Oligonucleotide-Based Drugs and Therapeutics
Preclinical and Clinical Considerations for Development
1. Edition September 2018
576 Pages, Hardcover
Wiley & Sons Ltd
ISBN:
978-1-118-53733-6
John Wiley & Sons
Short Description
Antisense-based drugs are part of a growing number of pharmaceutical and biotech programs to treat diseases including cancer, diabetes, and ALS. Reviewing fundamentals and offering guidelines for drug discovery and development, this book is a practical guide from the safety assessment to clinical experience with antisense oligonucleotides. It provides key information about toxicology and chemistry, giving in-depth detail on innovative topics and their relation to antisense therapeutics. Overall, 20 chapters of detailed discussion review and connect recent developments in the field and provide room for further research and advancement.
Further versions
A comprehensive review of contemporary antisense oligonucleotides drugs and therapeutic principles, methods, applications, and research
Oligonucleotide-based drugs, in particular antisense oligonucleotides, are part of a growing number of pharmaceutical and biotech programs progressing to treat a wide range of indications including cancer, cardiovascular, neurodegenerative, neuromuscular, and respiratory diseases, as well as other severe and rare diseases. Reviewing fundamentals and offering guidelines for drug discovery and development, this book is a practical guide covering all key aspects of this increasingly popular area of pharmacology and biotech and pharma research, from the basic science behind antisense oligonucleotides chemistry, toxicology, manufacturing, to safety assessments, the design of therapeutic protocols, to clinical experience.
Antisense oligonucleotides are single strands of DNA or RNA that are complementary to a chosen sequence. While the idea of antisense oligonucleotides to target single genes dates back to the 1970's, most advances have taken place in recent years. The increasing number of antisense oligonucleotide programs in clinical development is a testament to the progress and understanding of pharmacologic, pharmacokinetic, and toxicologic properties as well as improvement in the delivery of oligonucleotides. This valuable book reviews the fundamentals of oligonucleotides, with a focus on antisense oligonucleotide drugs, and reports on the latest research underway worldwide.
* Helps readers understand antisense molecules and their targets, biochemistry, and toxicity mechanisms, roles in disease, and applications for safety and therapeutics
* Examines the principles, practices, and tools for scientists in both pre-clinical and clinical settings and how to apply them to antisense oligonucleotides
* Provides guidelines for scientists in drug design and discovery to help improve efficiency, assessment, and the success of drug candidates
* Includes interdisciplinary perspectives, from academia, industry, regulatory and from the fields of pharmacology, toxicology, biology, and medicinal chemistry
Oligonucleotide-Based Drugs and Therapeutics belongs on the reference shelves of chemists, pharmaceutical scientists, chemical biologists, toxicologists and other scientists working in the pharmaceutical and biotechnology industries. It will also be a valuable resource for regulatory specialists and safety assessment professionals and an important reference for academic researchers and post-graduates interested in therapeutics, antisense therapy, and oligonucleotides.
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
1.2 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 Oligonucleotidebased 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
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 Introduction91
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
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 Introduction137
5.2 Tissue Distribution 138
5.2.1 Dermal Delivery 138
5.2.2 Ocular Delivery 139
