989). Chromatography is the separation


technique mostly applied to modified antisense oligonucleotides with neutral backbone such as methylphosphonates and PNA. Among HPLC techniques, two main forms are distinguished according to their principle of separation: reversed-phase (RP) and anion-exchange chromatography. In RP-HPLC the oligonucleotide is bound by hydrophobic interaction to a nonpolar matrix and eluted with a gradient of increasing amount of organic solvent. For anion-exchange HPLC a positively charged ion-exchange material serves as stationary phase. The oligonucleotide binds to this material with the negatively charged phosphorodiester backbone, and elution is accomplished by a gradient of increasing ionic strength. 2.4.2.1 Reversed-phase HPLC Reversed-phase HPLC is most frequently used for the analysis of crude oligonucleotide synthesis mixtures. Since the separation is based on the hydrophobic interactions with the column material, it may be advisable to leave the terminal dimethoxytrityl (DMTr) protection group on the oligonucleotide. This unpolar group retards the migration and allows a good separation of the fulllength product from truncated sequences without a DMTr group. An important application of RP-HPLC is the analysis of backbone-modified oligonucleotides which cannot be accomplished by other techniques. Neutral oligonucleotide analogues do not bind to ion-exchange resins and do not migrate in an electric field, so that RP-HPLC is the only method for their separation at physiological pH. The modification of the backbone can introduce chirality at the phosphorus atom. Methylphosphonates or phosphorothioates consist of 2n diastereomers (n=number of modifications in the oligonucleotide) if no attempts at a diastereoselective synthesis were undertaken. This large amount of diastereoisomers can lead to a broadening of the peaks. A wide variety of RP columns for oligonucleotide analysis, differing in the type of packing material and particle diameter, is commercially available. For each of these columns a slightly different gradient is recommended. Furthermore, the choice of gradient depends on the nature and length of the oligonucleotide to be analysed. Triethylammonium acetate is most commonly used as elution buffer. This volatile compound can be removed in vacuo from the purified oligonucleotide upon preparative separations. 2.4.2.2 Anion-exchange HPLC Anion-exchange HPLC is a very effective analysis for nucleic acids (Drager and Regnier, 1985; Metelev and Agrawal, 1992). The resolution of n from n–1 chain length can be achieved for up to 30-mer oligonucleotides, and even attempts to increase the resolution up to 50-mer samples have been successfully undertaken.


Figure 2.13 Strong anion exchange HPLC of a crude oligonucleotide A-16-mer and a 15mer phosphorothioate

The individual resolution depends significantly on the column type, gradient and pH chosen. Strong anion-exchange material remain ionized at pH values up to 12, whereas weak exchangers possess lower pK values and are operated at neutral pH values. The elution of the sample is accomplished with a gradient of increasing salt concentration; sometimes organic solvent is added to the mobile phase which gives a better separation of certain analytes by minimizing hydrophobic interactions between the sample and the column packing material. Metal ions may poison the exchange resin of certain columns or hydrolyse RNA, so these should be used with metal-free HPLC systems. Short failure sequences with fewer negative charges elute before the fulllength product. The retention time is almost independent of the base composition of the oligonucleotide (Figure 2.13). Phosphorothioates bind strongly to anion-exchange columns. An elution can be obtained by increasing the ionic strength (e.g. 2.0 M NaCl) and by prolonged chromatography time. For these compounds weak anion-exchange columns have been employed successfully. The broadening of the peaks due to the diastereomeric nature of the phosphorothioates is not so pronounced as by RPHPLC. 2.4.3 Electrophoretic Techniques The electrophoretic analysis of oligonucleotides (Rickwood and Hames, 1990) is based on the migration of charged molecules under the influence of an electric field. During electrophoresis the molecules are separated due to their ratio of mass to charge and to interaction with a supporting medium. For DNA fragments


of the size of antisense oligonucleotides, cross-linked polyacrylamide is commonly used as supporting gel; for larger molecules agarose gels are suitable. Only molecules that carry a charge under electrophoresis conditions can be analysed by this technique. There have been few changes in the basic protocols for gel electrophoresis since the introduction of the method in the middle of the 20th century. In recent years capillary gel electrophoresis (CGE) arose as a new and potent alternative to the traditional electrophoretic techniques. 2.4.3.1 Polyacrylamide Gel Electrophoresis (PAGE) on slab gels Although the electrophoresis of nucleic acids on slab gels has been practised for several decades, it remains a powerful tool for the analysis of oligonucleotides. The main advantage of this method is a good resolution of the full-length oligonucleotide from truncated sequences. Furthermore, it combines the possibility of running several samples on one gel with a high sensitivity, especially in combination with radioactively labelled oligonucleotides. The interaction with the stationary polyacrylamide (PAA) matrix retards this migration, depending on size and shape of the moving species. For short oligonucleotides high concentrations of PAA (20%) are necessary, while lower concentrations allow the separation of oligonucleotides up to 1000 base pairs (3%). The electrophoresis is usually carried out in the presence of urea as denaturing agent. Under these conditions the oligonucleotides are separated by their length, and sequence-specific effects are minimized. Analysis at low temperature in the absence of denaturing agents may provide further information, such as secondary structure or duplex formation. Depending on the type and amount of oligonucleotide used, different techniques for the visualization of the molecules may be performed. A practical method for the detection of oligonucleotides is to stain the gel. Stains all (Green and Pastewka, 1974) is a suitable dye for the visualization of oligonucleotide samples in PAA gels. The most sensitive method for the detection is the autoradiography of radioactively labelled oligonucleotides (Sambrook et al., 1989). The irradiation of 32P is detected with a phosphorus imager or by covering the gel with an X-ray film which allows a quantification of the oligonucleotide. 2.4.3.2 Capillary Gel Electrophoresis (CGE) Capillary electrophoresis (CE) is a special form of electrophoresis where the charged molecules are separated in a capillary with 25 to 100 µm diameter (Cohen et al., 1988; Engelhardt et al., 1993; Khur and Moning, 1992). For the separation of oligonucleotides capillaries filled with a sieving gel (CGE) are used almost exclusively, although some publications have described the separation of


DNA in open capillaries. Several types of polymer have been described, such as the traditional cross-linked PAA, linear PAA without cross-linking agent, cellulose, agarose and hydroxyethylcellulose. The sample is injected electrokinetically by short immersion of one end of the capillary in the oligonucleotide solution and application of an electric field. The molecules migrate into the capillary, which is then shifted to a buffer reservoir, and electrophoresis is performed typically at 300 V/cm, corresponding to 12 kV, on a commercial 40 cm long PAA capillary. The sample molecules are resolved on their ratio of mass to charge by the same principles valid for PAGE on slab gels. At the end of the capillary, a window in the coating material allows direct detection of the oligonucleotides by UV-or laser-induced fluorescence (LIF). Coupling with an electrospray ionization (ESI) mass spectrometer is possible and can provide valuable information about the chemical composition of the detected peaks. CGE is a good method for the analysis of all phosphorothioate oligonucleotides, since these molecules bind strongly to ion-exchange HPLC columns. The main advantage of this method is the high resolution (single base resolution up to 300 nucleotides), and chemically modified oligonucleotides such as phosphorothioates, 5′-aminohexyl or biotinylated oligonucleotides are well resolved from unmodified molecules. The analysis is rapid and automation is possible, but the method is limited by salt or buffer ions present in the sample. 2.4.4 Mass Spectrometry of Oligonucleotides Mass Spectrometry (MS) is a valuable analytical technique that can provide additional information to chromatographic and electrophoretic data. The determination of the molecular mass allows the verification of length and base composition of the molecule. Fragmentation of the sample and analysis of the resulting pattern permit direct sequencing of shorter (10>9>15>11>12>13, as reflected by their HPLC retention time under the same conditions (Crooke et al., 1996) (Table 3.4). Stability of 6 to 15 was evaluated in TCE and in the presence of PLE using an HPLC on-line cleaning method (Pompon et al., 1992) (Table 3.4). Only 9, 14 and 15 were substrates for PLE. These three compounds have in common a structure with several successive SATE in a row and a lipophilicity in the same range. In contrast, 10 possesses a lipophilicity in the same range but alternating SATE groups, and was not a substrate. Our results suggest that the esterase activities of PLE are very sensitive to the presence of negative charges adjacent to the SATE groups, since all alternating pro-oligos (10 to 13) were not substrates while gap pro-oligos (14 and 15) and fully Me-SATE (8 and 9) were


hydrolysed. This discrepancy could be explained by the fact that only in gap structures can carboxyesterases hydrolyse a SATE group surrounded by neutral phosphotriesters. Hence, the difference of structure appears to be determinant for a pro-oligo to be a PLE substrate. This is not surprising, since it was reported that most carboxyesterases have higher affinity for lipophilic esters than for polar or charged substrates (Satoh, 1987; Satoh and Hosokawa, 1998). In addition, it appears that PLE does not accept substrates with too high a lipophilicity, since 6 and 7 are not substrates. In TCE only 6 and 7 were not substrates for esterases, probably because of their excessively high lipophilicity and their poor hydrosolubility. The other prooligos may be classed in two series: the fully SATE (8–9) and gap (14–15) on one hand, and the alternating ones (10–13) on the other. The half-life values for the first hydrolysis, reported in Table 3.4, brought out the difference of substrate capacity between these two series of pro-oligos. Thus, for each series, the higher the lipophilicity of pro-oligo, the lower is the rate of hydrolysis for the first SATE group. Our results suggest that when a pro-oligo possesses too high a lipophilicity, its substrate capacity for carboxyesterases decreases. We reported similar results with pro-oligos bearing pivaloyloxymethyl (POM) enzymolabile groups with either neutral or charged wings (see section 3.3.2) (Morvan et al., 1997; Tosquellas et al., 1997). It is noteworthy that compounds 8 and 9 with Me-SATE are fully deprotected to the parent oligo with a half-life of 20 h and 22 h respectively, and that compounds 14 and 15 with tBu-SATE are fully deprotected with a half-life of 33 h and 38 h respectively. This difference is probably due to the nature of the enzymolabile group (Me- versus tBu-SATE). In addition, the ex vivo stability studies herewith presented should not fully reflect the in vivo behaviour of the prooligos. We have further observed that the esterase activity in total cell extracts decreases after 3–4 h at 37°C (data not shown). Thus, demasking of prooligos in intact cells should proceed more rapidly. In addition, 9 was not degraded by snake venom and calf spleen phosphodiesterases (Tosquellas et al., 1998a). The behaviour of compounds 8 and 9 was also evaluated in human serum and in human gastric juice. No degradation was observed upon seven-day incubation in gastric juice, which is in agreement with the expected stability of phosphotriesters in acidic media. In human serum, no degradation was observed for 8 and the half-life of 9 was 3 h. 3.4.6 Preliminary Data of Pro-oligos′ Cell Uptake Four pro-oligos with different lipophilicity (Figure 3.8) were labelled with fluorescein and their uptake was studied on HeLa cells (unpublished data). Results showed (Figure 3.9) that whereas the control T12 phosphorothioate 16 was not taken up, the other pro-oligos were easily internalized in the HeLa cells, and that the higher the lipophilicity of pro-oligos, the higher was the uptake. It is

94 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Figure 3.8 Structure of the fluorescein labelled pro-oligos

noteworthy that compounds 16–19 contain respectively 12, 6, 6 and 4 negative charges. 3.5 Conclusion In conclusion, we have shown that SATE-prododecathymidylates (fully SATE or mixed SATE and phosphodiester) can be efficiently synthesized on solid support. As expected, the fully SATE pro-oligos are not degraded by nucleases (Tosquellas et al., 1998a) and present lower affinity to proteins as compared to the parent oligonucleotides (Vlassov et al., unpublished results). In addition, the pro-oligos are selectively hydrolysed to the parent oligonucleoside phosphodiester or phosphorothioate in total cell extract, but are much more stable in human serum. It is noteworthy that substrate capacity of each pro-oligo was dependent on its global lipophilicity, but also on the surroundings of the thioester to hydrolyse. The fact that some pro-oligos are substrates for esterases present in TCE and not for PLE is not surprising, since a large variety of carboxyesterases from various origins but with different substrate specificity have been isolated and characterized (Heymann, 1980; Mentlein and Heymann, 1984; Satoh, 1987; Aldridge, 1993; Satoh and Hosokawa, 1998). One can hypothesize that the bioavailability of such neutral or partially charged but still lipophilic pro-oligos will be different from that of the phosphodiester oligos. Furthermore, their pharmacokinetics may be modulated according to the nature of the enzymolabile protecting group, i.e. Me-SATE versus tBu-SATE, and the number and the disposition of the charges. It appeared that lipophilicity and hydrosolubility are two major components to be optimized.


Figure 3.9 Microscopy fluorescence of labelled pro-oligos incubated for 1 h at 10 µm with HeLa cells and retention time on reverse phase HPLC C18

The validation of the pro-oligo approach must be envisaged in vivo and experiments to this end are in progress. It is now possible to synthesize pro-oligos partially substituted with tBu-SATE protecting groups in any given positions. After labelling, such compounds, of various lipophilicities, are under evaluation for their ability to be taken up into intact cells. Preliminary data showed that a very important uptake was obtained when the lipophilicity of the pro-oligos was increased. Much more work will have to be done in this area before any definitive conclusion is reached, but we hope that the pro-oligo approach herewith presented could be the basis for the


development of a new generation of antisense oligonucleotides that could easily enter into intact cells. Acknowledgements We thank Dr I.Barber, Dr N.Mignet and Dr G.Tosquellas, who have worked on this approach, and K.Alvarez, C.Dell’Aquila, J.-C.Bologna and A.Meyer, who still are involved in this project. We thanks T.Beltran for mass spectrometry analysis. This project was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), the Association pour la Recherche sur le Cancer (ARC), and the CNRS. We thank ISIS Pharmaceuticals for financial support. References AGRAWAL, S. and IYER, R.P., 1997, Perspectives in antisense therapeutics, Pharmacol. Ther.,76, 151–160. ALDRIDGE, W.N., 1993, The esterases: perspectives and problems, Chem.-Biol. Interactions, 87, 5–13. ALVAREZ, K., TWORKOWSKI, I., VASSEUR, J.J., IMBACH, J.L. and RAYNER, B., 1998, A reinvestigation of sulfenyl groups as amino protecting groups for the synthesis of oligonucleotides on solid support by phosphoramidite chemistry, Nucleosides Nucleotides, 17, 365–378. BEAUCAGE, S.L. and IYER, R.P., 1992, Advances in the synthesis of oligonucleotides by the phosphoramidite approach, Tetrahedron, 48, 2223–2311. CROOKE, S.T., GRAHAM, M.J., ZUCKERMAN, J.E., BROOKS, D., CONKLIN, B.S., CUMMINS, L.L., GREIG, M.J., GUINOSSO, C.J., KORNBRUST, D., MANOHARAN, M., SASMOR, H.M., SCHLEICH, T., TIVEL, K.L. and GRIFFEY, R.H., 1996, Pharmacokinetic properties of several novel oligonucleotide analogs in mice, J. Pharmacol. Exp. Ther., 277, 923–937. DELL’AQUILA, C., IMBACH, J.L. and RAYNER, B., 1997, Photolabile linker for the solidphase synthesis of base-sensitive oligonucleotides, Tetrahedron Lett., 38, 5289–5292. DEVLIN, T., IYER, R.P., JOHNSON, S. and AGRAWAL, S., 1996, Mixed backbone oligonucleotides containing internucleotidic primary phosphoramidate linkages, Bioorg. Med. Chem. Lett., 6, 2663–2668. FODOR, P.A., LEIGTHON-READ, J., PIRRUNG, M.C., STRYER, L., LU, A.T. and SOLAS, D., 1991, Light-directed spacially addressable parallel chemical synthesis, Science, 251, 767–773. HASAN, A., STENGELE, K.P., GIEGRICH, H., CORNWELL, P., ISHAM, K.R., SACHLEBEN, R.A., PFLEIDERER, W. and FOOTE, R.S., 1997, Photolabile protecting groups for nucleosides: synthesis and photodeprotection rates, Tetrahedron, 53, 4247–4264. HAYAKAWA, Y., UCHIYAMA, M. and NOYORI, R., 1986, Nonaqueous oxidation of nucleoside phosphite to the phosphates, Tetrahedron Lett., 27, 4191–4194.


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MIGNET, N., CHAIX, C., RAYNER, B. and IMBACH, J.L., 1997a, Synthesis and evaluation of glucuronic acid derivatives as alkylating agents for the reversible masking of internucleoside groups of antisense oligonucleotides, Carbohyd. Res., 303, 17–24. MIGNET, N., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1997b, The prooligonucleotide approach. 5. Influence of the phosphorus atom environment on the hydrolysis of enzymolabile dinucleoside phosphotriesters, Bioorg. Med. Chem. Lett., 7, 851–854. MIGNET, N., TOSQUELLAS, G., BARBER, I., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1997c, The pro-oligonucleotide approach—synthesis and stability of chimeric pro-oligonucleotides in culture medium and in total cell extract, New J. Chem., 21, 73–79. MORVAN, F., TOSQUELLAS, G., MIGNET, N., BARBER, I., RAYNER, B. and IMBACH, J.L., 1997, The pro-oligonucleotide approach—chimeric dodecamers bearing six bioreversible protecting groups, Nucleosides Nucleotides, 16, 1213–1214. PÉRIGAUD, C., GOSSELIN, G. and IMBACH, J.-L., 1997, Minireview: from the pronucleotide concept to the SATE phosphate protecting groups, Curr. Topics Med. Chem., 2, 15–29. PÉRIGAUD, C., GOSSELIN, G., LEFEBVRE, I., GIRARDET, J.L., BENZARIA, S., BARBER, I. and IMBACH, J.L., 1993, Rational design for cytosolic delivery of nucleoside monophosphates—SATE and DTE as enzyme-labile transient phosphate protecting groups , Bioorg. Med. Chem. Lett., 3, 2521–2526. POMPON, A., LEFEBVRE, I. and IMBACH, J.L., 1992, On-line internal surface reversedphase cleaning—the direct HPLC analysis of crude biological samples— application to the kinetics of degradation of oligonucleotides in cell culture medium. Biochem. Pharmacol., 43, 1769–1775. SASTRY, J.K., NEHETE, P.N., KHAN, S., NOWAK, B.J., PLUNKETT, W., ARLINGHAUS, R.B. and FARQUHAR, D., 1992, Membrane-permeable dideoxyuridine 5′-monophosphate analogue inhibits human immunodeficiency virus infection, Mol. Pharmacol., 41, 441–445. SATOH, T., 1987, Role of carboxyesterases in xenobiotic metabolism, Rev. Biochem. Toxicol., 8, 155–181. SATOH, T. and HOSOKAWA, M., 1998, The mammalian carboxylesterases: from molecules to functions, Annu. Rev. Pharmacol. Toxicol., 38, 257–288. SEKINE, M. and SEIO, K., 1993, Synthesis and properties of N-tritylthio nucleoside derivatives and reductive removal of the tritylthio group by use of tributyltin hydride and tris(trimethylsilyl)silane, J. Chem. Soc. Perkin Trans., 1, 3087–3093. TOSQUELLAS, G., ALVAREZ, K., DELL’AQUILA, C., MORVAN, F., VASSEUR, J.J., IMBACH, J.L. and RAYNER, B., 1998a, The pro-oligonucleotide approach—solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates, Nucl. Acids Res., 26, 2069–2074. TOSQUELLAS, G., BARBER, I., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1996, The prooligonucleotide approach. 3. Synthesis and bioreversibility of a chimeric phosphorodithioate prooligonucleotide, Bioorg. Med. Chem. Lett., 6, 457–462.


