Table Of ContentOligonucleotide‐Based
Drugs and Therapeutics
Oligonucleotide‐Based Drugs and
Therapeutics
Preclinical and Clinical Considerations for Development
Edited by
Nicolay Ferrari and Rosanne Seguin
This edition first published 2018
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Library of Congress Cataloging‐in‐Publication Data
Names: Ferrari, Nicolay, 1969– editor. | Seguin, Rosanne, editor.
Title: Oligonucleotide-based drugs and therapeutics : preclinical and clinical
considerations for development / edited by Nicolay Ferrari, Rosanne Seguin.
Description: Hoboken, NJ : John Wiley & Sons, 2018. | Includes bibliographical
references and index. |
Identifiers: LCCN 2018006576 (print) | LCCN 2018009683 (ebook) |
ISBN 9781119070290 (pdf) | ISBN 9781119070306 (epub) | ISBN 9781118537336 (cloth)
Subjects: LCSH: Oligonucleotides–Therapeutic use. | Antisense nucleic acids–Therapeutic use.
Classification: LCC RM666.N87 (ebook) | LCC RM666.N87 O445 2018 (print) |
DDC 572.8/5–dc23
LC record available at https://lccn.loc.gov/2018006576
Cover Design: Wiley
Cover Images: © nechaev-kon/Getty Images;
© Michal Sanca/Shutterstock
Set in 10/12pt Warnock by SPi Global, Pondicherry, India
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
v
Contents
List of Contributors xvii
Preface xxi
Acknowledgments xxii
1 Mechanisms of Oligonucleotide Actions 1
Annemieke Aartsma‐Rus, Aimee L. Jackson, and Arthur A. Levin
1.1 Introduction 1
1.2 Antisense Oligonucleotide Therapeutics 2
1.2.1 Antisense Activity Mediated by RNase H 2
1.2.2 The RNase H Mechanism 2
1.2.3 Chemical Modifications to Enhance RNase H‐mediated
Antisense Activity 3
1.3 Oligonucleotides that Sterically Block Translation 5
1.4 Oligonucleotides that Act Through the RNAi Pathway 5
1.4.1 The RISC Pathway 5
1.4.2 Mechanisms of RISC‐mediated Gene Silencing 8
1.5 Chemical Modification of siRNAs and miRNAs 10
1.5.1 Delivery of Therapeutic siRNAs or miRNAs 12
1.6 Clinical Use of Oligonucleotides that Act through the RNAi
Pathway 14
1.7 Oligonucleotides that Modulate Splicing 17
1.7.1 Pre‐mRNA Splicing and Disease 17
1.7.2 Mechanisms of Oligonucleotide‐mediated Splicing Modulation 17
1.7.3 Chemical Modifications that Enhance Activity of Oligonucleotide‐
based Splicing Modulators 21
1.7.4 Clinical Applications of Splicing Modulators 22
1.8 Conclusions 22
References 22
2 The Medicinal Chemistry of Antisense Oligonucleotides 39
Jonathan K. Watts
2.1 Introduction: The Antisense Approach and the Need for
Chemical Modification 39
vi Contents
2.1.1 How Does Medicinal Chemistry Apply to Oligonucleotides? 40
2.1.2 Chemistry and Toxicity 41
2.2 Why Chemically Modify an Oligonucleotide? 42
2.2.1 Medicinal Chemistry Can Increase Nuclease Stability 42
2.2.2 Medicinal Chemistry Can Tune Binding Affinity and Specificity 43
2.2.3 Medicinal Chemistry Can Change Interactions
with Cellular Factors 44
2.2.4 Medicinal Chemistry Can Modulate Immunostimulation 45
2.2.5 Medicinal Chemistry Can Improve RNase H
Cleavage Specificity 46
2.2.6 Medicinal Chemistry Can Improve Cellular Uptake and Subcellular
Trafficking 47
2.3 Chemical Modifications of Current Importance by Structural
Class 48
2.3.1 Sugar Modifications 48
2.3.1.1 2′‐Modified Ribose Sugars 48
2.3.1.2 2′‐Modified Arabinose Sugars 50
2.3.1.3 2′,4′‐Difluorinated Nucleosides 50
2.3.1.4 Constrained Nucleotides 50
2.3.1.5 Sugars with Expanded Ring Size 53
2.3.2 Phosphate Modifications 54
2.3.2.1 Phosphorothioate 54
2.3.2.2 Other Charged Phosphate Analogues 58
2.3.2.3 Neutral Mimics of the Phosphate Linkage 58
2.3.2.4 Metabolically Stable 5′‐Phosphate Analogues 60
2.3.3 Total Replacement of the Sugar‐Phosphate Backbone 61
2.3.4 Nucleobase Modifications 62
2.3.4.1 Sulfur‐Modified Nucleobases 63