5.2.3 Oral Administration 139
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
7.3.1 Confirmation of Unintended Targets within Introns 195
7.4 Mechanism of OTE 196
7.5 Determining the Extent that Accessibility, Affinity and, Mismatch Tolerance Contribute to Off-target Activity 198
7.5.1 Accessibility 198
7.5.2 Affinity 199
7.5.3 The Interaction of RNase H1 with the RNA/ASO Duplex 200
7.5.4 Mismatch Tolerance 202
7.6 Consequences of Unintended Transcript Knockdown: In Vivo and In Vitro Toxicity 203
7.7 Identification and Evaluation of Putative OTEs 207
7.7.1 Computational Prediction of Unintended Targeting 207
7.7.1.1 Database Creation 209
7.7.1.2 Sequence Alignments 209
7.7.1.3 Cross-species Off-target Homology 210
7.7.1.4 Results Filtering and Annotation 211
7.7.1.5 RNA Structure and Target Accessibility 211
7.7.1.6 ASO-Target Duplex Thermodynamics 213
7.7.1.7 Computational Framework for OTEs 214
7.7.1.8 In Vitro Screening for OTEs 214
7.7.1.9 Methods for Measuring Gene Expression 216
7.8 Summary 216
Acknowledgments 217
References 218
8 Class-Related Proinflammatory Effects 227
Rosanne Seguin
8.1 Introduction 227
8.2 Proinflammatory Effects of ASO for Consideration in Drug Development 228
8.2.1 Activation of the Complement Cascade in Monkeys 228
8.2.2 Cytokine Release 229
8.2.3 Mononuclear Cellular Infiltrate 232
8.2.4 Hematological Changes 236
8.2.5 Immunogenicity 237
8.3 Conclusions 238
References 239
9 Exaggerated Pharmacology 243
Alain Guimond and Doug Kornbrust
9.1 Introduction 243
9.2 Regulatory Expectations 244
9.3 Scope of EP Assessment 245
9.3.1 Species Selection 245
9.3.2 Determination of Pharmacologic Relevance 247
9.4 EP Evaluation Strategies 248
9.4.1 Concerns About the Use of Animal-active Analogues 248
9.4.2 Animal-active Analogues in Reproductive and/or Carcinogenicity Studies 250
9.4.3 Other Considerations for Use of Animal Analogues 250
9.4.4 The Use of Inactive Analogues as Control Articles 250
9.4.5 The Role of Formulations 251
9.4.6 Aptamer Oligonucleotides 251
9.4.7 Immunostimulatory Oligonucleotides 252
9.4.8 MicroRNA 253
9.5 Conclusions 254
References 255
10 Genotoxicity Tests for Novel Oligonucleotide-Based Therapeutics 257
Cindy L. Berman, Scott A. Barros, Sheila M. Galloway, Peter Kasper, Frederick B. Oleson, Catherine C. Priestley, Kevin S. Sweder, Michael J. Schlosser, and Zhanna Sobol
10.1 Introduction 257
10.1.1 History of Regulatory Guidance on Genotoxicity Testing 259
10.1.2 Relevance of the Standard Genotoxicity Test Battery to ONs 260
10.2 Experience with ONs in the Standard Battery 262
10.2.1 ON Chemical Classes Tested for Genotoxicity 264
10.2.2 Conclusions Based on the Database 265
10.3 OSWG Recommendation for Genotoxicity Testing of ONs 266
10.3.1 Recommended Test Battery 266
10.3.2 Requirement for Evidence for Uptake 270
10.3.3 Need for Testing of ONs 271
10.3.3.1 Nonconjugated ONs in Simple Aqueous Formulations 271
10.3.3.2 ONs in Complex Formulations or Conjugates 272
10.3.4 Recommended Test Conditions 273
10.3.4.1 Top Concentration for In Vitro Tests 273
10.3.4.2 Use of S-9 in In Vitro Tests 273
10.3.4.3 In Vivo Tests 274
10.4 Triplex Formation 275
10.4.1 Biochemical Requirements for Triplex Formation 275
10.4.2 Assessment of New ONs for Triplex Formation 277
10.5 Impurities 278
10.5.1 ON-Related Impurities 278
10.5.2 Potentially Mutagenic Impurities 278
10.6 Conclusions 279
Acknowledgments 280
References 280
11 Reproductive and Developmental Toxicity Testing Strategies for Oligonucleotide-Based Therapeutics 287
Tacey E.K. White and Joy Cavagnaro
11.1 Introduction 287
11.2 General Design of Reproductive and Developmental Toxicity Studies 289
11.3 Product Attributes of Oligonucleotide Drugs 291
11.4 The Role of Intended Pharmacology in Reproductive and Developmental Effects 293
11.5 Selection of Animal Species 294
11.5.1 Design and Use of Animal-active Analogues 294
11.6 Justification of Dosing Regimen 296
11.7 Exposure Assessment 297
11.8 Subclass- specific Considerations 298