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4 Peptide Nucleic Acids P.E.NIELSEN

4.1 Introduction

Figure 4.1 Chemical structures of DNA and PNA: B is a nucleobase

Peptide nucleic acids (PNAs) are not oligonucleotides and not even DNA analogues, but rather DNA mimics. Chemically PNA is a pseudopeptide in which the individual nucleobase units are amino acids joined together by amide bonds (Figure 4.1) (Nielsen et al., 1991; Egholm et al., 1992). Therefore, although PNA is a very good structural mimic of DNA in terms of its ability to form stable and sequence-selective complexes with complementary DNA or RNA oligonucleotides, it has unique chemical and physicochemical properties (Hyrup and Nielsen, 1996; Good and Nielsen, 1997). These can be exploited advantageously for development of pharmaceuticals, but also limit the degree to


which results, such as bioavailability and pharmacokinetics, obtained with oligonucleotides or their close chemical analogues can be used in developing PNA. It is therefore not surprising that ‘PNA drug development’ is lagging behind the much larger field of oligonucleotide (phosphorothioate) drug development. Nonetheless, many exciting results have been reported within the past five years, and some of these are highly relevant for discussing pharmaceutical aspects of PNA and also hold promise that highly efficient PNA-based gene therapeutic drugs may not be too far away. 4.2 Antisense Antisense inhibition of in vitro translation by PNA has been demonstrated in a variety of systems (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996; Gambacorti-Passerini et al., 1996; Good and Nielsen, 1998a). Not surprisingly, PNA—RNA duplexes are not substrates for RNase H, and thus antisense inhibition must take place by an RNase H-independent mechanism (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Therefore, sensitive sequence targets on specific mRNAs identified by phosphorothioates are not necessarily good targets with PNA. Indeed, it has been found that sensitive PNA targets are found at the AUG initiation codon (Knudsen and Nielsen, 1996; Gambacorti-Passerini et al., 1996) or upstream from this (GambacortiPasserini et al., 1996), but no thorough ‘gene-walks’ (Monia et al., 1996) have so far been reported for PNA. It is also worth noting that triplexforming PNA decamers are able to cause translation elongation arrest, and homopurine targets, even if these are situated within the coding region, should be prime targets for PNA antisense (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Recently a very exciting study reported antisense downregulation of the galanin receptor activity in cells in culture using PNAs conjugated to membranepenetrating small peptides (e.g. the 16 amino acid homeodomain of the antennapedia protein from Drosophila) (Pooga et al., 1998), and these authors were even able to demonstrate downregulation of the galanin receptor activity in live rats upon intrathecal injection of the PNA. In another, less thorough, study, ‘naked’ PNAs targeted to either the mRNA of the opioid(mu)- or the neurotensin-receptor were injected into the brain of rats, and decreased ligand-binding activity of the respective receptor was reported (Tyler et al., 1998). The latter results are surprising in view of the fact that it is generally observed that PNAs are taken up very poorly by eukaryotic cells in culture (Bonham et al., 1995). However, as with oligonucleotides, cells in tissues and in particular neurons may well behave differently from cells in culture. These reports demonstrate that it is possible to find ways and/or systems in which PNAs exhibit antisense compatible effects in vivo. Formally, these two

102 PEPTIDE NUCLEIC ACIDS

Figure 4.2 Secondary structure of E.coli 23S RNA indicating the positions of the peptidyl transferase centre and the α-sarcin loop. The targets for the PNAs, H-Lys-TTJTJJJTTTJT(eg1)3-TCTTTCCGTCTT-LysNH2 and H-Lys-JTJTJJT-(eg1)3-TCCTCTC-LysNH2 are shown in bold below (positions 2051–2062 and 2659–26659). J is pseudoisocytosine (cf. Figure 4.4)

studies have not shown that the biological effects on the rats are bona fide antisense. and therefore more studies are warranted.


4.3 Antimicrobials Quite surprisingly, PNAs are to a limited extent taken up by E.coli bacterial cells, and by designing triplex forming bis-PNAs targeted to essential loop regions of 23S ribosomal RNA (Figure 4.2), PNAs with antibiotic-like properties were obtained (Good and Nielsen, 1998b). Specifically, it was found that two such PNAs (Figure 4.2) inhibited translation in vitro at nanomolar concentrations (Good and Nielsen, 1998b) and were able to inhibit the growth of both wild type E.coli K12, and, especially, a permeable mutant (AS 19) at micromolar concentrations. Provided the bacterial uptake of such PNAs can be improved by chemical modification and/or formulation and that similar or improved effects can be demonstrated with pathogenic strains of bacteria, these results open the way for the development of novel PNA-based antibacterial drugs. Along similar lines, it was demonstrated that antisense downregulation of gene expression in E.coli is possible with PNAs (Good and Nielsen, 1998a). Specifically, PNAs targeted to the AUG-region of the mRNA of the β-lactamase gene were found to resensitize formerly penicillin-resistant (due to β-lactamase production) bacteria to penicillin by six to seven orders of magnitude using micromolar concentrations of the PNAs. Thus the antisense principle could also be a means for development of ‘antibiotics’ by targeting essential genes in pathogenic bacteria. 4.4 Anti-telomerase The enzyme, telomerase, which is responsible for maintaining telomere ends on chromosomes during mitosis, and which appears to be overexpressed in at least some cancer cells, is an RNA—protein complex in which the RNA is essential for actitivity. The RNA actually provides the template for the new telomere. PNAs targeted to this RNA are very potent inhibitors of the enzyme in vitro (C50 in the sub-nanomolar range) (Norton et al., 1996), and, provided telomerase activity is indeed required for malignant growth, such PNAs could be developed into anticancer drugs. 4.5 Antigene Homopyrimidine PNAs bind to sequence complementary homopurine targets in double-stranded DNA by invasion into the DNA duplex by forming an internal PNA2—DNA triplex in a strand displacement complex (Figure 4.3) (Nielsen et al., 1994a). Such PNA strand displacement complexes are extremely stable and inhibit protein (transcription factor) binding to an overlapping (or even adjacent) binding site (Nielsen et al., 1993; Vickers et al., 1995), and can even arrest


Figure 4.3 Schematic drawing of a PNA—dsDNA strand displacement complex (right) consisting of two PNA strands (composed of thymines (T) and cytosines (C)) that invade the DNA duplex and bind to the complementary DNA strand (composed of adenines (A) and guanines (G)) by Watson-Crick and Hoogsteen base pairing (left). The noncomplementary DNA strand is extruded as a singlestranded loop

Figure 4.4 Hoogsteen recognition of guanine by pseudoisocytosine, a ‘permanently protonated’ cytosine mimic

transcription elongation (Hanvey et al., 1992; Nielsen et al., 1994b; Vickers et al., 1995; Praseuth et al., 1996). The most stable complexes form with two antiparallel PNAs, of which one binds antiparallel by Watson-Crick base pairing to the DNA target and the other binds antiparallel by Hoogsteen base pairing. Thus even more potent PNAs are obtained by chemically joining such two PNAs into a bis-PNA (Egholm et al., 1995). Furthermore, pH independent binding can


be accomplished by exchanging cytosines in the Hoogsteen PNA strand with pseudoisocytosines (Figure 4.4) (Egholm et al., 1995). Once formed, the PNA2—DNA strand invasion complexes are exceedingly stable and are formed with exquisite, kinetically controlled sequence selectivity (Demidov et al., 1995; Kuhn et al., 1997). However, because the DNA double helix must be opened (denatured) in the binding process, the binding rate is very sensitive to elevated ionic strength, and binding is in fact very slow at physiological ionic strength (140 mM K+). Thus, most in vitro experiments demonstrating antigene effects were done using PNA—dsDNA complexes that were preformed at low ionic strength and then transferred to higher ionic strength buffers. It might therefore be feared that biological antigene effects of PNA would be very weak. None the less, a recent study reported mutagenic effects of a bis-PNA target to a gene in mouse cells (Faruqi et al., 1998). Although it is not possible from these data to determine the degree of PNA binding to the dsDNA target, the result clearly indicates that some level of binding was achieved. Indeed, two observations from in vitro experiments could provide an explanation for facilitated binding in vivo. It has been found that the transcription process itself, presumably via the single-stranded transcription bubble, catalyses PNA binding (Larsen and Nielsen, 1996), and transcription may also indirectly facilitate PNA binding via induction of negative supercoiling upstream from the transcription complex, because it has been found that negative supercoiling can accelerate PNA binding up to 200-fold at 140 mM K+ (Bentin and Nielsen, 1996). 4.6 Anti-HIV In vitro studies have shown that purine/pyrimidine mixed sequence, duplexforming PNAs as well as homopyrimidine triplex-forming PNAs targeted to, for example, gag-RNA are very potent inhibitors of HIV reverse transcriptase by blocking the elongation by the enzyme (Koppelhus et al., 1997; Lee et al., 1998). Thus further developments in this area, especially regarding cellular uptake and bioavailability, may lead to new anti-HIV drugs. 4.7 Pharmacology Very little is known about the phamacological behaviour of PNA. It has been established that PNAs are inherently very stable in biological fluids such as serum and cellular extracts (Demidov et al., 1994), but thorough studies on bioavailability, pharmacokinetics and pharmacodynamics are still very much needed. As already indicated (Norton et al., 1995; Pardridge et al., 1995; Pooga et al., 1998; Aldrian-Herrada et al., 1998), PNA peptide conjugates could be of high interest in this connection.


Figure 4.5 Comparison of a diaminopurine-thymine and a guanine-cytosine base pair. The ‘extra’ 2-amino group of diaminopurine is shown in bold

Figure 4.6 Examples of a backbone functionalized PNA using the amino acid lysine, leucine, aspartic acid or asparagine instead of glycine in the PNA backbone

4.8 Further Developments Besides favourable hybridization (Egholm et al., 1993) and stability (Demidov et al., 1994) properties, PNA distinguishes itself from other gene therapeutic drug leads by what may be termed ‘chemical flexibility’: it is fairly straightforward and easy to synthesize PNA oligomers with modified backbones and/or nucleobases. For instance, substituting adenine with diaminopurine (Figure 4.5) increases the thermal stability of PNA—DNA duplexes by 2–4 K per substitution (Haaima et al., 1997). Furthermore, chemical functionality can easily be introduced in the PNA backbone by using α-amino acids other than glycine (Figure 4.6) (Haaima et al., 1996; Püschl et al., 1998) without seriously affecting the hybridization efficiency. Such modifications could be extremely valuable in tuning the pharmacokinetic and pharmacodynamic properties of PNA drug leads, and they might also improve PNA bioavailability. Acknowledgement This work was supported by the Danish National Research Foundation.


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NORTON, J.C., WAGGENSPACK, J.H., VARNUM, E. and COREY, D.R., 1995, Synthesis and membrane permeability of PNA—peptide conjugates, Bioorg. Med. Chem., 3, 437. PARDRIDGE, W.M., BOADO, R.J. and KANG, Y.-S., 1995, Vector-mediated delivery of a polyamide (‘peptide’) nucleic acid analog through the blood—brain barrier in vivo, Proc. Natl Acad. Sci. USA, 92, 5592–5596. POOGA, H., SOOMETS, U., HÄLLBRINK, M., VALKNA, A., SAAR, K., REZAEI, K., KAHL, U., HAO, J.-X., XU, X.-J., WIESENFELD-HALLIN, Z., HÖKFELT, T., BARTFAI, T. and LANGEL, Ü., 1998, Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo, Nat. Biotechnol., 16, 857. PRASEUTH, D., GRIGORIEV, M., GUIEYSSE, A.L., PRITCHARD, L.L., HARELBELLAN, A., NIELSEN, P.E. and HELENE, C., 1996, Peptide nucleic acids directed to the promoter of the α-chain of the interleukin-2 receptor, Biochim. Biophys. Acta, 1309, 226–238. PÜSCHL, A., SFORZA, S., HAAIMA, G., DAHL, O. and NIELSEN, P.E., 1998, Peptide nucleic acids (PNAs) with a functional backbone, Tetrahedron Lett., 39, 4707–4710. TYLER, B.M., MCCORMICK, D.J., HOSHALL, C.V., DOUGLAS, C.L., JANSEN, K., LACY, B.W., CUSACK, B. and RICHELSON, E., 1998, Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo, FEBS Lett., 421, 280–284. VICKERS, T.A., GRIFFITH, M.C., RAMASAMY, K., RISEN, L.M. and FREIER, S.M., 1995, Inhibition of NF-kappa B specific transcriptional activation by PNA strand invasion. Nucl. Acids Res., 23, 3003–3008.

PART THREE Delivery

5 Peptide-mediated Delivery of Oligonucleotides E.VIVÈS AND B.LEBLEU

5.1 Delivery Vehicles for the Improved Uptake of Nucleic Acids: a Survey Synthetic oligonucleotides offer interesting prospects for the control of gene expression through specific interactions with RNA (antisense oligonucleotides, ribozymes), DNA (triple helix-forming oligonucleotides) or even proteins (decoy oligonucleotides, aptamers). These strategies, and in particular ribozymes and antisense ON, have been the object of numerous studies in various in vitro and in vivo (including several human clinical trials) biological models. Whatever the successes encountered with a first generation of phosphorothioate antisense ON and synthetic ribozymes, it is generally admitted that greatly improved pharmacological properties will have to be provided. The most frequently encountered problems are degradation by nucleases, insufficient affinity for the target, lack of specificity and, more importantly, poor bioavailability (for a review see Gewirtz et al. (1998)). Synthetic oligonucleotides are negatively charged and therefore do not freely cross biological membranes, a problem also encountered with mononucleotides in antiviral chemotherapy or with plasmid DNA in gene delivery. Mere neutralization of the internucleotidic linkages does not lead to improved cellular uptake as experienced with methylphosphonates (Giles et al., 1993) or with peptide nucleic acids (Wittung et al., 1995). The synthesis of prooligonucleotides (see Chapter 3) might, however, lead to a breakthrough in this field. Somewhat unexpectedly antisense ONs are taken up by various cell types through as yet poorly explored mechanisms involving fluid phase and receptormediated endocytosis (Yakubov et al., 1989; Geselowitz and Neckers, 1992; Beltinger et al., 1995). The efficiency of these endocytotic processes for oligonucleotides is generally poor, and material remains for a large part segregated in endocytotic vesicles. Some notable exceptions have been reported, for instance primary keratinocytes or keratinocyte cell lines which efficiently

112 PEPTIDE-MEDIATED DELIVERY OF OLIGONUCLEOTIDES

internalize antisense ONs and concentrate them in the nuclei (for a review see Lebleu et al., 1997). Successes of in vivo studies with synthetic ribozymes or antisense phosphorothioate analogues have been assigned partly to efficient uptake of nucleic acids in primary cells, but the underlying mechanisms have not been explored to our knowledge. Although much attention has been paid to the cellular uptake of synthetic ON, intracellular trafficking or crossing through biological barriers such as the bloodbrain barrier will be of the utmost importance for the future of nucleic acid-based drugs. Many strategies have been proposed to improve cellular delivery of synthetic oligonucleotides. Direct microinjection (Leonetti et al., 1991) and permeabilization of the plasma membrane by physical or enzymatic agents, such as electroporation (Bergan et al., 1996) or streptolysin (Giles et al., 1993), have been successfully adapted to antisense ON but cannot be applicable for in vivo experimentation. Association or encapsidation of nucleic acids to particulate delivery vehicles such as liposomes or nanoparticles (reviewed in Lebleu et al., 1996) has been the object of many developments and is reviewed in Chapters 6 and 7. Chemical conjugation or physical association to cationic lipids or cationic peptides has been explored for the delivery of synthetic ONs and plasmid DNA. The conjugation of plasmid DNA or antisense ONs to various cationic lipid formulations (Zelphati and Szoka, 1996) or to polyethyleneimine (Boussif et al., 1995) allows their efficient delivery in many cell types, at least in in vitro experiments, and is the most widely used strategy so far. Studies in our group have established the potential of cationic polyaminoacids for the delivery of nucleic acids (see below). The chemical conjugation of nucleic acids to cell ligands, such as transferrin (Gotten et al., 1992) or neoglycoproteins (Midoux et al., 1993; Hangeland et al., 1995) represents an interesting strategy allowing specific recognition by cognate receptors at the cell surface and subsequent internalization. None of these strategies is devoid of problems, and most studies in this emerging field are still in an early phase. Which strategy would be preferable in a particular therapeutic application cannot be decided at the present time. Among the most commonly encountered problems in drug delivery are capture of particulate material by cells of the reticulo-endothelial system, immunogenicity or toxicity of the delivery vehicle for certain cell types, and segregation in the endocytic vesicles. Remarkably, however, cationic lipids (Zelphati and Szoka, 1996) and polyethyleneimine (Boussif et al., 1995) are taken up by endocytosis but efficient release from the endosomes is allowed through lipid fusion or endosome destabilization.


5.2 The Potential of Peptides for Nucleic Acids Delivery So far, none of the DNA transfection protocols have approached the efficiency with which viruses internalize (and express) their nucleic acids payload in an infected cell. Many efforts have therefore been devoted to engineering virus mimics for the delivery of plasmid DNA, antisense ONs or ribozymes. Two main strategies have been evolved by viruses to bypass cellular membranes: fusion of their envelope at neutral pH with the cell plasma membrane, and endocytosis (mainly receptor-mediated) followed by escape from the endocytotic compartments. In both cases the efficiency of nucleic acids release largely relies on fusogenic and/or membrane-destabilizing events driven by fusogenic domains in viral proteins. Interestingly, several cellular or viral proteins can pass cellular membranes efficiently, as demonstrated for the Antennapaedia protein (Derossi et al., 1994) and for the HIV-1 Tat protein (Vivès et al., 1997a). Whatever the physiological relevance of these properties, they might be exploited for the delivery of nucleic acids, as will be discussed in section 5.5. Similar goals are sought for gene and synthetic oligonucleotides transfer, e.g. nucleic acids protection from nucleases in biological fluids, selective recognition by targeted cells or tissues when possible, efficient cellular uptake, and delivery and accumulation in the appropriate intracellular compartment. However, significant differences can also be found. For example, a single expressed gene is in principle sufficient for cell transformation, while many copies of a synthetic antisense ON are required in order to exert a significant biological activity. On the other hand, the large size of plasmid DNA requires compaction prior to delivery, which is not the case for antisense ONs. Finally, short ONs released in the cytoplasm will diffuse to the nucleus through the nuclear pores (Leonetti et al., 1991), while nuclear uptake is a major problem for plasmid DNA in resting cells in particular. ON analogues with improved pharmacological properties can be engineered, as discussed in several other chapters. We will restrict ourselves to a review of the literature dealing with peptide (or polypeptide)-mediated delivery of antisense ON, with emphasis on our own work dealing with poly (L-lysine)mediated delivery (reviewed in Lebleu et al., 1996), and on attempts to exploit the fusogenic properties of influenza haemagglutinin (Bongartz et al., 1994) or the membrane translocating activity of the HIV-1 Tat protein (Vivès et al., 1997a). Although most recent efforts have been focused on attempts to harness the potential of peptides for increased cellular uptake and release from (or avoidance of) the endocytotic compartment, other strategies might be beneficial. Short cellular recognition motifs could potentially be introduced to restrict uptake by certain cell types, as already demonstrated in same studies (Leonetti et al., 1990b; Hangeland et al., 1995).