2.3.4.2 5‐Modified Pyrimidines 63
2.3.4.3 Nucleobases with Expanded Hydrogen Bonding Networks 65
2.3.5 Assembly of Oligonucleotides into Multimeric Structures 66
2.4 Conclusion 67
References 69
3 Cellular Pharmacology of Antisense Oligonucleotides 91
Xin Ming
3.1 Introduction 91
3.2 Molecular Mechanisms of Antisense Oligonucleotides 92
3.2.1 Classic Antisense Oligonucleotides 92
3.2.2 siRNA 94
3.2.3 Splice Switching Oligonucleotides 94
3.2.4 microRNA Antagomirs 95
3.2.5 lncRNAs Antagomirs 95
Contents vii
3.3 Cellular Pharmacology of Antisense Oligonucleotides 96
3.3.1 Endocytosis of Free Oligonucleotides 98
3.3.2 Endocytosis of Oligonucleotide Conjugates 98
3.3.3 Uptake and Trafficking of Oligonucleotides Incorporated into
Nanocarriers 100
3.4 Conclusion 101
References 101
4 Pharmacokinetics and Pharmacodynamics of Antisense
Oligonucleotides 107
Helen Lightfoot, Anneliese Schneider, and Jonathan Hall
4.1 Introduction 107
4.2 Pharmacokinetic Properties of Antisense Oligonucleotides 108
4.2.1 Protein Binding 109
4.2.2 Dose Dependency of ASO Pharmacokinetics 110
4.2.3 Absorption 110
4.2.4 Distribution 111
4.2.5 Metabolism and Excretion 112
4.3 Pharmacodynamic Properties of Antisense Oligonucleotides 113
4.3.1 ASO Target Selection and Validation 114
4.3.2 Mechanisms of Action 117
4.3.3 Biomarkers and PD Endpoints 118
4.4 PD and PK Results and Strategies of ASOs in Clinical Development 119
4.4.1 Genetic Diseases 122
4.4.1.1 Mipomersen, Apolipoprotein B‐100, and
Hypercholesterolemia 122
4.4.1.2 Drisapersen, Dystrophin, and Duchenne Muscular
Dystrophy (DMD) 123
4.4.2 Infectious Diseases 125
4.4.2.1 Miravirsen, miR‐122, and Hepatitis C Virus (HCV) 125
4.4.3 Cancer 126
4.4.3.1 Custirsen, Clusterin, and Cancer 126
4.4.3.2 LY2181308 (ISIS‐23722), Survivin, and Cancer 127
4.5 Summary and Conclusions 128
References 130
5 Tissue Distribution, Metabolism, and Clearance 137
Mehrdad Dirin and Johannes Winkler
5.1 Introduction 137
5.2 Tissue Distribution 138
5.2.1 Dermal Delivery 138
5.2.2 Ocular Delivery 139
5.2.3 Oral Administration 139
viii Contents
5.2.4 Intrathecal Delivery 141
5.2.5 Intravesical Administration 142
5.2.6 Pulmonary Administration 142
5.2.7 Distribution to Muscular Tissue 143
5.2.8 Intravenous Administration 144
5.3 Cellular Uptake 146
5.4 Metabolism and Clearance 148
5.4.1 Phosphorothioates Including 2′‐Modifications 148
5.4.2 Phosphorodiamidate Morpholino Oligonucleotides 149
5.5 Conclusion 150
References 151
6 Hybridization‐Independent Effects: Principles and Specific
Considerations for Oligonucleotide Drugs 161
Nicolay Ferrari
6.1 Background 161
6.2 Mechanisms of Hybridization‐independent Toxicities 162
6.2.1 Effects Related to Oligonucleotide Sequence 162
6.2.1.1 Unmethylated CpG Motifs 162
6.2.1.2 Poly‐G Sequences 163
6.2.1.3 DNA Triplex‐forming Oligonucleotides 164
6.2.1.4 Other Motifs 164
6.2.2 Effects Related to Oligonucleotide Chemistry 164
6.2.2.1 Phosphorothioate Oligonucleotides 165
6.2.2.2 Effects of Other Chemical Modifications 171
6.3 Hybridization‐independent Effects Following Local Delivery
of Oligonucleotides 171
6.3.1 Pulmonary Toxicity of Inhaled Oligonucleotides 171
6.3.1.1 Specific Considerations for Inhaled Oligonucleotides 173
6.3.2 Approaches to Reduce Hybridization‐independent Class Effects
of Inhaled Oligonucleotides 175
6.3.2.1 Mixed Phosphorothioate/Phosphodiester Oligonucleotides 175
6.4 Conclusion 180
References 180
7 Hybridization‐Dependent Effects: The Prediction, Evaluation,
and Consequences of Unintended Target Hybridization 191
Jeremy D. A. Kitson, Piotr J. Kamola, and Lauren Kane
7.1 Introduction 191
7.1.1 Scope of this Review: RNase H1‐dependent ASOs 192
7.2 Specificity Studies with ASOs 192
7.3 Implications of the Nuclear Site of Action of RNase H1 194