11.8.1 Single-stranded DNA Antisense Oligonucleotides 299
11.8.2 CpG and Immunostimulatory (IS) Oligonucleotides 300
11.8.3 microRNA Mimetics/Antagonists and siRNAs 301
11.8.4 Aptamer Oligonucleotides 303
11.9 Conclusions 304
Acknowledgments 305
References 305
12 Specific Considerations for Preclinical Development of Inhaled Oligonucleotides 311
Nicolay Ferrar
12.1 Background 311
12.2 Oligonucleotide Delivery Systems 312
12.2.1 Inhalation Exposure Systems 312
12.2.2 Intratracheal Aerosol Instillation 313
12.3 Repeat-dose Toxicity 314
12.3.1 General Principles 314
12.3.2 Recovery Phase 317
12.4 Toxicokinetics 319
12.5 Safety Pharmacology 322
12.5.1 Respiratory System 323
12.5.2 Cardiovascular and Central Nervous Systems 324
12.6 Additional Testing 326
12.6.1 Complement Activation 326
12.6.2 Proinflammatory Effects 327
12.7 Conclusion 328
References 328
13 Lessons Learned in Oncology Programs 331
Cindy Jacobs, Monica Krieger, Patricia S. Stewart, Karen D. Wisont,and Scott Cormack
13.1 Introduction 331
13.2 Clinical Development of First-generation ASOs 332
13.2.1 Aprinocarsen 332
13.2.2 Oblimersen 334
13.2.3 Challenges Associated with First-generation ASOs 335
13.3 Clinical Development of Second-generation ASOs 336
13.3.1 Custirsen 337
13.3.2 Lessons Learned from Custirsen Clinical Development 343
13.3.3 Apatorsen 344
13.3.4 Bladder Cancer 346
13.3.5 Lung Cancer 346
13.3.6 Pancreatic Cancer 347
13.3.7 Prostate Cancer 347
13.4 Regulatory Considerations 348
13.5 Future Opportunities for ASOs as Therapeutic Agents for Cancer Treatment 349
References 349
14 Inhaled Antisense for Treatment of Respiratory Disease 355
Gail M. Gauvreau, Beth E. Davis, and John Paul Oliveria
14.1 Introduction 355
14.2 Atopic Asthma 355
14.2.1 Pharmacotherapy of Asthma 356
14.2.2 Anti-IL-5 Monoclonal Antibodies 357
14.2.3 Anti-IL-4/13 Monoclonal Antibodies 359
14.3 Antisense Oligonucleotides in Animal Models 361
14.3.1 CpG Immunostimulatory Sequences 361
14.3.2 Antisense to Receptors on Eosinophils 366
14.3.3 Antisense to IL-4 and IL-13 Receptors 368
14.3.4 Summary of Antisense Oligonucleotides in Animal Models 368
14.4 Clinical Data 369
14.4.1 Allergen Challenge: A Model of Asthma Exacerbation 369
14.4.2 Allergen Challenge for Evaluation of Efficacy 369
14.4.3 1018 Immunostimulatory Sequence 370
14.4.3.1 Study Design for 1018 ISS 370
14.4.3.2 Results for 1018 ISS 371
14.4.4 AIR645 372
14.4.4.1 Study Design for AIR645 373
14.4.4.2 Results for AIR645 373
14.4.5 TPI ASM8 374
14.4.5.1 Mechanism of TPI ASM8 374
14.4.5.2 Study #1 for TPI ASM8 375
14.4.5.3 Study #2 for TPI ASM8 377
14.5 General
Conclusion 378
References 378
15 Antisense Oligonucleotides for Treatment of Neurological Diseases 389
Rosanne Seguin
15.1 Introduction 389
15.1.1 Delivery of ASO to Central Nervous System 389
15.2 Potential ASO Therapies in Neurodegenerative Diseases 390
15.2.1 Spinal Muscular Atrophy (SMA) 390
15.2.2 Amyotrophic Lateral Sclerosis (ALS) 393
15.2.3 Huntington's Disease (HD) 396
15.2.4 Muscular Sclerosis (MS) 399
15.2.5 Alzheimer's Disease (AD) 401
15.3 Conclusion 403
References 403
16 Nucleic Acids as Adjuvants 411
Kevin Brown, Montserrat Puig, Lydia Haile, Derek Ireland, John Martucci, and Daniela Verthelyi
16.1 Introduction 411
16.1.1 TLR as Nucleic Acid-Sensing Pathogen Recognition Receptors (PRR) 412
16.2 Categories of Nucleic Acid Adjuvants 413
16.2.1 DNA-Based Adjuvants and Vaccine Studies in Mice 417
16.2.2 Classes of CpG ODN that Activate Human TLR9 421
16.2.3 Preclinical Studies with Human CpG ODN 422
16.2.4 Safety Issues Raised in Animal Models 424
16.2.5 Clinical Trial Experience 425
16.2.6 Safety Issues from Human Clinical Trials 427
16.2.7 Novel Delivery Systems for CpG ODN as Adjuvants 427
16.3 Conclusion 429
Acknowledgments 429
References 430
17 Splice-Switching Oligonucleotides 445
Isabella Gazzoli and Annemieke Aartsma-Rus
17.1 Introduction of Splice Switching 445
17.1.1 Correct Cryptic Splicing 446
17.1.1.1 ß-Thalassemia 446
17.1.1.2 Cystic Fibrosis 450