Once delivered in the cytoplasm, antisense ONs are rapidly translocated to the nuclei and bind tightly to nuclear proteins (Clarenc et al., 1993). If potential synthetic ON targets such as pre-mRNA or DNA obviously exist in the nuclei, tight binding to nuclear proteins might reduce the free intracellular ON pool. Reducing nuclear proteins trapping by appropriate chemical modification of the ON or favouring nucleo-cytoplasmic transport by conjugation to appropriate peptidic motifs might therefore be required to target cytoplasmic mRNAs. In this respect, studies with ribozyme-expressing genes have clearly demonstrated the benefits of co-localizing the effector ribozyme with the target RNA (Bertrand et al., 1997). The appending of basic peptides to oligonucleotides (or their analogues) increases affinity for their nucleic acids target, as shown in several cell-free models, and should also favour invasion of highly structured DNA or RNA targets (Iyer et al., 1995). Whether any of the strategies discussed here will be able to meet some or all of these requirements without unacceptably increasing cost and complexity is far from certain. Finally, it should be pointed out that such peptide-based delivery vectors could also be useful to improve the bioavailability of other potential drugs, and in particular of non-permeant peptides. 5.3 Strategies for the Coupling of Peptides to Oligonucleotides Several chemical routes have been proposed for the conjugation of a peptide to an ON. The most convenient method would in principle be to synthesize entirely the chimeric molecule by stepwise synthesis on a single solid support. Although feasible, this strategy is still under development, mainly to circumvent problems arising from incompatibilities of the protection schemes required for the solidphase synthesis of ONs and peptides. Several low molecular weight chimeric molecules have been synthesized by such strategies, but the final products were obtained with poor yields (Haralambidis et al., 1990; Truffert et al., 1994). It is therefore likely that the direct synthesis of larger antisense ON—peptide conjugates will lead to further synthesis and/or purification problems (Peyrottes et al., 1998). It should be recalled here that both entities require a minimal length to retain their desired activities, i.e. 10 to 15 amino acids for an efficient translocating activity of the peptide, and 12 to 15 nucleotides for maintaining a sequence-specific binding of the ON. A solid-phase strategy for the synthesis of longer ON—peptide conjugates at a scale suitable for antisense studies has, however, been described (Soukchareun et al., 1995). It concerned a fusion peptide located in the N-terminus of the HIV transmembrane gp41 glycoprotein. In this method, the peptide (11 or 16 amino acids) was assembled first by the Nfluorenylmethoxycarbonyl (Fmoc) solid-phase strategy on a derivatized controlled pore glass (CPG) resin. After derivatization of the amino terminus to a hydroxy group, the standard phosphoramidite method was used for the synthesis


of the ON moiety (20-to 27-mer). Although this method could circumvent some peptide solubility problems sometimes encountered during a classical block conjugation scheme, the overall yield was in the range of 6–14% and will certainly have to be improved. In addition, new side-chain protection moieties will probably be required for the efficient stepwise synthesis of longer peptide— ON conjugates. It therefore appeared easier to couple separately prepared and purified entities. Peptide and nucleotide chemistries allow a large panel of derivatization schemes for both molecular synthons to allow their specific coupling. One convenient strategy for coupling appears to be disulphide bond formation between the peptide and the ON. The incorporation of a sulphhydryl group in a peptide sequence is obvious through a cystein residue available from the native sequence or appended to the N-terminal or to the C-terminal end of the peptide. Likewise, several chemical protocols allow the efficient introduction of a sulphhydryl group at either the 5′ or the 3′ end of an ON (Connolly and Rider, 1985; Li et al., 1987; Sinha and Cook, 1988; Zuckerman et al., 1987; Gupta et al., 1991). The condensation of two sulphhydryl groups containing molecules through disulphide bridge formation could lead to the loss of substantial amounts of starting material through homodimers synthesis. It is therefore worth activating first one of the thiol-containing molecules. Since it is generally easier to synthesize peptides in larger quantity (synthesis scale of several hundred micromoles) than ON (synthesis scale of some micromoles), an initial activation of the ON moiety (a reaction which is easily brought to completion) should be preferred. A two-to three-fold molar excess of the peptide over the activated ON would then lead to chimeric molecules with very good yields (Chu and Orgel, 1988; Vivès and Lebleu, 1997; Eritja et al., 1991). Several thiol-activating groups such as 2, 2-dipyridyldisulphide, 3-nitro-2pyridinsulphenyl (Npys) and 2-nitrophenyl are available, and lead to the activated molecule with very good yields (Chu and Orgel, 1988). Alternatively, it is possible to synthesize directly the peptide with an activated thiol group by incorporating a Boc-Cys(Npys) monomer (Eritja et al., 1991). The intracellular release of the transported material will be preferred in many applications, since this would prevent possible interferences with the delivery vector for target recognition. In this respect, it is generally anticipated that the intracellular reductive environment will reduce disulphide bridges and release the conjugated drug (Derossi et al., 1998; Prochiantz, 1996), although no direct evidence has been provided to our knowledge. Along these lines, a 15-mer antisense ON coupled to a KDEL peptide through a stable thioether bond was 10 times fold more efficient than the disulphide-linked chimeric molecule (Arar et al., 1995). An early reduction of the disulphide bond of the ON—peptide conjugate was proposed to explain that difference (Arar et al., 1995). Early studies on the fate of radioiodinated tyramine conjugated to peptide carriers through a disulphide bond suggested the Golgi apparatus as the most probable site of


reduction (Feener et al., 1990). However, such mechanisms should be relevant only for conjugates which are internalized by endocytosis. In some applications, however, a stable linkage between the conjugated material and the delivery vector might be sought, for example in cases in which the conjugated peptide is also involved in intracellular trafficking or in target recognition. Several stable bonds between the peptide and the ON have been described, e.g. thioether (Arar et al., 1995) or maleimide linkages (Zhu et al., 1993; Tung et al., 1991; Eritja et al., 1991; Ede et al., 1994). For instance, a procedure for the selective coupling of an ON to a peptide was based on the reactivity of a maleimide group bound to the ON with the thiol group of a cysteine residue of the peptide (Tung et al., 1991). Following the same chemistry a series of positively charged (D-ornithine)n-cysteine peptides was coupled to an ON (Zhu et al., 1993). Binding of the nucleic acid—peptide conjugate to its complementary DNA target showed that Tm increased with the net charge on the conjugated peptide. The coupling of highly basic peptides such as Antennapaedia or Tat to an ON should improve the binding kinetics and the stability of the chimeric molecules to their target. Moreover, site-directed cleavage with RNase H demonstrated that the peptide-modified ON hybridized specifically with its RNA target sequence. 5.4 Poly (L-lysine)-based Delivery Systems The efficiency of poly (L-lysine) (PLL) conjugation to deliver ON was first established using 2–5A tetramers, a generic term for the 2′–5′ linked activators of RNase L. Activation of RNase L (see also Chapter 2) was achieved when incubating various cell lines in culture with PLL-conjugated 2–5A or 2–5A analogues at nanomolar doses, while free 2–5A oligomers were totally inactive even in the micromolar concentration range (Bayard et al., 1986). Likewise, PLL-conjugated short antisense ON were shown to be 10 to 50 times more active than unconjugated oligomers in various biological models (reviewed in Lebleu et al., 1996). For example, a PLL-conjugated 15-mer unmodified antisense ON complementary to the translation initiation site of HIV-1 tat mRNA exerted a dose-dependent antiviral activity (with an IC 50 of 150 nM) in de novo infected T cell lines, while unconjugated material was barely active (Degols et al., 1994). Moreover a sequence-specific effect could be demonstrated with PLL-conjugated material but not with unconjugated material, as often encountered when using antisense ON as antivirals (Degols et al., 1992). Likewise, short antisense ON complementary to the U5 or to the pre-PBS regions of HIV-1 genomic RNA exerted a sequence-specific reduction in viral DNA production and an antiviral activity at submicromolar concentrations (Bordier et al., 1995). The mechanism through which PLL increases the biological activity of antisense ON is not fully understood. The positive net charge of these conjugates


should facilitate binding to the negatively charged cell membrane and subsequent internalization through adsorptive endocytosis. Proteolysis of the polyaminoacids carrier in the endocytic vesicle is probably involved in the release of the conjugated material, since poly (D-lysine)-conjugated material is inactive (Leonetti et al., 1990a). Protection against nucleases in cell culture media as well as in cells themselves should also contribute to the increased efficiency of PLL-conjugated material. Indeed, the antisense (or 2–5A) oligomers were oxydized at their 3′ end before being conjugated to ε-amino groups of the carrier polypeptide. Whatever the fate of the carrier moiety after cell uptake, such 3′ modification will confer increased resistance to phosphodiesterases which are known to play a major role in the catabolism of nucleic acids. Various modifications of these PLL carriers have been introduced in order to confer cell-targeting properties. In these studies the antisense ON was covalently linked or was complexed by electrostatic interaction with the polycationic carrier. For example, glycosylated PLL was used to allow recognition by membrane lectins. The PLL entity has also been covalently linked to polypeptidic ligands as transferrin (Citro et al., 1993) or asiologlycoproteins (Nakazono et al., 1996), thus allowing preferential capture by cells expressing transferrin or galactose receptors, respectively. Whether such PLL-based delivery vectors will be applicable for the in vivo delivery of nucleic acids remains questionable, although extensive studies have been conducted in the perspective of gene transfer. Molecular complexity of these conjugates, cytoxicity for certain cell types, immunogenicity and complement activation, as well as segregation in endocytotic compartments, will limit in vivo applications. Methods for the covalent attachment of ONs to alternative cationic polypeptides such as polyarginine (Wei et al., 1994) or polyornithine (Zhu et al., 1993) have been described, but the biological activity of these conjugates in intact cells has not been reported. More complex structures known as L-oligomers have recently been proposed as drug carriers. They consist of an oligolysine scaffold branched with basic amino acid sequences consisting of a lysine pentapeptide extended by a nuclear localization signal. In contrast to PLL, these L-oligomers were efficiently translocated to cell nuclei apparently through an active process (Sheldon et al., 1995). Their use for the transport of nucleic acids material has not been reported, however. 5.5 Conjugation to Fusogenic Peptides Allowing Membrane Fusion or Membrane Translocation As mentioned in section 5.2, most of the peptide sequences described here are short native sequences belonging to various proteins showing translocating, fusogenic or intracellular trafficking properties. Some of these sequences have


been further studied to point out the determinants that could be involved in the observed translocating process and used for internalizing different molecular entities as reviewed below. 5.5.1 Influenza Virus Haemagglutinin Fusogenic Peptide The fusogenic properties of the influenza virus haemagglutinin protein have been extensively studied. They are currently assigned to a pH-dependent conformational change of the viral haemagglutinin occurring in the endosomal compartment leading first to the exposure of its hydrophobic N-terminal region, and second to the fusion of the viral and endosomal membranes (Plank et al., 1994). A short peptide (24 amino acids) corresponding to the fusogenic region of this protein retains the conformational change at acidic pH, and therefore its membrane-destabilizing potential. A HIV-1 Tat m-RNA specific antisense ON that has been covalently bound to this fusogenic peptide by a disulphide bridge has an increased antiviral activity in vitro, probably as a result of improved intracellular release (Bongartz et al., 1994). The increment of activity remained low, however, and the majority of the conjugated ONs remained segregated in endocytic vesicles (Bongartz, Milhaud and Lebleu, unpublished observations). The topological organization of the haemagglutinin proteins at the viral membrane surface is critical for fusion and is not maintained in these conjugates. Interestingly, such peptides increased the delivery of plasmid DNA complexed with transferrin-PLL conjugates (Wagner et al., 1992) and were found to be much more efficient when used as dimers. 5.5.2 HIV-1 gp41 Fusogenic Peptide A chimeric 27 residues peptide combining the HIV gp41 fusion sequence and the SV40 T-antigen nuclear localization sequence was used for the intracellular delivery of antisense ON and for the transfection of mRNA (Morris et al., 1997; Vidal et al., 1997; Chaloin et al., 1998). The HIV gp41 peptide was also shown to facilitate the intracellular delivery of covalently linked antisense ON (Soukchareun et al., 1995). 5.5.3 Antennapaedia Peptide Studies of the biological role of the Drosophila Antennapaedia protein led to the serendipitous observation of its capacity to be internalized in nervous cells. A 16 amino acid long peptide derived from the Antennapaedia homeodomain third helix was still able to translocate through the plasma membrane and to reach the nucleus. Extensive structure-relationship studies have been performed in order to


define the motifs responsible for cell uptake. Such studies have excluded the involvment of an amphipathic α-helix as initially proposed by reference to the structure of several viral fusogenic peptides. The translocating activity does not involve a chiral receptor, as the uptake of a retro-inverso form of the Antennapaedia peptide was fully maintained (Derossi et al., 1996; Brugidou et al., 1995). Interestingly, the peptide was also internalized at 4°C, a temperature at which endocytosis does not occur. The formation of inverted micellar structures at the membrane level after electrostatic interactions of negatively charged phospholipids and the positively charged amino acids of the peptide has been proposed as a mechanism for internalization (Derossi et al., 1996). The tryptophan residue at position 48 might play a crucial role in the destabilization of these structures, thus leading to the release of the peptide at either side of the membrane. The intranuclear accumulation of the peptide probably involves its passive diffusion (as the phenomenon was also described for incubation at 4°C), followed by as yet unexplained concentration in this compartment. The Antennapaedia peptide has been extensively used as a vector for ONs’ and for peptides’ cellular delivery, and is commercially available under the trade name of Penetratin® (Appligen). Up to now, over 20 different ONs and peptides have been coupled and vectorized by the complete Antennapaedia homeodomain or by its shorter version corresponding to the third helix. The size of the vectorized molecule is a limiting factor for the uptake, as peptides over 100 amino acids cannot be carried across the plasma membrane by this strategy (Prochiantz, 1996). This can probably be explained by a steric hindrance which could prevent access to the plasma membrane and impair inverted micelles formation. Exogenous antigens with length in the range of 30–40 amino acids were taken up efficiently when coupled to the Antennapaedia peptide (Schutze et al., 1996). Concerning nucleic acids delivery, most of the experiments were dedicated to an antisense effect and the ON size mainly ranged between 15- and 25-mer. Plasmid DNA was not internalized by the Antennapaedia peptide (Prochiantz, 1996). The conjugate exerted an antiviral activity against vesicular stomatitis virus when added in the cell culture at doses as low as 100 nM. Likewise, an antisense ON directed against the amyloid precursor protein (APP) was coupled to the Antennapaedia peptide and inhibited neurite outgrowth at extracellular concentration ranging between 40 and 200 nM (Allinquant et al., 1995). The conjugation of an antisense PNA complementary to galanin-receptor 1 mRNA with penetratin allowed PNA internalization and reduced selectively GalRl expression in melanoma cell cultures and, excitingly, in vivo after intrathecal administration (Pooga et al., 1998) (see also Chapter 4). As mentioned above, structure-activity relationship studies of the translocating activity of the Antennapaedia peptide have led to the synthesis of several analogues. One of them contained only arginine and tryptophan residues and exhibited a stronger propensity to translocate through the plasma membrane. Tryptophan residues are known to be a key element in the transmembrane


domain of proteins, as they are in most cases located at the interface between the lipidic and the hydrophilic medium. Additional studies based on artificial membrane models could lead to the discovery of new peptide sequences or peptide mimetics showing an increased ability to translocate as well as a higher metabolic stability. 5.5.4 Tat Basic Domain The purified HIV-1 Tat protein is able to translocate efficiently through the plasma membrane and to reach the nucleus, as attested by its ability to transactivate the viral genome (Frankel and Pabo, 1988). This translocation activity has been assigned to a region of the Tat protein centred on a cluster of basic amino acids. Indeed, the chemical coupling of a few Tat-derived peptides extending from residues 37 to 72 (Tat 37–72) to large proteins such as horseradish peroxidase allowed their functional internalization into several cell lines or tissues (Fawell et al., 1994). Tat-37–72 linked proteins were the most efficiently internalized, although all Tat peptides retaining the basic domain promoted uptake with a variable and cargo-dependent efficiency (Fawell et al., 1994). The intracellular delivery of the human papillomavirus E2 transactivator by Tat-derived peptides was also established, and biological activity was documented (Pepinsky et al., 1994). Both a genetic fusion between Tat peptide and E2 transactivator and the chemical E2 transactivator Tat-conjugate were active in inhibiting the E2 transactivation at micromolar doses. A fusion protein containing the Tat basic domain only (residues 47 to 58) was more potent than the chemically linked chimera comprising the Tat 37–72 sequence. As it is generally admitted that the ends of a protein are exposed at the protein surface, the latter results, combined with those obtained by Fawell et al. (1994), could indicate that the exposure of the basic region of the peptide at the surface of the conjugates is an important determinant for efficient delivery. A short Tat peptide (residues 49 to 57) has been used to internalize proteins into the MHC class I pathway of CD8+ T cells (Kim et al., 1997). The uptake of the proteins was observed after chemical coupling of two to three Tat peptides per molecule of protein. The expected biological response was already observed at micromolar doses. In this study, PLL-protein conjugates did not induce any biological response when used at equivalent doses. Another Tat-derived peptide sequence (residues 37 to 62) was successfully used for the intracellular delivery of anti-tumor Fab antibody fragments (Anderson et al., 1993). Taken together, these data suggested that the Tat basic cluster was responsible for its translocating activity. In order to confirm this hypothesis, we have studied the main determinants required for Tat translocation (Vivès et al., 1997a). We unambigously established that the basic cluster was both necessary and sufficient in order to cross the plasma membrane. Peptide internalization was assessed by


direct labelling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain were taken up by cells within less than 5 min when incubated at concentrations as low as 100 nM with various cells in culture. A peptide harbouring a deletion of three arginine residues (sequence Tat 37–53) completely lost its ability to translocate. The full translocation activity was retained in a 13 amino acids long peptide which contains a nuclear localization signal (NLS) and four additional basic amino acids (Table 5.1). We later established that activity remained significant when the three C-terminal amino acids were deleted (Vivès et al., 1997b). In addition, the cellular uptake of the Tat peptide can be assigned to the minimized sequence Tat 48–57. A sharper structure-activity relationship study has been performed to define the determinants which were important in the observed activity. One or more basic charges have been either deleted or substituted within the short basic peptide. A direct correlation between translocation efficiency and the density of positively charged amino acids was highlighted (Vivès et al., 1997b) (Table 5.1). As also observed for the peptide derived from the Antennapedia homeodomain (Derossi et al., 1994), the internalization of the Tat basic peptide does not involve an endocytic pathway. No sign of endocytic accumulation has been found even in short-term experiments, and no inhibition of the uptake was observed at 4°C. Because of that result and the similar cationic nature of Tat and Antennapaedia peptides, an internalization mechanism based on inverted micelles formation at Table 5.1 Structure-activity relationship for the translocation of Tat peptides (Vivès et al., 1997a, 1997b): greater efficacy of translocation is indicated by a greater number of V symbols. The NLS sequence [Ruben, 1989 #980] is indicated in bold face Sequence

Name

Internalization efficacy

CFITKALGISYGRKKRRQRRRPPQC C--------LGISYGRKKRRQRRRPPQC C----------------GRKKRRQRRRPPQC CFITKALGISYGRKKRR------------C GRKKRRQRR--PPQC GRKKRRQR---- PPQC GRKKRRQ-----PPQC GRKKRRQRARPPQC GRKKRRQARAPPQC GRKKRRQRRR-----C

Tat 37–60 Tat 43–60 Tat 48–60 Tat 37–53 Tat EV-1 Tat EV-2 Tat EV-3 TatRAR Tat ARA Tat EV-4

++ +++ ++++ − ++ + − ++ + ++++

the membrane level (Derossi et al., 1996) appears attractive but requires further investigation. A possible internalization of the Tat basic peptide through potocytosis was also excluded (Vivès et al., 1997a).


A putative cytotoxicity of the Tat basic peptides was investigated for doses up to 100 µM and for incubation times up to 24 h. No significant toxicity was observed for the more potent peptide, i.e. the peptide with the full basic domain (Vivès et al., 1997a). The ability of the basic Tat peptide to promote the internalization of peptides and antisense ON was investigated in our laboratory. The efficient internalization of a non-permeant peptide was established by indirect immunofluorescence with a monoclonal antibody directed against the vectorized peptide (Vivès et al., 1997b). The cellular uptake of a rhodamin-labelled ON coupled through a disulphide bridge to the Tat carrier peptide was also demonstrated. No cellular uptake was detected when the ON or the non-permeant peptide was coupled to an inactive Tat peptide (Tat 37–53), when non-covalently bound to the translocating Tat basic peptide or when the chimeric molecule was reduced before incubation with cells (Vivès et al., 1997b and unpublished results). As shown in Figure 5.1, Tat 48–60 is able to drag antisense ON into cells. Briefly, the ON was first labelled with rhodamin on its 3′ end and then coupled to the Tat peptide through a 5′-activated sulphhydryl group as previously described (Vivès and Lebleu, 1997). When used at high concentration (50 µM), rhodamin was not taken up by cells after a 30 min incubation time at 37°C (Figure 5.1 A). At doses of 1.6 µM unconjugated rhodamin-labelled ON was very poorly taken up by cells (Figure 5.1B), but once coupled to the Tat basic peptide and incubated with cells (dose at 1 µM), the peptide—ON conjugate was rapidly detected in nuclei with a nucleolar accumulation. This translocating property was abolished when the conjugate was previously reduced by DTT treatment. Uptake of the conjugate was also efficient at 4°C (Figure 5.1E), in agreement with our previous observations showing the free peptide uptake at low temperature (Vivès et al., 1997a). Moreover, no labelling was observed after incubation of cells with 1 µM rhodamin-labelled ON coupled to the Tat 37–53 peptide, a peptide which does not translocate in cells (see Table 5.1, Figure 5.1F and Vivès et al., 1997a). In these experiments, cell uptake and intracellular distribution were monitored by fluorescence microscopy after cell fixation, but similar data were obtained in unfixed cells. 5.6 Conclusions Our increasing knowledge of the molecular determinants of polypeptides uptake and intracellular trafficking has been exploited with some success for the delivery of information-rich drugs such as antisense ON, peptides or proteins in cell culture models, as outlined in this chapter. Molecular modelling and combinatorial chemistry (see also Chapter 14) will most probably allow the definition of a new generation of shuttle peptide delivery vectors with improved pharmacological properties. Much remains to be done, however, to overcome problems which might arise in vivo. Immunogenicity could potentially cause


Figure 5.1 Tat 48–60 can internalize rhodamin-labelled antisense ON into cells when incubated for 30 min at 37°C. A: Free rhodamin (50 µM). B: Unconjugated rhodaminlabelled ON (1.6µM). C: Tat basic peptide conjugated to the rhodamin-labelled ON (1 µM). D: Same molecule as in (C) after DTT treatment. E: Uptake of the Tat-ON conjugate at 4° C. F: Incubation of cells with 1 µM rhodamin-labelled ON coupled to the Tat 37–53 peptide, a peptide which does not translocate in cells (see Table 5.1 and Vivès et al. (1997a))

problems despite the use of short peptides. The addition of targeting moieties allowing the concentration of these new drugs at the target level will certainly be preferable. The manufacturing cost of such conjugates will obviously require progress in synthetic chemistry.


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TUNG, C.H., RUDOLPH, M.J. and STEIN, S., 1991, Preparation of oligonucleotide— peptide conjugates, Bioconjug. Chem., 2, 464–465. VIDAL, P., MORRIS, M.C., CHALOIN, L., HEITZ, F. and DIVITA, G., 1997, New strategy for RNA vectorization in mammalian cells. Use of a peptide vector, C.R. Acad. Sci. III, 320, 279–287. VIVÈS, E., BRODIN, P. and LEBLEU, B., 1997a, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus , J. Biol Chem., 272, 16010–16017. VIVÈS, E., GRANIER, C., PREVOT, P. and LEBLEU, B., 1997b, Structure activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein, Lett. Peptide Sci., 4, 429–436. VIVÈS, E. and LEBLEU, B., 1997, Selective coupling of a highly basic peptide to an oligonucleotide, Tetrahedron Lett., 38, 1183–1186. WAGNER, E., PLANK, C., ZATLOUKAL, K., GOTTEN, M. and BIRNSTIEL, M.L., 1992, Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin—polylysine–DNA complexes: toward a synthetic viruslike gene-transfer vehicle, Proc. Natl Acad. Sci. USA, 89, 7934–7938. WEI, Z., TUNG, C.H., ZHU, T. and STEIN, S., 1994, Synthesis of oligoarginine— oligonucleotide conjugates and oligoarginine-bridged oligonucleotide pairs, Bioconjug. Chem., 5, 468–474. WITTUNG, P., KAJANUS, J., EDWARDS, K., HAAIMA, G., NIELSEN, P.E., NORDEN, B. and MALMSTROM, B.G., 1995, Phospholipid membrane permeability of peptide nucleic acid [corrected and republished with original paging, article originally printed in FEBS Lett. 1995 May 22; 365(1): 27–9], FEBS Lett., 375, 27–29. YAKUBOV, L.A., DEEVA, E.A., ZARYTOVA, V.F., IVANOVA, E.M., RYTE, A.S., YURCHENKO, L.V. and VLASSOV, V.V., 1989, Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc. Natl Acad. Sci. USA, 86, 6454–6458. ZELPHATI, O. and SZOKA, F.J., 1996, Mechanism of oligonucleotide release from cationic liposomes, Proc. Natl Acad. Sci. USA, 93, 11493–11498. ZHU, T., WEI, Z., TUNG, C.H., DICKERHOF, W.A., BRESLAUER, K.J., GEORGOPOULOS, D.E., LEIBOWITZ, M.J. and STEIN, S., 1993, Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA, Antisense Res. Dev., 3, 265–275. ZUCKERMAN, R., COREY, D. and SCHULTZ, P., 1987, Efficient method for attachment of thiol specific probes to the 3′-ends of synthetic oligodeoxyribonucleotides, Nucl. Acids Res.. 15. 5305–5321.

6 Polymeric Nanoparticles and Microparticles as Carriers for Antisense Oligonucleotides E.FATTAL AND P.COUVREUR

6.1 Introduction Antisense oligonucleotides (ONs) displaying base sequences complementary to a specific RNA are able to modulate selectively the expression of an individual gene (Helene and Toulme, 1990; Zon, 1988). Due to this mechanism of action, ONs were proposed for the treatment of several diseases such as viral infections or cancers resulting from oncogenes’ activation (Cohen, 1991; Cooke, 1992; Milligan et al., 1993; Stein and Cheng, 1993). However, crucial problems such as the poor stability of ONs versus nuclease activity in vitro and in vivo and their low intracellular penetration have not yet been solved (Loke et al., 1989; Yakubov et al., 1989). To overcome these obstacles, different delivery approaches were proposed: electropermeabilization, microinjection, chemical modifications, and particulate delivery systems. Chemical modifications of the natural phosphodiester ONs’ backbone (see also Chapters 2 and 4) have been performed with success inducing the protection of ONs against enzymatic degradation and improving their cellular uptake (see also Chapter 9). However, in some cases, especially with the phosphorothioates’ derivatives, non-specific effects were observed (Stein, 1996). Techniques such as electropermeabilization (Mir et al., 1988) and microinjection (Dash et al., 1987) were also proposed to increase oligonucleotide penetration into cells. Both methods are supposed to introduce ONs directly into cell cytoplasm, skipping the passage across intact cell membrane. However, these methods cannot be easily applied in therapies for humans. Use of particulate carriers such as liposomes (see Chapter 7) or nanoparticles may be a more realistic approach to deliver ONs, because colloidal carriers are able to protect natural unmodified phosphodiester ONs against degradation and, since they are taken up by endocytosis, they can increase cell penetration of the ONs. Among drug carriers, nanoparticles, biodegradable or not, have shown interesting potentialities to bind and deliver ONs (Fattal et al., 1998). Indeed, nanoparticles were able to protect ONs against degradation in vitro and in vivo and also to enhance significantly their pharmacological activity in cell

130 POLYMERIC NANOPARTICLES AND MICROPARTICLES

culture conditions, but also after in vivo administration (Fattal et al., 1998). More recently, slow delivery systems were proposed for ONs administration, including biodegradable microparticles (Lewis et al., 1998). These particles can be implanted very close to the target site, thus releasing the oligonucleotide in situ. This chapter will thus focus on the use of polymeric drug carriers for the delivery of ONs, the liposomal systems being reviewed in Chapter 7. 6.2 Nanoparticles 6.2.1 Rationale of Using Nanoparticles for the Delivery of ONs Nanoparticles are defined as being submicrometre (heart>liver>lung>spleen (radiolabel in spleen peaked at 1 h and remained elevated for 24 h). At 2 h, the concentration in all organs, except spleen, was equal to that in blood. For the phosphorothioate, kidney uptake was high for 24 h (autoradiographic studies). No intact compound could be detected in the urine (excreted compounds were degradation products). However, in rats, Iversen et al. (1994) found that a 27-mer oligodeoxynucleotide complementary to the rev gene mRNA of HIV-1 was excreted unchanged in the urine (electrophoretic analysis), with a complete

236 PHARMACOKINETICS OF OLIGODEOXYNUCLEOTIDES

elimination in the urine within 3 days. They characterized the plasma profile by a min and . There two-compartment model with appears to be a contradiction between a complete elimination in 72 h and half-lives longer than 24 h! A 36-mer phosphorothioate oligonucleotide (targeting the tat gene of HIV-1) was studied in rats (Boado et al., 1995). The reported pharmacokinetic ml/min per kg; steady-state volume of parameters were: clearance, ml/kg; distribution half-life, min; and distribution, min. elimination half-life, Tissue distribution was described in tumor-bearing mice, after injection of a 15-mer oligonucleotide targeting the translation/initiation of the c-myc mRNA (Plénat et al., 1995), by autoradiography and immunohistochemistry, as well as with fluorescence microscopy. The findings can be summarized as follows: (a) within minutes, oligonucleotides permeate all cells and tissues with the exception of erythrocytes and intervertébral discs; (b) concentration of oligonucleotides is higher within the connective tissue cells than in the interstitial matrix; (c) after uptake, oligomers partition throughout all of the cellular compartment, including, at the highest intracellular concentrations, in the nuclei; (d) oligonucleotides penetrate easily the tumor cell compartments, oligonucleotide diffusion being impeded by the extracellular matrix. GEM® 91 (a 25-mer phosphorothioate targeting HIV-1 gag mRNA) was injected to rats, as a bolus of 30 mg/kg (Zhang et al., 1995a). The half-lives were ; . The main estimated to be: elimination process was renal excretion, with 26.7 ± 6.5% of the radioactivity ). recovered in the urine, within 24 h, compared with faecal excretion ( During the initial 30 min, the highest concentrations were found in the kidney, liver, spleen, lungs, and heart. Another pharmacokinetic study was carried out in cynomolgus monkeys (Lee et al., 1995) with an oligonucleotide (GS-522) developed as an inhibitor of thrombin. After a constant intravenous infusion (0.1, 0.3 and 0.5 mg/kg per min) for 60 min or a bolus injection (11.25 and 22.5 mg/kg), the half-lives were: min and min. The apparent steady-state volume of distribution (Vss) was superimposable with the blood volume of the monkey. Almost similar results were found with ISIS 3082 (a 20-mer phosphorothioate that inhibits the expression of mouse intercellular adhesion molecule-1) and analogues (ISIS 9045, ISIS 9046, ISIS 9047), where little intact drug could be recovered in the urine or faeces for any analogue. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 h, most of the radioactivity in plasma was due to metabolites but, in tissues, intact ISIS 3082 was present for much longer times (Crooke et al., 1996). During the development of an animal model to study the effect of photoreceptorderived debris accumulation on the normal function of the retina in vivo, Rakoczy et al. (1996) described the uptake, distribution and persistence of an antisense oligonucleotide injected into the vitreous of 7-week-old RCS-rdy +


rats. Following intravitreal injection, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 h and in the photoreceptor and pigment epithelial layers 3 days later. It was also demonstrated that the fluoresceinlabelled oligonucleotide present in the retinal pigment epithelial cells was not degraded and retained its original 19-mer length. Deverre et al. (1997), using their competitive enzyme hybridization assay, determined the pharmacokinetics of a 15-mer phosphodiester oligonucleotide, after intravenous injection of 120 nmol/kg to mice. They found a very short halflife of ~4.8 min, an approximate area under the plasma-time curve (AUC) of 114 ng min/ml, and a clearance (CL) of about 4.4 l/kg per min, for the unchanged drug. When two phosphorothioate bounds were introduced in the oligonucleotide, the pharmacokinetic parameters were: half-life of 7.4 min, AUC of 188 ng min/ ml and CL of 2.7 l/kg per min. After replacement of all phosphodiester bonds by phosphorothioates, the AUC increased by a factor of 29 (AUC = 3257 ng min/ ml), the clearance decreased by a factor of 22 (CL = 0.2 l/ kg per min), but the elimination half-life was not significantly modified (5.2 min). Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues was also studied in vivo using three histological methods: immunohistochemistry, direct fluorescence microscopy and autoradiography (Butler et al., 1997). Proximal tubule cells in the kidney and Kupfer and endothelial cells in the liver were among the most heavily labelled, at all doses and time-points. At 2 h post-injection, the oligonucleotides were detectable in the extracellular matrix, although by 24 h the label was predominantly intracellular; they were not detected in erythrocytes, cartilage, compact bone and skeletal muscle. In spleen, white pulp was negative, whereas cells surrounding the sinusoids and nucleated cells in the red pulp were strongly positive. In nude mice, DeLong et al. (1997) also studied the fate of 15-mer modified (phosphorothioate, phosphorodithioate and methylphosphonate) 14Coligodeoxynucleotides complementary to the AUG region of K-ras, after intravenous injection to nude mice harbouring a K-ras-dependent human pancreatic tumor. Plasma concentration profiles appeared to be biphasic, with a rapid distribution characterized by a half-life, T½(α), of 1 min or less and an elimination half-life, T½(β), in the range of 24–35 min. The apparent volumes of distribution were 6.3 ml (phosphorothioate), 3.2 ml (phosphorodithioate), and 4.8 ml (methylphosphonate), compared with 3.6 ml, the volume of distribution for glucose (as a fluid-phase marker). Tissue distribution was highest in kidneys, followed in decreasing relative values by liver, spleen, tumor and muscle. Some intact oligonucleotide was detectable in all tissues studied, including tumor. Kidney and liver appeared to be the main clearance organs. Geary et al. (1997) focused their work on two phosphorothioate oligonucleotides designed as inhibitors of PKC-α (ISIS 3521 or CGP 64128A) and C-raf kinase (ISIS 5132 or CGP 69846A), before entering phase I/II clinical trials (see also Chapter 12). Their findings suggest that the pharmacokinetics of

238 PHARMACOKINETICS OF OLIGODEOXYNUCLEOTIDES

phosphorothioate oligodeoxynucleotides is independent of sequence and that the pattern of distribution to organs is similar across species and independent of the route of administration. For example, the plasma clearance of ISIS 3521 was 14. 5 ml/min per kg in mouse, 1.6 ml/min per kg in rat, and 2.1 ml/min per kg, in cynomolgus monkey, at comparable doses (0.6–1.0 mg/kg), although the relationship between dose and exposure of tissues varies non-linearly. The toxicological properties of phosphorothioate oligodeoxynucleotides were described by Henry et al. (1997), such as prolongation of clotting time, complement activation, proximal tubule degeneration and hepatotoxicity. Again, a pharmacokinetic and distribution study, in nude mice, of a 20-mer phosphorothioate DNA-methyl-transferase antisense oligodeoxynucleotide (Qian et al, 1997) displayed the same features concerning: (a) the amount of drug found in tissues (kidney>liver>tumor>lung>muscle>brain); (b) the elimination halflife, varying between 46 min (at 30 mg/kg) and 240 min (at 300 mg/kg). Wallace et al. (1997) found that AR177 (zintevir, a 17-mer oligonucleotide, with just two phosphorothioate bonds, one at the 5′-end and one at the 3′-end), which has anti-HIV activity in vitro, displayed a very long half-life, either in blood (367 h) or in plasma (271 h), with distribution in the liver (40%), in the bone marrow (17%) and even in the brain cortex (15%), 8 h after intravenous injection to rats. These authors also report that more than 90% of the [33P]radioactivity in the tissues represented intact AR177. In contrast with previous reports, those results were attributed to the tight binding of AR177 to tissues. With new and more specific analytical procedures, the degradation products, chain-shortened oligonucleotides, could be identified by capillary electrophoresis and electrospray mass spectrometry (Phillips et al., 1997). Positron emission tomography (PET) is also a very potent tool for in vivo imaging to study tissue distribution and kinetics. This technique was used by Tavitian et al. (1998) to trace the fate of an 18-mer oligonucleotide complementary to nucleotides 1209–1227 of the env sequence of the Lilly and Steeves strain of murine SFFV Friend erythroleukemia virus. PET scan was performed with 18F (fluorine)-labelled oligonucleotides at the 3′-end (which has no effect on the hybridization with its target complementary sequence). Then, these authors compared the body distribution of the phosphodiester oligonucleotide and the phosphorothioate and 2′-O-methyl-RNA analogues in baboons. They observed that, in fact, the kinetics are highly variable with the nature of the oligonucleotide backbone. They could measure the concentration of [18F]-labelled metabolites in plasma and confirmed that the oligonucleotides were mainly distributed in kidneys and liver, the phosphodiester being rapidly excreted in the bladder while the phosphorothioate was eliminated more slowly in the urine but higly concentrated in the kidney tissues. The 2′-O-methyl-RNA analogue has a behaviour which falls between the phosphodiester and the phosphorothioate (urinary excretion and kidney concentration). This technique should be preferred to whole-body autoradiography.


Other modes of administration of antisense oligonucleotides have been used to deliver those drugs locally. A pharmacokinetic study has been reported (Leeds et al., 1998) after intravitreal administration of ISIS 2922, a 21-mer phosphorothioate that has shown antiviral activity against the cytomegalovirus, by inhibition of the expression of the major immediate-early gene. It has undergone phase III studies as a treatment for CMV-induced retinitis. Concentrations in the vitreous were proportional to the dose administered (11, 57 or 115 µg/eye), which ranged between 80 nM and 1.5 µM, 3 days after dosing. The uptake by the retina was reported to be saturated with average (n = 4, at the 57 µg dose) and concentrations of (n = 3, at the 115 µg dose). Here again, shorter oligonucleotides were detected in the vitreous and retina, according to the electrophoretic profiles. Subcutaneous, intra-peritoneal and intra-tracheal (pulmonary) routes were also investigated (Nicklin et al., 1998). Bioavalability was estimated by comparison of the intravenous administration in rats: subcutaneous (~31%), intra-peritoneal (~28%) and gastrointestinal (1 µM) is required to turn off protein synthesis—for instance, if a non-accessible RNA sequence or a highly abundant message is targeted—one essentially sees a ‘chemistry-driven’ anti-malarial effect due to the well-known non-specific binding of phosphorothioate oligomers to proteins. The two effects might eventually co-exist: P. falciparum proliferation was inhibited by a phosphorothioate 18-mer antisense to the AUG initiator region of the superoxide dismutase (SOD) mRNA as well as by control sequences, but only the antisense sequence induced a reduction of SOD synthesis (Dives, personal communication). A slightly different strategy was successfully used against P. falciparum demonstrating that sequence-dependent effect can be achieved with phosphorothioate sequences. Hammerhead ribozymes bear a catalytic RNA motif flanked by two oligonucleotide stretches complementary to a pre-selected RNA. Upon binding of the ribozyme wings to the intended RNA target site the catalytic core is brought in the vicinity of the substrate, leading to a selective cleavage of the target RNA, hence the inactivation of the corresponding message. Chemically synthesized ribozymes made of an RNA core inserted between two wings containing 10 phosphorothioated RNA residues were targeted to the Plasmodium mRNA coding for the carbamoyl—phosphate synthetase (Flores et al., 1997). The Plasmodium viability was reduced by about 50% following exogenous delivery of these ribozymes to parasite cultures (at 0.5 µM). Control sequences (either ribozymes with non-complementary flanks or the antisense sequence composed of the two wings in tandem without the catalytic motif) displayed a modest effect (5–15% reduction), even though these molecules contained 20 to 28 phosphorothioate ribonucleotides. This supported a sequence-dependent anti-plasmodial activity of phosphorothioate sequences at low concentration (≤0.5 mM), and indicated that some genes which do not respond to antisense oligomers on a sub-micromolar concentration range could be efficiently targeted with ribozymes. 14.3.2 Antisense Oligonucleotides against Trypanosomatids Trypanosomatids are protozoans which comprise several species responsible for severe or even fatal diseases in human beings. Trypanosoma cruzi is the pathogen responsible for Chagas disease in South America; in Africa sleeping sickness results from infection by Trypanosoma brucei. Different species of Leishmania are responsible for cutaneous, muco-cutaneous or visceral diseases


in a large inter-tropical area. Transmitted by insects, these parasites multiply intracellularly (Leishmania, T. cruzi) or in the bloodstream (T. brucei). The expression of genes in these organisms involves a trans-splicing step between two pre-RNA species: one contains the coding region whereas the second one brings a cap structure at the 5′ end of the so-called mini-exon sequence, which is unique for a given trypanosomatid species. This results in the presence of a 39 nt long common sequence at the 5′ end of every mature mRNA (Agabian, 1990; Ullu and Tschudi, 1991) which has been extensively used as a target for oligonucleotides (see Toulmé et al. (1997a) for a review). It was tempting to target this mRNA region, as a single complementary sequence can prevent the synthesis of every protein of the parasite. Moreover, this sequence is present neither in the human (the physiological host) nor in the mouse genomes (a model host). The potential interest of this strategy firstly demonstrated with T. brucei mRNA was then confirmed for other species (Cornelissen et al., 1986; Walder et al., 1986). Anti-mini-exon oligonucleotides were also shown to prevent the translation of Leishmania (Pascolo et al., 1993) or Crithidia mRNA (Gabriel et al., 1987) in cell free extracts. The anti-mini-exon strategy led to the in vitro inhibition of protein synthesis from Ascaris suum and Haemoncus contortus (Bektesh et al., 1988). It was then demonstrated that a trans-splicing mechanism generated a 22 nucleotide long spliced-leader on a subset of mRNA from these worms, and that such a process was also used in Fasciola hepatica (Davis et al., 1994) and Schistosoma mansoni (Rajkovic et al., 1990), two flat worms responsible for human diseases. Trans-splicing might be an important form for gene expression in early metazoa. Therefore, this makes the anti-miniexon approach potentially valuable also for controlling the development of nematodes and trematodes which comprise major human pathogenic parasites. The studies on trypanosomatids were further extended from cell-free extracts to cultured parasites. In a series of studies to investigate the splicing mechanism in trypanosomes, Tschudi and Ullu made use of antisense oligomers to delineate accessible regions in the pre-mini-exon RNA and to shed some light on the role played by U2, U4 and U6 snRNAs (Tschudi and Ullu, 1990; Ullu and Tschudi, 1993). In this work, lysolecithin permeabilized trypanosomes were used to allow oligonucleotide entry into the cell. However, no such treatment was necessary to observe antisense properties: a nonamer (Acr9mer) complementary to the T. brucei mini-exon sequence, which was shown to reduce selectively the protein synthesis in a cell-free assay, induced drastic morphological changes of cultured procyclics (the trypanosome stage corresponding to the insect form) upon addition to the growth medium. Large vacuoles appeared in the cells after 24–30 h incubation and parasites exhibited a limited mobility (Verspieren et al., 1987; Verspieren et al., 1988). Then the cells took a round shape, suggesting a loss of osmotic pressure control, and subsequently died. A high (>100 µM) oligonucleotide concentration was required to observe such effects, probably due to the presence of the mini-exon sequence on every mRNA in the cell leading to a high intracellular concentration of the target.

302 OLIGONUCLEOTIDES AS ANTIPARASITE

A non-modified (phosphodiester) backbone was used but the anti-mini-exon Acr9mer was linked at its 3′ end to an acridine derivative which aimed at increasing the affinity of this short oligomer for the complementary RNA sequence: the intercalation of the dye in the oligonucleotide/RNA duplex was shown by physicochemical measurement to stabilize the sense/antisense complex (Toulmé et al., 1986). Moreover, this acridine residue brought two additional benefits. Firstly, it prevented the attack of the oligomer by 3′ exonucleases which constitute the major activity responsible for the degradation of the oligonucleotides (Verspieren et al., 1987). (However, the experiment was performed in a serum-free medium to reduce the amount of nucleases.) Secondly, the acridine ring promoted the uptake of the oligonucleotide in the parasite, probably through hydrophobic interactions between the dye and the cell membrane (Toulmé et al., 1997a). The presence of the dye was crucial for the killing of the parasites: the unconjugated 9-mer did not induce any change on cultured trypanosomes. Reduced affinity, shortened lifetime and limited uptake might account for this lack of effect, but the first reason is less likely as longer unmodified complementary oligomers did not show any activity either. The trypanocidal property of Acr9mer was sequence-dependent and therefore probably reflected a true antisense effect: non-complementary acridineconjugated 9-mers and a shortened antisense sequence (Acr6mer) with a low affinity for its target did not display any effect (Verspieren et al., 1987). These negative results ruled out a direct effect of the acridine residue which might have been released following the degradation of the oligonucleotide by intracellular nucleases. These experiments pointed out the requirement for chemically modified oligonucleotides. Indeed, unmodified oligomers complementary to the mini-exon RNA of Leishmania amazonensis were without any effect on either promastigotes (the free stage) or amastigotes (the intracellular form of the parasite). Phosphorothioate derivatives, in which a non-bridging oxygen of the phosphodiester bond was substituted by a sulphur atom, were shown to exhibit an increased nuclease resistance. These oligomers bind to the complementary RNA sequence, although with a reduced affinity compared to regular DNA, and elicit RNase H activity (Hélène and Toulmé, 1990; Toulmé and Tidd, 1998) (see also Chapter 1). The addition, to infected murine peritoneal macrophages in culture, of a 16mer phosphorothioate oligonucleotide (16PS) targeted against the mini-exon sequence of Leishmania amazonensis cured about 40% of infected cells after a 48 h treatment at 25 µM (Ramazeilles et al., 1994). The cured cells displayed highly fragmented and empty parasitophorous vacuoles. In some cases parasite remnants were detected. For those cells which contained more than one live leishmania, the parasitic load was considerably reduced: about 15% of treated cells contained a single parasite compared to 3% for untreated ones, whereas less than 10% of treated macrophages were infected by more than 10 parasites compared to 40% for the control of untreated ones. We were never able to cure


more than 50% of the cells, even at high oligonucleotide doses. This might be related to the multiplicity of infection when the oligonucleotide is added, or to the properties of the host cell (the phagocytic activity, for instance), but not to the susceptibility of the parasite itself. In particular one can exclude that some leishmania were resistant to the oligonucleotide treatment. The leishmanicidal effect fulfilled the criteria expected for an antisense mechanism: (i) the parasiticidal efficiency was dose-dependent, (ii) shorter sequences which bind with a lower affinity to the target RNA exhibited a reduced effect, and (iii) non-complementary phosphorothioate 16-mers (inverted, random, scrambled, sense) did not induce such an effect (Ramazeilles et al., 1994). Not only anti-mini-exon sequences have been evaluated: oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also shown to induce a lethal effect on L. amazonensis, demonstrating firstly that antisense oligonucleotides can reach the nucleus of the parasite (Ramazeilles et al., 1994), and secondly that they can interfere with trans-splicing (and/or transport of the mature mRNA). The uptake of oligonucleotides by live cell is a limiting step as discussed in a previous section. In a preliminary attempt to improve the efficacy of antiminiexon sequences, PS oligonucleotides conjugated to a palmitate chain were delivered to the cultured cells associated with low density lipoproteins (Mishra et al., 1995). Forty per cent of infected macrophages were cured at 2.5 µM oligonucleotide, i.e. at a 10-fold lower concentration than naked PS oligomers. This improved efficacy was strictly dependent on the oligonucleotide sequence and was ascribed to an increased uptake of the antisense oligonucleotide. But negative results were obtained with delivery vehicles such as poly L-lysine or polyethylenimine which proved to be efficient with other target cells (Toulmé and Bourget, unpublished results). The properties of other derivatives were tested with respect to Leishmania. In particular, 2′-O-methyl, oligoribonucleotides (OMe) were re-evaluated in order to determine the role of RNase H in the effect of anti-mini-exon oligomers. These enzymes were shown to play a key role in the antisense effect, in particular when the target sequence is located downstream of the initiation AUG codon (see Toulmé and Tidd, 1998, for a review). Both fully modified 2′-Omethyl and OMe/PO/OMe sandwich 16-mers were used. In sandwich oligomers, the central part made of phosphodiester DNA is flanked by OMe stretches, thus restricting the RNase H activity to the RNA region bound to the central PO part of the oligonucleotide (for PO windows larger than five nucleotides). Contrarily to a previous report (Toulmé et al., 1997a), OMe and sandwich 16-mers induced the selective killing of L. mexicana amastigotes in cultured macrophages (Bourget and Toulmé, unpublished results). A similar result was obtained with morpholino, phosphorodiamidate derivatives (Bourget et al., unpublished). As neither 2′-O-methyl nor morpholino analogues elicit RNase H activity, this


demonstrated that the leishmanicidal properties of anti-mini-exon oligomers are controlled by an RNase H-independent mechanism. The use of reactive oligonucleotides is a way to circumvent the possible requirement of RNase H activity. In this case the oligomer is tethered to a chemical agent which, upon binding of the carrier oligonucleotide, induces a chemical modification of the RNA target. This strategy was used against the mini-exon sequence of trypanosomatids. An alkylating group borne by an oligonucleotide complementary to the T. brucei mini-exon led to the expected covalent cross-link (Boiziau et al., 1991). In the case of Leishmania a 12-mer carrying a psoralen derivative (Psol2mer) induced an RNA—oligonucleotide photo-adduct upon UV irradiation, which was responsible for a specific inhibition of in vitro protein synthesis in rabbit reticulocyte lysate (Pascolo et al., 1994). This experiment also demonstrated a restricted access of the target sequence, suggesting that the L. amazonensis mini-exon RNA adopted a secondary (tertiary) structure (see below). 14.3.3 Other Parasites A limited number of studies involving antisense sequences have been performed in the framework of investigations dealing with the control of the development of other parasites. These include the targeting of host genes which play a role in the infection. One example is provided by a study about the effect of the bovine casein kinase II (CKII), serine/threonine protein kinase, on the permanent proliferation of Theileria, a protozoan parasite, in bovine lymphoblastoid cells (Shayan and Ahmed, 1997). It was reported that the expression of the CKII a subunit is closely related to the presence of the parasites in the host cell. The treatment of the infected cells by buparvaquone, a theilericidal drug, leads to the inhibition of the CKII a mRNA expression. Conversely, an antisense oligonucleotide complementary to the host CKII a mRNA reduced [3H]thymidine incorporation by Theileria-infected cells by about 50%. These results were confirmed in an independent study performed with a 30-mer complementary to the translation initiation region. On the contrary, antisense oligonucleotides targeted to the CKII a of the parasite did not show a specific effect: both sense and antisense sequences inhibited the proliferation of the parasite (Chaussepied and Langsley, personal communication). In this latter study the authors used unmodified oligonucleotides. In order to reduce the degradation by nucleases, foetal calf serum—preheated at 65°C instead of 56°C —was added (at only 2% instead of 10%), allowing the development of Theileria-infected bovine lymphocytes in culture. As discussed previously, the uptake of antisense sequences is of key importance. Limited information about this topic is available in the case of parasites. The uptake and compartmentalization of phosphorothioate oligomers have been studied on the trematode Schistosoma mansoni. A minor fraction of


the oligonucleotide accumulated in the tegumental coat of the worm (Tao et al., 1995). The uptake of 35S-labelled oligonucleotides was time-dependent, reaching a maximum after about 40 h. The oligomer was shown to be more stable in nuclei than in cytosol fractions. 14.4 RNA Structures are Valid Targets for Regulatory Oligonucleotides In an effort to optimize the antisense sequence against mini-exon RNAs, a series of phosphodiester oligonucleotides complementary to trypanosome or leishmania mini-exon sequences were evaluated both for affinity for the target and for inhibition of in vitro protein synthesis. A fair relationship between the two parameters was obtained: as expected, the longer the oligomer, the more important is the inhibitory effect. Thermal elution of filter-bound oligonucleotide —RNA complexes (Toulmé et al., 1996a) allowed one to demonstrate that the critical temperature of half elution (i.e. the affinity) was proportional to the logarithm of l+x (where l is the length and x is the GC content of the oligonucleotide). However, the two investigated sequences complementary to L. amazonensis mRNA exhibited an abnormally low affinity for their target, suggesting a restricted access of this RNA region (Verspieren et al., 1990). This was confirmed by the fact that Pso12mer, a psoralen oligonucleotide conjugate, exhibited a very weak photo cross-linking efficiency with the full length miniexon RNA compared to that observed with the 5′ half of this sequence (Pascolo et al., 1994). This can be explained by a competition between intramolecular RNA folding and intermolecular oligonucleotide-RNA complex formation. This constitutes a very general problem for antisense applications: even if RNA is a single-chain nucleic acid, more than 50% of a messenger RNA is doublestranded (Wyatt and Tinoco, 1993) due to intramolecular pairing between complementary stretches. Predicting structure for long RNA molecules is far from accurate. Therefore, targeting an ‘open’ region with an antisense sequence is essentially a hit-and-miss process. Systematic screening of a target mRNA from the 5′ to the 3′ end has been undertaken, and methods have been devised to identify non-structured RNA portions used (Milner et al., 1991: Monia et al., 1996). In the case of the Leishmania mini-exon there is almost no freedom for shifting due to the short length of the target. It was recently confirmed by RNase mapping that this RNA fragment adopted a non-perfect hairpin structure in which most of the sequence was engaged (Figure 14.2; Compagno et al., 1999). It was also demonstrated that the precursor of the miniexon RNA in trypanosomatids folds into stem-loop structures (Lecuyer and Crothers, 1993). Therefore targeting either the mature or the unspliced mini-exon RNA implies that secondary structures have to be taken into account. Designing strategies to this aim is of general interest, as the number of RNA structures playing a key role in gene expression is growing. These RNA motifs


Figure 14.2 Sequence of the mini-exon RNA from Leishmania amazonensis (Miller et al., 1986). In addition to 7mGppp, the terminal part of the 5′ sequence contains four modified nucleosides (in brackets) designed as the ‘cap 4 structure’ (Freistadt et al., 1987). Moreover, the sixth residue is a 2′-O-methyl riboadenosine (Perry et al., 1987). The target sequence of the phosphorothioate oligonucleotide 16PS which shows leishmanicidal properties (Ramazeilles et al., 1994) extends from G(8) to G(23). The L. amazonensis mini-exon secondary structure, deduced from enzymatic and chemical footprinting studies (Compagno and Toulmé, 1999), shows that for binding to the miniexon RNA 16PS has to compete with intramolecular RNA interactions

are frequently selectively recognized by proteins which contribute to regulatory processes. Numerous functional RNA structures have been identified in viruses; the trans activating response RNA element of the human immunodeficiency virus is one of the many examples available (Gait and Karn, 1993). Ligands able to bind to such structured regulatory RNA domains would allow interference with the biological processes that they mediate. Several possibilities have been considered to this end (Figure 14.3; see Toulmé et al. (1996b) for a review): (i) designing oligomers able to invade the RNA structure, (ii) forming triple helices on double-stranded RNA regions of appropriate sequence, (iii) selecting oligonucleotides able to recognize the folded RNA structure. The design of RNA structure invaders can be achieved in two different ways. Firstly, minimizing the energy required for unfolding a large enough portion of the RNA takes advantage of structural peculiarities (bulges, internal or apical loops). The interest of this approach has been demonstrated for the TAR RNA


Figure 14.3 Strategies for targeting RNA structures. (1) High-affinity oligomers such as SBC oligonucleotides can strand-invade RNA hairpins, i.e. shift the equilibrium towards the unfolded RNA (Compagno and Toulme, 1999). (2) Oligopyrimidines or oligopurines can form triple helical structures following binding to the double-stranded stem of an RNA hairpin. This requires an oligopyrimidine-oligopurine stem. A similar approach generates ‘double hairpin’ complexes from clamp oligonucleotides (Pascolo and Toulme, 1996; see text). (3) Aptamers can be selected from randomly synthesized DNA or RNA libraries. The structure of the complex between the aptamer and the target RNA is not known a priori and may involve non-canonical interactions

element of HIV-1 and for a portion of the H-Ras mRNA (Ecker et al., 1992; Lima et al., 1992) (see also Chapter 12). The binding of an oligonucleotide to an RNA double-strand would be favoured if we were able to design modified nucleic acid bases (let's say A′ and T ′) which would result in an increased stability of hybrid pairs compared to normal RNA pairs (i.e. A′-U, T′-A>AU) and which could not pair with each other (A-U >> A′-T′). This last criterion prevents intramolecular folding of the antisense sequence. 2-aminoadenine and 2-thio thymine (Figure 14.1) satisfy these conditions and have been shown to strand-invade a double-stranded DNA (Kutyavin et al., 1996). The process is favoured both kinetically and thermodynamically. Complementary oligonucleotides containing these modified A and T residues, called selectively binding complementary (SBC), have been shown to invade the Leishmania miniexon RNA hairpin and to bind with a 50-200-fold higher affinity than the normal base (NB)-containing oligomer with either a phosphodiester or a phosphorothioate backbone (Compagno et al., 1999). A complementary 25-mer was a much more efficient inhibitor of in vitro translation in the SBC than in the NB version: half inhibition was observed at 0. 08 µM with 25SBC whereas 25NB induced only a 25% decrease of Leishmania


protein synthesis at 1 µM. These SBC oligomers retained the ability to elicit RNase H activity. However, SBC 15-mers displayed only a modest improvement of the leishmanicidal properties of phosphorothioate anti-mini-exon oligonucleotides, indicating that the target RNA structure did not constitute the major limitation to the antisense efficacy in the cell. Triple-stranded structures can be formed from a purine—pyrimidine doublestrand. The third strand binds to the purine strand of the double helix through socalled Hoogsteen hydrogen bonds. Clamp oligomers have been designed which first anneal with a single-stranded homopurine region giving rise to a WatsonCrick double helix and secondarily fold back on themselves to generate a local triple helix (Giovannangeli et al., 1991). The use of clamp oligonucleotides has been extended to the recognition of hairpin motifs in which the stem is a homopurinehomopyrimidine duplex, leading to a double hairpin complex (Brossalina et al., 1993; Brossalina and Toulmé, 1993). Similar complexes have been observed by targeting the Leishmania mini-exon hairpin: the antisense oligomer was designed to form 10 base-pairs with nucleotides 5–14 (Figure 14.2). A four T connector allowed the 15 nt long 3′ portion of the antisense to fold back on the duplex and on the double-stranded stem of the mini-exon hairpin, thus forming a 16 triplet triple helix. The third strand (the 3′ part of the oligomer) was derived from the ‘pyrimidine motif’, i.e. was designed to form TA*T and C-G*C+ triplets; six inverted pairs were read by either G or T to minimize the destabilization. Both electrophoretic mobility shift assays and footprinting studies demonstrated that a double hairpin complex was formed (Pascolo and Toulmé, 1996). However, this required an acidic pH and therefore could not be evaluated with respect to translation inhibition. More appropriate sequences or the use of modified bases which allow the formation of stable triple helices at physiological pH can be considered (Povsic and Dervan, 1989). Recently, combinatorial approaches have been blossoming to identify molecules of interest in randomly synthesized libraries. In the case where the library is made of nucleic acids, up to 1015 different sequences can be screened at a time in a so-called ‘SELEX’ experiment to generate ‘aptamers’ (Ellington and Conrad, 1995; Gold et al., 1995). Either RNA or DNA sequences have been characterized which recognize different target molecules (proteins, antibiotics, etc.) with both high affinity and high selectivity. Such a strategy has been used against nucleic acids, and in particular against structured DNA or RNA motifs. It has been reported that DNA oligomers obtained through an in vitro selection experiment were able to bind to a structure derived from the DNA version of the Leishmania mini-exon sequence (Mishra et al., 1996; Mishra and Toulmé, 1994). The candidates were designed according to the model described above for clamp oligomers: a fixed region complementary to the single-strand at the bottom of the hairpin anchored the oligonucleotide onto the target through Watson Crick base-pairing, whereas the 3′ part constituted the randomized region. In vitro selection led to the identification of aptamers whose 3′ sequence interacted with the target hairpin through uncharacterized bonds, contributing to the stabilization


of the hairpinaptamer complex. Interestingly, the aptamers selected against the ‘DNA miniexon hairpin' were shown to recognize the RNA version of this structure and to prevent selectively the in vitro translation of a luciferase mRNA in which the target hairpin was inserted upstream of the initiator AUG (Le Tinévez et al., 1998). Similar results were obtained in the frame of a selection performed in a DNA library using a non-perfectly structured DNA motif as a target (Boiziau et al., 1997). More recently RNA and DNA libraries have been successfully screened to identify aptamers against a 59 nt long RNA motif (the TAR element) involved in the trans-activation of transcription of the HIV RNA (Boiziau et al., 1999; Toulmé et al., 1997b, Ducongé and Toulmé, 1999). In both cases the selection identified a class of candidates which adopted a stem-loop structure, the top of which displayed a consensus octameric sequence, complementary to the apical part of the TAR RNA element. The TAR RNA—aptamer complexes were able to give rise to six base pairs through loop-loop interactions. Such ‘kissing’ hairpin complexes were previously characterized for natural RNA and were shown to be responsible, for instance, for the control of some plasmid replication in E.coli (Tomizawa, 1990). In the case of the anti-TAR sequences the affinity of the aptamers for the RNA hairpin did not reside only in the six Watson-Crick pairs; short oligomers containing the sequence complementary to the TAR loop, unable to fold into a hairpin, exhibited a 50-fold reduced binding constant (Boiziau et al., 1999). The stem region, next to the aptamer loop, probably acted as a scaffold to pre-organize the bases complementary to the target loop in such a way that a minimal reorganization (i.e. a minimal thermodynamic cost) was required for complex formation. Interestingly, the aptamers selected in DNA and RNA libraries did not lead to the same consensus sequence: even though six base pairs can potentially be formed in both TAR RNA-DNA and TAR RNA-RNA aptamer complexes, the complementary motif is shifted by one nucleotide in the former case compared to the latter one (Boiziau et al., 1999; Ducongé and Toulmé, 1999). It has also been demonstrated that the aptamers identified are highly dependent on the ionic conditions: the consensus sequence surrounding the six base motif was different for a selection performed at high (10 mM) or low (3 mM) magnesium (Boiziau et al., 1999; Sekkai et al., unpublished results). The aptamers selected in 10 mM Mg2+ could not bind at 3 mM, demonstrating the key role played by the bases next to the interacting nucleotides to minimize electrostatic repulsions between the two partners in loop-loop complexes. Other classes of high-affinity ligands have been isolated for which the interaction pattern is not identified yet. Similar experiments have been undertaken using the mini-exon RNA of Leishmania or its precursor as a target.


14.5 Conclusion The investigations undertaken so far with antisense oligonucleotides in the world of parasites underline the high potential of this strategy. Despite the small number of organisms studied and the limited sampling of RNA targets, antisense oligonucleotides have been shown to constitute promising antiparasite molecules. Two major groups of protozoan parasites—Plasmodium falciparum and the trypanosomatids, Trypanosoma brucei and Leishmania amazonensis— were demonstrated to be sensitive to antisense oligonucleotides. For the trypanosomatids the parasiticidal activity of oligonucleotides targeted to the mini-exon RNA sequence exhibited the characteristics of true antisense effects; in particular, non-complementary sequences did not induce a lethal consequence. For Plasmodium, even though the results are still controversial, it is clear that a significant part of the anti-malarial properties of phosphorothioate oligonucleotides are sequenceindependent and probably result from interference with the invasion process of the red blood cell by the parasite. Whatever the mechanism, for T. brucei, L. amazonensis and P. falciparum antisense oligonucleotides allowed one to control the multiplication of the pathogens. However, independently of any further problems, the cost of oligonucleotides and the geographical area in which these parasites constitute a major health problem make it unlikely that antisense oligomers will be considered as therapeutic agents of interest. Up to now the potential of antisense oligonucleotides as tools in molecular genetics of parasites has not been demonstrated. The non-specific effects of phosphorothioate analogues hamper their use to dissect gene function in Plasmodium. The use of other oligonucleotide analogues (many are available) might circumvent this problem. The target worked out in trypanosomatids—the universal mini-exon sequence—did not allow the selectivity of the antisense approach to be explored in these organisms. (The author is not aware of any attempt to turn down the expression of a single gene in trypanosomatids by antisense oligonucleotides.) Moreover, no in vivo experiment has been reported yet, in contrast to viral pathologies, for instance, for which model studies and even clinical trials have been performed. Recently, however, ‘nucleic acid therapies’ have demonstrated an interest in the field of parasitology. The role of paraflagellar rod (PFR)—a large structure contained in the flagellum—in the motility of the trypanosomes has been demonstrated with the help of an antisense construct. The PFR is made of two closely related proteins: PFR-A and PFR-C. A trypanosome transformant obtained with the PFR-A gene in the antisense orientation led to a reduced expression of PFR-A at both the mRNA and protein levels (Bastin et al., 1998). No effect was seen on PFR-C expression. The transformed cells grew normally but were paralysed. However, this effect was due to the integration of the antisense construct at one of the two PFR-A loci. No phenotype was observed when the


antisense construct was targeted to another site, indicating that position in the genome and not antisense production was critical in this case. Double-stranded (ds) RNA was also shown to give rise to genetic interference in trypanosomes: the in vivo simultaneous expression of sense and antisense RNA led to the selective degradation of the homologous RNA sequence, thus invalidating the expression of the corresponding gene. It was demonstrated that dsRNA homologous to the 5′ untranslated region of the α-tubulin message led to morphological alteration of the parasites (Ngo et al., 1998). Either strand (sense or antisense) did not individually induce the same changes. It was demonstrated that dsRNA-mediated RNA degradation takes place in the cytoplasm, and might be associated with mRNA translation. The enzymes involved in such a process, which has also been described in nematodes and plants, are not identified and the physiological function of ds RNA is still unknown, but might constitute a mechanism for regulating gene expression in ancient eukaryotic organisms. This adds one more opportunity to use the genetic information itself to interfere selectively with the expression of a gene of interest. Acknowledgements The author wishes to thank C.Bourget, S.Chabas, M.Chaussepied, D.Compagno, D.Dives, F.Ducongé, G.Langsley, R.Le Tinévez, D.Sekkai, H.Vial and H.J.Yeo for sharing unpublished results. The research projects developed in the author's laboratory are supported by INSERM, the Conseil Régional Aquitaine, the Pôle Médicament Aquitaine, the Agence Nationale de Recherche sur la SIDA and the European Community (Biotechnology Programme). References AGABIAN, N., 1990, Trans splicing of nuclear pre-mRNAs, Cell, 61, 1157-1160. ALVING, C.R., STECK, E.A., CHAPMAN, W.L., WAITS, V.B., HENDRICKS, L.D., SWARTZ, G.M. and HANSON, W.L., 1978, Therapy of leishmaniasis: superior efficacies of liposome-encapsulated drugs, Proc. Natl Acad. Sci. USA, 75, 2959–2963. BARKER, R.H., METELEV, V., RAPAPORT, E. and ZAMECNIK, P., 1996, Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides, Proc. Natl Acad. Sci. USA, 93, 514–518. BASTIN, P., SHERWIN, T. and GULL, K., 1998, Paraflagellar rod is vital for trypanosome motility [letter], Nature, 391, 548. BEKTESH, S., VAN DOREN, K. and HIRSH, D., 1988, Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes, GenesDev., 2, 1227–1283. BOIZIAU, C., BOUTORINE, A.S., LOREAU, N., VERSPIEREN, P., THUONG, N.T. and TOULMÉ, J.J. , 1991, Effect of antisense oligonucleotides linked to alkylating


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Index

α4β7 (LPAM-1) 265 α-anomeric oligonucleotides 51–2 αdβ2(CD11d/CD18) 265 A-raf 247 AAT motifs 37 abasic sites 56 absorbance (OD) 60 absorption (A) 227 accessible site, p53–248 18 acridine 57, 58, 294 acute myelogenous leukaemia 246 acute renal transplant rejection 274 acyloxyalkyl 82 acyloxyaryl 82 adamantoyl chloride 45 adaptor proteins 244 addressin 265 adenosine Al and A2 receptors 278 adenosine receptors 277–8 2′, 5′-adenylate-dependent RNase L 47 adhesion of leukocytes 264–8 adhesion molecules, table 265–6 ADME 227 aggregation 151 AIDS patients 38, 234–5 albumin 133 2′-O-alkyl-oligoribonucleotide/ phosphodiester 17 2′-O-alkylation 16, 41, 52 alkylation, post-synthesis 83 alkylphosphonates 48–9 2′-O-alkylribonucleotides 47, 52 all-phosphorothiaote modification 38 allergic diseases 277–8 allergic inflammatory response 279

alpha-anomeric compounds, translation blocking 12–13 alpha-anomeric oligonucleotides 51–2 alpha-globin gene 17 9-amino-6-chloro-2-methoxy-acridine 5 8 2′-aminoadenine 300 2′-O-aminoalkylribonucleoside phosphoroamidites 58 Γ-aminobutyroyloxyethyl (GABOE) 81 2′-aminonucleoside triphosphates 46 amphipathic helix peptides 118, 219–20 amyloid precursor protein (APP) 119 analysis of oligonucleotides 59–68 analytical chemistry 227–8 angiotensinogen 155, 156 animal models cytotoxic dermatitis in SCID mice 270 mouse cardiac allograft rejection 271 mouse tumor xenograft 249 rodent allografts 270–2 SCID mice cytotoxic dermatitis 270 G3139 antisense inhibition of lymphoma 245–6 tat gene of HIV-l 231 anion-exchange HPLC 63, 235 anionic lipidic vesicles 148 anionic lipids, flip-flop 153 anionic liposomes 147, 149 Antennapaedia peptide 116, 118–19 Antennapaedia protein 99, 113, 219 anti-c-myc oligonucleotides 150 anti-c-myc phosphorothioate 148 anticancer therapy 101, 150, 243–59 antigene 101–3 triplex, schema 36 318

Index

antigene strategy, comb-type copolymers 172–91 anti-H2K antibody 162 anti-HER2 Fab’ 152, 162 anti-HIV 103 antimalarial activity 291–2 antimicrobials 101 anti-mini-exon oligonucleotides 291 structure 298 anti-myb oligonucleotides 161 antiparasite compounds 286–303 anti-ras oligonucleotide 139 antisense, schema 36 antisense cancer therapy 101, 150, 243–59 approach against ras 250–8 Bcl-2 245–6 C-myb 246–7 clinical trials of oligonucleotides 244 conclusions and future prospects 259 protein kinase A 250 protein kinase C-α 249–50 raf kinase 247–8 antisense chimeric structures 50, 53–5 antisense effect 207–10, 217–18 intracellular locations 205 antisense inhibition gene expression mRNA destruction 14–21 steric blocking 12–14 PNA translation 99 antisense inhibitors 251–4 analogues, structure 288 antisense oligonucleotides (ASOs) 244, 263–80 antiparasite use 286–303 clinical studies 38 coupled to a KDEL peptide 115 defined 35 delivery in vitro 201–20 cell surface oligonucleotide-binding proteins 202–4, 216 delivery reagents 206–7 experimental considerations 207–10 intracellular compartmentalization 205–6 ras, antitumour activity 257–8 gene expression inhibition 3 mechanisms of action 3–21

319

RIα antitumour activity 250 antisense ribozymes, triplex 39–40 anti-telomerase 101 apoptosis, CpG motifs 12 apoptosis in epithelial cells 248 aptameric binding 3 aptameric effects, G-quartet oligonucleotides and analogues 7–10 aptamers 299, 301 schema 36 aPTT 273–4 AR-177 38, 226, 228, 233 A-raf 247 arylphosphonates 48–9 Ascaris suum 294 asialoglycoprotein-PLL conjugates 184–5 asthma 277–8 model 279 attenuation of angiotensinogen protein 155– 6 AUC 232 AUG initiation coding region 101, 247 AUG initiation codon 14, 99, 155, 156 AUG region of K-ras 233 automated synthesis on solid support 86 autoradiography 64, 231–2 β1 VLA4 integrins 264 β2 integrins LFA-1 264, 266–7 B lymphocytes 245 murine 278–9 B-cell activation 10 CpG motifs 11 B-cell lymphoma 245 B-cells, murine 278–9 β-globin gene 13 β-globinmRNA 17 B-raf 247 β-thalassaemia 13 Bcl-2 155 antisense approaches for cancer 226, 244–6 Bcl-X 244 bcr-abl antisense oligomers 18, 20 leukaemia cell inhibition 6 bcr-abl mRNA 18, 150 Beaucage's reagent 48, 52, 87, 89

320 INDEX

Beer’s law 60 beta-cyanoethyl group 44 beta-globin gene, splice sites 13 beta-globin mRNA 17 beta-thalassemia 13 bicyclo-[3.2.1]-DNA 53 biotin 57 2, 3-bis(oleoyl)propyl trimethyl ammonium chloride (DOTMA) 231 bladder carcinoma 139 boranophosphate, structure 48 bovine casein kinase II (CKII) 297 bradykinin B2 receptors 278 breast carcinoma 6, 152, 162, 249 brochoalveolar fluid, adenosine receptors 277–8 Brome mosaic virus mRNA 291 buparvaquone 297 C+GC motifs 37 C5 propynyl pyrimidines 55–6, 157 C5 trifluoroacetyl-protected aminoalkenyl pyrimidine 58 C7-aminoalkynyl-7-deazapurine neucloside phosphoramidites 58 c-myb antisense approaches for cancer 246–7 smooth muscle cell hyperplasia inhibition 7, 8 c-myc 8, 13, 18, 19, 20 antisense oligodeoxynucleotide 16 breast and lung cancer cell line inhibition 6 mRNA 231 smooth muscle cell hyperplasia inhibition 7 C-raf 247 C-raf kinase 226, 233, 247 C-raf mRNA 247–8 calcein molécules 159, 160 cancer 128, 140, 148 anticancer therapy 101, 150, 243–59 cancer cells, telomerase 101 capillary gel electrophoresis (CGE) 64–5, 228 carboxyesterase activation 80, 85 carcinoembryonic antigen (CEA) 250, 257

cardiac allografts 271 CaSki cells 151, 155 cationic amphiphilic molecule 135 cationic hydrophobic peptides 129 cationic lipids 43, 112, 136, 152, 206–7, 214, 218, 275 delivery mechanisms 218–19 cationic liposomes 147, 149, 150–7 intracellular delivery and distribution 151–3 PEG modified 152, 162 pharmacological efficacy, in vitro/in vivo 139, 154–7 cationic molecules, hydrophobic peptides 129 cationic peptides 112 cationic polyaminoacids 112 cationic polystyrene nanoparticles 132, 136 cationic porphyrin delivery vehicles 59 CD4, phosphorothioate oligodeoxynucleotide analogue binding 7 CD4 monoclonal-antibody-targeted liposomes 160 CD molecules 265–6 cell, schema 36 cell adhesion molecules, table 265–6 cell proliferation arrest, deoxynucleoside release 4–6 cell surface heparin-binding proteins 202–4 cell surface oligonucleotide-binding proteins 202–4, 216 cellular capture of oligonucleotides 153 cellular pharmacokinetics 229-30 cellular uptake 54, 58, 103, 113, 128, 213– 15 cetyltrimethylammonium bromide (CTAB) 135, 137, 139 CG motif 41 CGP 64128A 139, 156, 226, 233, 249–50 CGP 69846A 226, 233, 247 CHEMS (cholesterol hemisuccinate) 157, 160, 164 chimeric formacetal-phosphodiester oligonucleotides 50 chimeric oligomers 41, 53–5 CHO cell line 155

INDEX 321

cholesterol(3-imidazol-lyl propyl) carbamate 157 cholesterol 57, 136, 138, 147, 148, 159, 160, 161, 163, 209 cholesterol hemisuccinate (CHEMS) 157, 160, 164 cholesterol-coupled oligonucleotides 58, 161, 214, 229 3′-cholesteryl oligonucleotides 58 5′-cholesteryl oligonucleotides 58 choline phosphate cytidyl transferase mRNA 292 chromatography 59, 63, 132 chronic myelogenous leukaemia 246 cell line 150, 246 chronic myeloid leukaemia 18, 20 circular dichroism 175–7 G-quartet identification 7 clinical pharmacokinetics 234–6 clinical trials lymphoma 24 non-Hodgkin’s lymphoma 38, 245–6 oligonucleotides, antisense cancer therapy 244 CMV retinitis, cytomegalovirus 234 colitis 272–3 colon carcinoma 249 colon carcinoma cell line 251 comb-type copolymer 172–91 as cell specific DNA carrier 184–91 as stabilizer for DNA duplex and triplex 172–84 combinatorial chemistry 301 competitive enzyme hybridization assay 228–9, 232 conjugates, types 57–9, 229 conjugation cationic lipids or peptides 112 with cholesterol 58, 161, 214, 229 fusogenic peptides 117–22 internucleoside linkages 57–8 lipophilic molecules 58 oligonucleotides 56–9, 229 positively charged polypeptides 58 copolymers, comb-type copolymers 172– 91 copolymers of poly(l-lysine), with polysaccharide 174

CpG motifs 11, 210, 278, 289 apoptosis 12 immune stimulation 3, 10–11 tyrosine kinase inhibition 11–12 Crithidia 294 Crohn’s disease 38, 272, 274 CTAB (cethyltrimethylammonium bromide) 135. 137. 139 2-cyanoethyl phosphoramidite chemistry 52 cyclosporin A 271 cytokines see interleukins and other named cytokines cytomegalovirus (CMV) 38 CMV retinitis 234 cytoplasm 43, 113, 129, 148 schema 36 cytosine phosphate guanine see CpG cytotoxic dermatitis, SCID mice 270 DDAB (dimethyldioctadecyl ammonium bromide) 151, 152, 155 DEAE-dextran 136, 137 7-deazaguanine, GGGG-motifs 55, 56 degradation, RNA 12 delivery peptides 111–27, 217, 219–20 dendritic cells 279 2′-deoxy-2′-fluoro oligonucleotide 52 (3′S, 5′R)-2′-deoxy-3′, 5′-ethano-beta-Dribofuranosyladenine 53 deoxynucleoside release, cell proliferation arrest 4–6 2′-deoxynucleoside triphosphates 45–6 deoxynucleosides 41–2 monodeoxynucleotide hydrolysis 5 dephospho oligonucleotide analogues 50–1 dextran sulphate 272–3, 292 1, 2-diacyl-3-trimethylammonium propane (DOTAP) 151, 152, 158, 159 dialkylsilyl internucleoside linkages 50 diapedesis 264 dicyanoimidazoles 44 diethylaminoethyl dextran, lipofectin(r) 229 dihydrofolate reductase-thymidylate synthase (DHFR-TS) 291 diisopropylsiloxane likages 50

322 INDEX

dilauroylphosphatidylcholine (DLPC) 151 dimer stability, biological media 81–3 4′, 4′-dimethoxytrityl (Dmt) group 45 dimethyldioctadecyl ammonium bromure (DDAB) 151, 152, 155 dimyristoylphosphatidylglycerol (DMPG) 147 dinitrophenyl 57 1, 2-dioleoyl-3-succinylglycerol (1, 2DOSG) 159 2, 3-dioleoyl)propyl trimethyl ammonium chloride (DOTMA) 231 dioleyl phosphatidyl ethanolamine (DOPE) 151, 153, 154, 155, 156, 157, 158, 159, 160, 162, 164 2, 3-dioleyloxy-N-(sperminecarboxamino) ethyl-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) 152 1, 2-dipalmitoyl-3-succinylglycerol (1, 2DSPG) 159, 160 dipalmitoyl-Dl-alpha-phosphatidyl-Lserine (DPPS) 163 dipalmitoylphosphatidylcholine (DPPC) 147 dipalmitoylsuccinylglycerol (DPSG) 162 DLPC (dilauroylphosphatidylcholine) 151 DMPG (dimyristoylphosphatidylglycerol) 147 DNA bacterial, methylation 11 chemical structure 98 melting curves 61–2 schema 36 DNA carriers, antigène strategy, comb-type copolymers 172–91 DNA mimics see peptide nucleic acids (PNA) DNA mini-exon hairpin 299–300 DNA receptor protein, 30 kDa 202 DNA-PNA chimeras 53–5 5′-DNA-PNA chimeras 41, 54 DOGS (polyamines) 136, 151, 173–84, 206 DOPE (dioleyl phosphatidyl ethanolamine) 151, 153, 155, 156, 157, 158, 159, 160, 162, 164 DOSPA (2, 3-dioleyloxy-N(sperminecarboxamino)ethyl-N, N-

dimethyl-1-propanaminium trifluoroacetate) 152 DOTAP (1, 2-diacyl-3trimethylammonium propane) 151, 152, 158, 159 DOTMA (2, 3-bis(oleoyl)propyl trimethyl ammonium chloride) 231 double-stranded (ds) RNA, PNA-dsDNA schematic drawing 102 DPPC (dipalmitoylphosphatidylcholine) 147 DPPS (dipalmitoyl-Dl-alpha-phosphatidylL-serine) 163 DPSG (dipalmitoylsuccinylglycerol) 159, 160, 162 drug carriers, polymeric nanoparticles 128– 45 DU 145 prostate cancer cells 206 E-selectin antisense oligonucleotide 265, 275–6 egg phosphatidylcholine 147 ELAM-1 (E-selectin) 265 electropermeabilization 128 electrophoresis 59, 64 CGE 64–5 PAGE, slab gels 64 electrophoretic mobility shift assays 64–8 electroporation 14, 112, 214 intracytoplasmic delivery 4 electrospray ionization (ESI) 65 electrospray mass spectrometry (ESI-MS) 65–6, 228, 234 elimination or excretion (E) 227 emulsion polymerization 131, 133 encapsulation 137, 149–1 3′, 5′-end capped modification, plus protection at internal pyrimidines 38 5′-end conjugates 57 3′-end conjugates 57 3′-end-capped modification 38 3′, 5′-end-capped modification 38 end-capping 38, 41 endocytosis 4, 42, 111, 112, 113, 115, 116, 128, 129, 146, 148, 152, 157, 161, 163, 207, 213 endonuclease degradation 5, 15, 39, 41

INDEX 323

endosomal membrane penetration 205, 218 endosomes 4, 43, 112, 152, 153, 218 endothelial-leukocyte adhesion molecules 265–6, 275–6 env mRNA complementary oligonucleotide 158 env sequence, SFFV virus 234 enzymatic synthesis of oligonucleotides 45 enzymolabile groups 80–3 dimers, stability 81–3 hydrolysis, mechanism 81 structure 80–1 epidermal growth factor receptors (EGFR) 162, 164 A431 cell inhibition 6 ERK MAP kinase stimulation 248, 255 erythrocytic merozoite 291–2 erythroleukaemia cells, K562 206, 246 Escherichia coli 16–17, 100, 101 23S RNA 100 ESI-MS, mass spectrometry, oligonucleotide analysis 65–6 ethylcellulose 134 ethylcellulose nanospheres 134 europium, single-stranded RNA 59 exocytosis 4 5′-exonucleases 42 3′-exonucleases 15, 48, 57, 140 degradation 5, 39, 41, 42, 53 extinction coefficient 60 farnesyltransferases 243 Fasciola hepatica 294 FGF signal sequence 220 fibrinogen 203 fibronectin-antisense phosphorothioate oligonucleotides 153 analogue binding 7 flip-flop of anionic lipids 153 fluid phase endocytosis see endocytosis fluid phase pinocytosis 4 fluorescein 57, 92, 120, 153 fluorescence correlation spectroscopy 214 fluorescence labelling 46, 92–3, 163–4 fluorescence microscopy 43, 93, 122, 140, 153, 231–2

fluorescence resonance energy transfer (FRET) 153 fluorescently-labelled nanoparticles 138 fluorescently-labelled oligomers 4, 59, 148, 205–6. 209. 213–14. 250 2′-fluoro oligonucleotides 52 2′-fluoronucleoside triphosphates 46 folate 161–2, 164, 291 folate receptor 162 footprinting studies 37, 50, 301 formacetal linkages 50 formacetal-phosphodiester oligonucleotides 50 fos Friend retrovirus 158, 234, 290 fusion 117, 151 fusion protein 163 fusogenic liposomes 163–4 fusogenic peptides 117–22, 214 fusogenic proteoliposomes 163–4 G-3139 155, 226, 244–6 antisense oligonucleotides, antilymphoma clinical trials 38, 244–6 G-quartet oligonucleotides 203, 210 aptameric effects 7–10 defined 7 formation, relA antisense effector 9 intermolecular 8 G-SATE (glucuronoyl-S-acylthioethyl) 81 G-tetrad see G-quartet GABOE (Γ-aminobutyroyloxyethyl) 81 GALA 219–20 galanin receptor 119 antisense downregulation 99 gap-mers 38, 41 GAPDH mRNA 19 GEM-92 38 GEM-132 38 GEM-231 244 GEM(r)-91 226, 232, 234–5 GGC motifs 37 glioblastoma 249, 258 gliomas 230 glucuronic acid with glucuronoyloxyethyl (GOE) 81 glucuronoyl-S-acylthioethyl (G-SATE) 81

324 INDEX

glyceraldehyde 3-phosphate dehydrogenase 19 P-glycoprotein 150 glycoproteins, variant surface 290 glycosylated PLL 173 GM-1 160 GM-CSF 275, 279 GOE (glucuronic acid with glucuronoyloxyethyl) 81 Gps0193 38 GS-522 226, 232 GTG2TG3TG3TG3T, HIV1 integrase inhibition 9 GTP regulatory domains 251 GTP-binding proteins 244 H9 cells 161 H mechanism. non-RNase 13 H-phosphonate method 44-5, 48, 57 H-ras (ISIS 2503) 226, 300 Ha-ras 138, 226, 251, 258 oncogenes 139 haemagglutinating virus of Japan (HVJ) 163–4 Haemonchus contortus 276, 294 hairpin motifs 299–300 hairpin ribozymes 35 hammerhead consensus sequence, conserved nucleotides 36 hammerhead ribozymes 35, 56, 293 Hep-G2 cells 139, 155, 204 heparin-binding integrin 203 heparin-binding protein 202–4 phosphorothioate oligodeoxynucleotide analogue binding 7 hepatitis B virus 154–5 herpes virus 220 heterocyclic bases, modification 55–6 hexagonal structure 153, 159 hexitol nucleic acids (HNA) 53 high-performance liquid chromatography (HPLC) 84, 91, 228, 235 anion-exchange 63, 235 oligonucleotide analysis 62–3 reversed-phase 62–3, 85, 89

HIV-1 envelope glycoprotein (gp120) 9, 204 mutation 10 phosphorothioate oligodeoxynucleotide analogue binding 7 HIV-1 gp41 fusogenic peptide 118 HIV-1 infection anti-HIV drugs 38, 103, 226 pharmacokinetics 234–5 HIV-1 integrase inhibition, GTG2TG3TG3TG3T 9 HIV-1 replication inhibition 8, 160–1 S-T2G4T2 octamer 9 HIV-1 tat gene 231 HIV-1 Tat protein 113, 116, 117 HIV-1 transmembrane gp41 glycoprotein 114 HIV-1 vpr gene 8 HL60 cells 206 HLA class I antigen, immunoliposomes directed 160 homopurine-homopyrimidine duplex 180, 300 Hoogsteen base pairing 42, 101, 102, 178– 9, 210, 300 structure 37 HPLC see high-performance liquid chromatography HPRT (hypoxanthine-guanine phosphororibosyl transferase) 291 human c-raf-1 kinase 226, 233, 247 human IgE, dissociation constants 10 human IgG, dissociation constants 10 human papillomavirus 119, 155 human xenografts 269–70 HVJ-liposomes 163–4 hyaluronic acid 187–91 hybrid arrest of translation, defined 12 hydrogen bonds, Hoogsteen base pairing 300 hydrolysis, mechanism 80–1 hydrophobic peptides 129 3′-hydroxylacceptor 46 hypochromicity 61 hypoxanthine-guanine phosphororibosyl transferase (HPRT) 291

INDEX 325

ICAM-1 (CD54) antisense oligodesoxyribonucleotide 156 antisense oligonucleotide 154, 264–75 ICAM-2 (CD 102) 265 ICAM-3 (ICAM-R, CD50) 265 IgE 10 antibody production 279 IGF-1 receptor 217 IgG 10 IL-1 277 IL-2 277 IL-4 279 IL-5 279 IL-6 277, 279 IL-8 277 IL-12 279 IL-13 279 imidazole-4-carboxamide 55, 56 immune response 278–9 immune stimulation 210 CpG motifs 3, 10–11 immunoglobulin secretion 10 immunoliposomes 146–8, 160–4 directed to HLA class I antigen 160 inflammatory bowel disease 38 inflammatory processes 263–80 modification with antisense oligonucleotides 263–80 influenza virus 154, 155 influenza virus haemagglutinin 113, 219 influenza virus haemagglutinin fusogenic peptide 117–18 initiation codon 12–14, 99, 155, 156, 296 initiator AUG 14, 99, 155, 156, 296 integrins 264–7 β1 VLA4 264 β2 LFA-1 264, 266–7 β2 sub-family 216–17 phosphorothioate oligodeoxynucleotide analogue binding 7 intercellular adhesion molecule-1 (ICAM-1) 232 interferon treatment 47 interferon-gamma 140, 279 interleukins IL-1 receptor 276–7

regulation by NF-kappaB 277 internucleotide linkage 79 boronated 50 conjugation 57–8 intracellular compartmentalization 113 intracellular delivery and distribution 42– 3, 58 138, 151–3 pharmacological efficacy 154–7 intracytoplasmic delivery 4, 6 intramolecular folding 298 intrathecal administration 119 intratumoral administration 139 intravitreal injection 232 introns 244 3′, 3′, 5′, 5′-inversions 46–7 ion spray 66 ISIS-1570 154 ISIS-1939 267–8 ISIS-2105 229 ISIS-2302 38, 235, 267–70, 273–4 ISIS-2503 38, 226, 244, 256, 300 ISIS-2922 28, 234 ISIS-3082 27, 232, 270–1 ISIS-3466 231 ISIS-3521 38, 139, 156, 226, 233, 244, 249–50 ISIS-4189 249 ISIS-4730 275 ISIS-5132 38, 226, 233, 244, 247–8 ISIS-5320 38 ISIS-9045 232 ISIS-9046 232 ISIS-9047 232 ISIS-9125 154, 270–1 isothermal titration calorimetry (ITC) 179– 80 JNK MAP kinase stimulation by TNF-α 248 K562 erythroleukaemia cells 206, 246 K-ras-dependent human pancreatic tumor 233 KDEL peptide, antisense oligonucleotide (ASOs) 115 keratinocytes 5, 111 Ki-ras 250, 258

326 INDEX

kidney transplant model 272 kidney transplant treatment 38 kissing hairpin complexes 299–300 L-olingomers 117 laryngeal carcinoma 248 laser-induced fluorascence (LIF) 65 lectin 117, 165 Leishmania 293–300 L. amazonensis 288, 295 L. major 289 L. mexicana 287, 295 leukaemia acute myelogenous leukaemia 246 chronic myelogenous leukaemia 246 chronic myeloid leukaemia 18, 20 leukemia cell inhibition, bcr-abl antisenseoligomers 6 leukaemia cell lines 6, 12–13, 16–20, 148– 50, 150, 161, 246 leukocytes 264, 267 adhesion 264–8 LFA-1 integrins 264, 266–7 ligation 46 lipid bilayer, interactions of oligonucleotides 215–16 lipids, cationic 43, 112, 136, 152, 206–7, 214, 218, 275 lipofectamine 13 lipofectin® 154, 229 lipofectin™ 43, 139, 155, 156, 207 lipofection, cationic liposomes 4, 14 liposomes 42, 112, 128, 129, 290 anionic 147–50 cationic 150–7 containing N-stearoylcysteamine 160 delivery of ONs 146–65 fusogenic 163–4 immunoliposomes 146–8, 160–4 intracellular delivery and distribution 151–3 ON delivery 146–65 PEG-coated modified 152, 162, 214 pH-sensitive 146, 149, 157–60 pharmacological efficacy, in vitro/vivo 154–7 poly(glycidol)-modified 159

proteoliposomes 163–4 liver sinusoidal endothelial cells 187 locked nucleic acid (LNA) 53 loop structures D-loop 39 P-loop 37 loop-loop complexes 301 low density lipoproteins 296 LPAM-1 266 lutetium, single-stranded DNA 59 lymphoma B-cell lymphoma 245 clinical trials 24 non-Hodgkin’s lymphoma 38, 245–6 lysine, poly(L-lysine) 113, 116–17, 120, 164, 173–91, 214, 230 lysine pentapeptide, extended by nuclear localization signal 117 lysolecithin 294 lysosomes 43, 138 degradation 129, 157, 163 MAC-1 (CD11b/CD18) 203–4, 216–17, 266 macrophages 159, 279 MadCAM-1 265 magnesium ions 35 malaria 291 MALDI (matrix-assisted laser desorption ionization) 65, 66–7, 228 MALDI-TOF 89 MAP kinase, inhibition by antisense ONs 255 MAP kinase phosphorylation cascade 248 mass spectrometry 59, 90 oligonucleotide analysis 65–7 oligonucleotide degradation 228–9 matrix-assisted laser desorption ionization (MALDI) 65, 66–7, 228 -TOF 89 MCF-7/ADR cells 150 MDA-MB231 tumors 249 mdr (multidrug résistance) 150 melanoma 119 metastasis 269–70 melting temperature (Tm) 16, 48, 49, 50, 51, 54, 60, 61, 116

INDEX 327

membrane-active peptides 219–20 18-mer phosphorothioate (G-3139) 155, 226, 245–6 metabolism (M) 227 metastasis 140 human melanoma 269–70 2′-O-methoxyethoxy 52 2′-O-methyl nucleoside triphosphates 46 2′-O-methyl oligoribonucleotides (OMe) 287, 296 morpholino phosphorodiamidates 287 splicing inhibition 13 2′-O-methyl RNA 16, 41, 52, 208, 234 methylene 48 methylphosphonamidites 49 methylphosphonate 15, 16, 48–9, 83, 147 compounds 215 HPLC 62 nuclease resistance 41 oligodeoxynucleotide protection 5 oligodeoxynucleotides 150, 214–15 oligonucleotides 230 phosphodiester linkage replacement 5 methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides 50, 53–5 microinjection 43, 112, 128, 214 microparticles 128–41 polylactide-co-glycolide (PLGA) 134, 140 microscopy, fluorescence microscopy 43, 93, 122, 140, 153, 231–2 minimal volume entrapment (MVE) 148, 150 ‘mismatched’ oligonucleotides 288 modification on heterocyclic bases 55–6 2′-modified oligoribonucleotides 55–6 molecular beacon 205 monensin 205–6 monoclonal antibodies Tat basic cluster 120 to ICAM-1 154 to Mac (-1) 203–4 morpholino compounds 215–16, 288 translation blocking 12–13 morpholino oligonucleotide 296 exon skipping 13–14 mouse cardiac allograft rejection 271

mouse tumor xenograft 249 mRNA, schema 36 mRNA degradation 43 5′-untranslated initiation codon region 12 multidrug resistance (mdr) 150 multiple emulsion (water-oil-water) solvent evaporation method 131, 133 murine B lymphocytes 278–9 MVE (minimal volume entrapment) 148, 150 myc probe 13 see also c-myc myeloma cells 155 N4, N4-ethano-5-methyl-cytosine 56 N4-(3-acetamidopropyl)-cytosine 56 N-(alpha-trimethylammonioacetyl) didodecyl-D-glutamate chloride (TMAG) 151 N-ras 250 N-stearoylcysteamine 160 nanocapsules 129, 132, 134 nanoparticles 42, 112, 128–41, 158 biodegradability 130 cationic polystyrene 132, 136 as drug carriers 128–41 fluorescently-labelled 138 in vivo studies 139 ON adsorption, in vitro stability 137 ON association 134–5 ON delivery, rationale 129–30 ON loaded cell interactions 138 in vitro pharmacological activity 138– 9 poly(alkylcyanoacrylates) (PACA) 229 poly(dialkylmethilidene malonate) 132 polylactic acid–PEG 136 preformed polymers 133–4 preparation, monomer polymerization 130–3 Stealth 130 nanospheres 129, 133, 134 neo gene 158 neointima formation 164 neurotensin-receptor 99

328 INDEX

NF-kappaB 277 tumor cell adhesion inhibition 8 NIH 3T3 cells 158 nitric oxide synthetase (NOS) 278 5-nitroindole 56 3-nitropyrrole 56 NK cells 203, 279 NMR, oligonucleotides 59, 67–8 non-antisense mechanisms 278–9 non-Hodgkin’s lymphoma 38, 245–6 G-3139 clinical trials 38, 245–6 non-RNase H mechanism 13 non-small cell lung carcinoma 6 NFS, protecting group on nucleobases 86, 87–9 nuclear accumulation of oligonucleotides 4 nuclear lamina nuclear localization signal (NLS) 120, 148 nuclear magnetic resonance 59, 67–8 nuclear pores 113 nuclease-resistant ON analogues 15 nucleases, degradation 111, 146 nucleic acid binding receptor-1 (NABR1) 202, 204 nucleic acids delivery, peptides 112–14 uptake, delivery vehicles 111–12 nucleobases conjugation 57–8 protecting groups 86, 87–9 nucleolytic degradation, deoxyribonucleoside release 3 nucleoside-2′-phosphoramidite building blocks 47 nucleoside-3′-beta-cy anoethyl N, Ndiisopropylphosphoramidite 44 nucleoside-3′-H-phosphonate 45 nucleoside-3′-phenylphosphonamidites 49 nucleoside-5′-O’phosphoramidites 47 nucleoside-5′-O’succinyl-supports 47 nucleus 43, 153 schema 36 ODNs see oligodeoxynucleotides 4-(2, 3-bis-oleoyloxy-propyl)-lmethyl-1H-imidazole 157

2, 3-bisoleoylpropyl trimethyl ammonium chloride (DOTMA) 154, 156, 157 2′, 5′-oligoadenylates 46, 47 oligodeoxynucleotides (ODNs) 38–9, 175, 247 analogues, nuclease-resistant 15 containing CPG motifs 10–11, 210, 278, 289 immune stimulation 3 ODN-698A 244 pharmacokinetics 226–36 phosphorothioates 232–3, 246, 271 oligolysine 117 oligonucleotide(s) 35–68 2′, 5′-linkages 46–7 2′-O-methyl-oligoribonucleotides 287 3′, 3′-/5′, 5′-end inversions 46–7 α-anomeric 51–2 analysis see oligonucleotide analysis as antiparasite compounds 286–303 antisense, mechanisms of action 3–21 carriers 208 conjugates 229 3′-end 57 5′-end 57 types 58–9 delivery by liposomes 146–65 dephospho linkages 50–1 design 39 in vivo 21 mismatched 288 modifications 43 2′-modified 52 alpha-anomeric 51–2 conjugates 56–9 degree 40–2 heterocyclic bases 55–6 universal base and abasic sites 56 internucleoside linkages, nucleobases and ribose 57–8 peptide nucleic acids 52–5 phosphorus 47–50 alkylphosphonates and arylphosphonates 48–9 phosphorothioates 47–8 sterically locked nucleic acid analogues 53 sugar modified 53

INDEX 329

sugar moiety 51–3 synthesis and properties 42–59 nanoparticle association 134–9 2′, 5′-oligoribonucleotides, synthesis 47 ON-cholesterol conjugates 229 ON-peptide conjugates 111–27, 220 peptide-mediated delivery 111–27 prodrugs 79–97 receptors and transporter proteins 216– 17 synthetic 111–12 unmodified 3′, 5′-phosphodiester linkages 42–6 chemical synthesis 44–5 enzymatic synthesis and ligation 45–6 see also antisense oligonucleotide analysis 59–68 electrophoretic techniques 64–5 CGE 64–5 PAGE on slab gels 64 HPLC analysis 62–3 anion-exchange 63, 235 reversed-phase 62 mass spectrometry 65–7 ESI-MS 65–6 MALDI 66–7 NMR 67–8 UV spectroscopy 60–2 DNA melting curves 61–2 quantification 60 see also oligonucleotide(s) oligonucleotide-DEAE-nanoparticle complexes 137 oligonucleotide-binding proteins, cell surface 202–4, 216 oligonucleotide-loaded nanoparticles 138– 9 2′, 5′-oligoribonucleotides, synthesis 47 oncogenes 5, 128, 139 open loops 18–19 opsonization 159, 165 ovarian cancer 156, 250 p53 antisense oligodeoxynucleotides 18 p53 mRNA 17, 20 p53–248, accessible site 18 p64 Myc protein 13, 14

p65 antisense oligodeoxynucleotide 277 P150/95 266 p210 Bcr-Abl protein 150 32P end-labelled oligomers 209 33P-pdT 16 139 PACA (polyalkylcyanoacrylate) 131, 134– 5, 229 PAGE 139 slab gels 64 2, 3-bis-palmitoyl-propyl-pyridin-4-ylamine 157 palmitoylhomocysteine (PHC) 157, 162 4-(2, 3-bis-palmitoyloxy-propyl)-1 methyl-1H-imidazole 157 pamamycin 57 pancreatic carcinomas 248 pancreatic tumor, K-ras-dependent 233 parasitic diseases 286–303 parasitophorous vacuole 291 particulate carriers 158 particulars delivery systems 112, 128 PCR, RLPCR (reverse ligation-mediated polymerase chain reaction) 15, 21 PE (phosphatidyl-ethanolamine) 157 PEC AM-1 266, 275 PEG see polyethyleneglycol PEG-modified cationic liposomes 152, 162 PEG-PE (polyethyleneglycolphosphatidylethanolamine) 151 Penetratin 118 peptide bond, PHONAs 55 peptide coupling, strategies 114–16 peptide nucleic acids (PNA) 39, 41, 47, 53– 5, 98–104, 215 chemical structure 54, 98 delivery 112–14 drug development 99 future 103–4 homopyrimidine 37 HPLC 62 oligomers 13 pharmacology 103 PNA-DNA chimeras 54 peptide-oligonucleotide conjugates 111– 27, 220 peptide-mediated delivery of oligonucleotides 111–27 peptides 217, 219–20

330 INDEX

synthesis 115 peptidyl transferase centre 100 pH-sensitive liposomes 146, 149, 157–60 pharmacodynamics 79 pharmacokinetics 226–36 ADME 227 cellular 229–30 clinical 79, 234–6 oligodeoxynucleotides 226–36 oligonucleotides 275 preclinical 230–4 and tissue distribution of liposomes 154 pharmacological efficacy in vivo 154–7 Phase I and II trials 38 PHC (palmitoylhomocysteine) 157, 162 phenoxazine 55 phenoxazine-substituted oligonucleotides 56 phenyl-S-acylthioethyls (Me-, tBu- or PheSATE) 80–1 PHONAs, peptide bond 55 phosphatidyl-ethanolamine (PE) 157 phosphatidylserine 148, 163 3′, 5′-phosphodiester linkages, unmodified oligonucleotides 42–6 phosphodiester oligodeoxynucleotides 228–9 phosphodiester oligonucleotides 214–15, 232, 234, 300 natural 86 3′-phosphodiesterase activity 5 phosphodiesterases 4, 83, 116 degradation 5 phospholipid flip-flop 153, 215 phosphoramidate, structure 48 phosphoramidite building blocks 44, 48, 87 method 44, 45 phosphorodiamidate derivatives 296 phosphorothioates 6, 47–8, 53 2′-O-methyl-oligoribonucleotides 287 18-mer (G-3139) 155, 226, 244–6 all-phosphorothiaote modification ‘gap-mer’ modification 38 antiproliferative effects 6 backbone 8, 208, 300 oligodeoxynucleotides 232-3, 246, 271 analogues 228, 231, 288

extracellular aptameric effects 6-7 oligonucleotides 4, 14, 208, 215-16, 228, 230 phosphorodithioate 15 ribonucleotides 293 side effects 40 structure 48 phosphorous environment modification 47-50 second generation pro-oligonucleotides 86 photolabile linker, solid support 86 PIBCA (polyisobutylcyanoacrylate) 135, 139 PIHCA (polyisohexylcyanoacrylate) 135, 136, 137, 138, 139 pinocytosis 158, 203 pivaloyl 45 pivaloyloxymethyl (POM) 79–80, 82, 83, 85, 91 PKC-alpha see protein kinase C plasma-time curve (AUC) 232 Plasmodium falciparum 289, 291–3 Plasmodium-infected erythrocytes 291 PLGA (polylactide-co-glycolide) microparticles 134, 140 PLL see poly (L-lysine) PNA see peptide nucleic acids polyacrylamide gel electrophoresis (PAGE), slab gels 64 polyacrylamide gels 139 polyalkyl chains 58 poly(alkylcyanoacrylates) (PACA) 131, 134, 135 nanoparticles 229 polyamines (DOGS) 136, 151, 173–84, 206 polyaminoacids 112 polyanionic molecules 217 polyanions 148 polyarginine 117 poly(beta-hydroxybutyrate) 133 polycations, comb-type 172–91 poly(dialkylmethylidene malonate), nanoparticles 132 poly(ethylene oxide) 185 polyethyleneglycol (PEG) 146, 150, 151, 159, 160, 162 -coated cationic liposomes 152, 162

INDEX 331

derivatives 130 polyethyleneglycol (PEG)– phosphatidylethanolamine (PEG-PE)) 151 polyethyleneimine 112, 296 poly(glycidol)-modified liposomes 159 polyisobutylcyanoacrylate (PIBCA) 135, 139 polyisohexylcyanoacrylate (PIHCA) 135, 136, 137, 138, 139 poly(L-arginine) 173 poly(L-lysine) 113, 116–17, 120, 173, 173– 91, 214, 230 PLL-g-Dex 174 polylactic acid 133, 134 -PEG nanoparticles 136 polylactide 134 polylactide-co-glycolide (PEGA) microparticles 134, 140 polylysine 164 see also poly(L-lysine) 3′-and 5′-polylysine-modified oligonucleotides 117, 174 polymeric drug carriers 128–45 polymeric nanoparticles 128–45 polymers biodegradable 140 preformed 133–4 polymethylmethacrylate 145 polyornithine 117 polysaccharide side chains comb-type DNA carrier 184–91 PLL-G-Dex 174 polystyrene 132, 134 polystyrene nanoparticles 132, 136 POM (pivaloyloxymethyl) 79–80, 82, 83, 85, 91 porphyrin delivery vehicles 59 positron emission tomography (PET) 227, 234 pre-mRNA 294 steric blockade 3, 13 preclinical pharmacokinetics 230–4 prion protein 10 prodrug approach 79–97 professional antigen presenting cells pro-oligonucleotides 79–97 alkylation synthesis 84

limitations 85 cell uptake 92 first models 83–6 stability in biological media 83–5 SATE, masking groups 89–92 second generation 86–92 stability in biological media 90–2 synthesis 111 protecting groups on nucleobases 86, 87–9 protein kinase A 250 protein kinase C (PKC) isoenzymes 156 phosphorothioate oligodeoxynucleotide analogue binding 6–7 PKC-α 139, 156, 226, 233, 249–50 protein kinases 244 proteins 154 aptameric binding 3 inhibition 101 phosphorothioate oligodeoxynucleotide analogue binding 6 30 kDa protein 202 47 kDa protein production 14 schema 36 viral 113 proteoliposomes 163–4 pseudoisocytosine 55 psi2neo cells 158 psoralen, conjugation 59 psoralen derivatives 57 psoriasis 38, 274 PTT 273–4 quantification of oligonucleotides 60 rabbit reticulocyte lysate 291, 296 raf kinase, antisense approaches for cancer 247–8 ras 250–8 antisense approach 250–1 antisense inhibitor specificity 252–4 antisense inhibitors 251–2, 255 antitumour activity 257–8 gene expression inhibition, cellular responses 254–7 on nanoparticles 139

332 INDEX

ras genes, (Ki-ras, Ha-ras and N-ras) 251 rat αl (I) collagen gene promoter receptor-mediated endocytosis see endocytosis receptors 216–17 counter-receptors 265–6 relA antisense effector, G-quartet formation 9 release of deoxynucleosides 4–6 renal ischaemia 272 renin-angiotensin system 156 resonance energy transfer (FRET) 153 reticuloendothelial system 112, 220 retinitis, cytomegalovirus 234 reverse ligation-mediated polymerase chain reaction (RLPCR) 15, 21 reversed-phase HPLC 62–3, 85, 89 rheumatoid arthritis 38, 204, 274 rhodamine-labelled lipids 153 rhodamine-labelled oligomers 121–2, 206 ribonuclease H see RNase H ribose, conjugation 57–8 ribosomes 292 schema 36 ribozymes 17, 293 for antisense, design criteria 39–40 development 35 hairpin 35 hammerhead 35, 56, 293 schema 36 RLPCR (reverse ligation-mediated polymerase chain reaction) 15, 21 mRNA 3′-fragment detection 21 RNA, double-stranded (ds) RNA 102 RNA folding 39–40, 298 RNase, mRNA ablation 3 RNase H 208, 244, 274 mRNA degradation 12, 47–8 non-RNase H mechanism, peptide chain elongation inhibition 13 non-targeted cleavage 18 partial oligonucleotide/RNA activation 40 RNase H-independent mechanism 99, 205 RNase L, 2′, 5′-adenylate-dependent 47, 116 rodent allografts 270–2 rodent xenografts 249 rolling on endothelial cells 187

Rous sarcoma virus 35 Rp configuration 45, 47, 49 S-acetylthiomethyl (SATM) 80–1 S-acylthioethyl see SATE 35S-labelled oligodeoxynucleotide 234–5 35S-labelled phosphorothioate oligonucleotide 231 S-T2G4T2 octamer, HIV–1 replication inhibition 9 α-sarcin loop 100 SATE (S-acetylthioethyl) 85, 86, 88–92 dimer protection, gastric juice stabilisation 82–3 pro-oligonucleotides 79–81, 82, 83 SATM (S-acetylthiomethyl) 80–1 scavenger receptors 217 Schistosoma mansoni 294, 297 schizonts 291 SCID mice cytotoxic dermatitis 270 G3139 antisense inhibition of lymphoma 245–6 selectins 264, 265 selectively binding complementary A and T analogues (SBC) 288, 300 SELEX 301 Sendai virus 163 serine/threonine protein kinase 249 enzyme activity 11–12 SH2 and SH3 proteins 244 sialyl lewis X 265 sinusoidal endothelial cells 187 SKVLB cells 150 smooth muscle cell hyperplasia inhibition, c-myb 7, 8 sodium azide 148, 201 solid support, photolabile linker 86 Sp configuration 47, 48, 49 Sp-isomer 45 spermidine 179 spermine 176–7, 179 splice sites, pre-mRNA 13 stability measurements of oligonucleotide degradation 228

INDEX 333

pro-oligonucleotides in biological media 90–2 starburst dendrimers 207–8 Stealth nanoparticles 130 stem-loop 155, 299 steric blocking mechanism gene expression 12–14 pre-mRNA 3 sterically locked nucleic acid analogues 53 streptolysin 4, 13, 14, 15, 17, 18, 19, 20, 112 reversible plasma membrane permeabilization 12 substance P (SP)/neurokinin-l receptor 156 sugar modifications 51–3 superoxide dismutase (SOD) 293 suramin 203 surface plasmon resonance (SPR) 180 SV40 T-antigen nuclear localization SW480 colon carcinoma cells 251 3′, 3′-switches 47 synthetic polymers 45–6 T4 DNA ligase 45–6 T7 polymerase 45–6 T helper-1 immune (Thl) response 279 T lymphocytes 279 TAR RNA element 300–2 Tat basic domain 119 TAT motifs 37 Tat peptides internalization promotion 121 translocation, structure-activity relationship 121 Tat polybasic sequence 220 Tat protein 116 Tat protein chimeras 220 Tb(DPA)33-compound 163 TCAACGTT 11 telomerase anti-telomerase 101 cancer cells 101 temperature, melting (Tm) 16, 48, 49, 50, 51, 54, 60,61, 116 tetraethylthiuram disulphide 48 texaphyrins 59 Th2 phenotype 279

β-thalassaemia 13 Theileria 297 2-thio thymine 300 3′-thioformacetal 47, 50 5′-thioformacetal 50 5′-thiophosphoryl 46 thrombin 226, 232 thrombin binding aptamer 9 time-of-flight (TOF), mass detection 66–7 tissue distribution (D) 227 TMAG (N-(alphatrimethylammonioacetyl) didodecyl-D-glutamate chloride) 151 TNF-α 203–4, 269, 277, 279 leukotriene B4 203 toxicity of cationic lipids 112, 218 toxicology antisense oligonucleotides 273 phosphorothioate oligodeoxynucleotides 233 toxins, cytoplasm entry 219 transcription 103 schema 36 transfer of oligonucleotides from endosomal to cytoplasmic compartments 205, 218 transferrin 112, 117, 185 translation, scheme 36 translation inhibition 12 transmembrane transport of oligonucleotides 213–20 transplantation tolerance 272 transporter proteins 216–17 triethylammonium acetate 62 triple helix 300 triplex 299 for antisense, design criteria 39–40 triplex DNA, triplex-forming ONs (TFOs) 172–84 Trypanosoma brucei 289, 293–7 Trypanosoma cruzi 291, 293–7 tryptophan residue 118, 119 (CGT[C]GA) tyrosine kinase inhibition 11– 12 ulcerative colitis 38, 272–4 universal base, defined 56

334 INDEX

5′-untranslated initiation codon region 12– 13 mRNA degradation 12 UV spectroscopy 60, 89 G-quartet identification 7 nucleic acid quantification 60 psoralen conjugation 59 UV-melting curves 59, 61–2 variant surface glycoproteins 290 vascular cell adhesion molecule 1 (VCAM-1) 264, 266, 275–6 vesicular stomatitis virus 119 viral infections 47, 128, 130, 140, 154, 158 vitamin E 58 vitravene 38 Watson-Crick double helix 35, 37, 39, 42, 46, 53, 54, 56, 101, 300–1 wheat germ extract 17 xenografts 269–70 Xenopus oocytes 12, 17 zintevir (AR177) 38, 226, 228, 233

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