17.1.2 Isoform Switching 451
17.1.2.1 Anticancer 451
17.1.2.2 Tauopathies 452
17.1.3 Induce Exon Inclusion 452
17.1.3.1 Tumorigenesis 452
17.1.3.2 Spinal Muscular Atrophy (SMA) 453
17.1.4 Reading Frame Correction 454
17.1.4.1 Duchenne Muscular Dystrophy 454
17.1.4.2 Dysferlinopathies 455
17.1.5 Knockdown 456
17.1.5.1 Atherosclerosis 456
17.1.5.2 Myostatin-Related Muscle Hypertrophy 457
17.2 Preclinical and Clinical Development of Splice-switching Oligos 457
17.2.1 Introduction to Different Chemistries to be Used for Splice Switching 457
17.2.2 AON Targets 459
17.2.3 AON Development for DMD 460
17.2.4 2'-O-Methyl Phosphorothioate AONs 461
17.2.4.1 Animal Studies 461
17.2.4.2 Human Studies 463
17.2.5 Phosphorodiamidate Morpholino Oligos 466
17.2.5.1 Animal Studies 466
17.2.5.2 Human Studies 467
17.2.6 Other Chemistries 468
17.2.6.1 Peptide-Conjugated PMOs 468
17.2.7 Preclinical and Clinical Studies for Other Diseases 470
17.2.7.1 Spinal Muscular Atrophy (SMA) 470
17.2.8 Biomarkers 472
17.3 Future Directions 474
Conflictof Interest 475
Acknowledgments 475
References 475
18 CMC Aspects for the Clinical Development of Spiegelmers 491
Stefan Vonhoff
18.1 Introduction 491
18.2 Technology (Mirror-imaged SELEX Process) Selected Pharmaceutical Properties 492
18.3 Preclinical Efficacy Data for Spiegelmers 494
18.4 Clinical Development 504
18.4.1 Emapticap Pegol: NOX-E36 504
18.4.2 Olaptesed Pegol: NOX-A12 506
18.4.3 Lexaptepid Pegol: NOX-H94 507
18.5 CMC Aspects for the Development of Spiegelmers 508
18.5.1 Discovery and Early Preclinical Stage 508
18.5.2 Generic Manufacturing Process 509
18.5.2.1 Solid-phase Synthesis 510
18.5.2.2 Deprotection 510
18.5.2.3 Purification of the Intermediate Spiegelmer Prior to Pegylation 510
18.5.2.4 Pegylation 510
18.5.2.5 Purification of the Pegylated Spiegelmer 510
18.5.3 CMC Aspects for the Selection of Development Candidates 511
18.5.4 GMP Production of Spiegelmers 514
18.5.4.1 Starting Materials 514
18.5.4.2 Drug Substance 516
18.5.4.3 Drug Product 516
18.5.5 Analytical Methods for the Quality Control of Spiegelmers 517
18.6 Future Prospects for Spiegelmer Therapeutics 521
References 521
Index 527
Preface xxi
Acknowledgments xxii
1 Mechanisms of Oligonucleotide Actions 1
Annemieke Aartsma-Rus, Aimee L. Jackson, and Arthur A. Levin
1.1 Introduction
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 Oligonucleotidebased 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
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 Introduction91
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
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 Introduction137
5.2 Tissue Distribution 138
5.2.1 Dermal Delivery 138
5.2.2 Ocular Delivery 139
5.2.3 Oral Administration 139
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
7.3.1 Confirmation of Unintended Targets within Introns 195
7.4 Mechanism of OTE 196
7.5 Determining the Extent that Accessibility, Affinity and, Mismatch Tolerance Contribute to Off-target Activity 198
7.5.1 Accessibility 198
7.5.2 Affinity 199
7.5.3 The Interaction of RNase H1 with the RNA/ASO Duplex 200
7.5.4 Mismatch Tolerance 202
7.6 Consequences of Unintended Transcript Knockdown: In Vivo and In Vitro Toxicity 203
7.7 Identification and Evaluation of Putative OTEs 207
7.7.1 Computational Prediction of Unintended Targeting 207
7.7.1.1 Database Creation 209
7.7.1.2 Sequence Alignments 209
7.7.1.3 Cross-species Off-target Homology 210
7.7.1.4 Results Filtering and Annotation 211
7.7.1.5 RNA Structure and Target Accessibility 211
7.7.1.6 ASO-Target Duplex Thermodynamics 213
7.7.1.7 Computational Framework for OTEs 214
7.7.1.8 In Vitro Screening for OTEs 214
7.7.1.9 Methods for Measuring Gene Expression 216
7.8 Summary 216
Acknowledgments 217
References 218
8 Class-Related Proinflammatory Effects 227
Rosanne Seguin
8.1 Introduction 227
8.2 Proinflammatory Effects of ASO for Consideration in Drug Development 228
8.2.1 Activation of the Complement Cascade in Monkeys 228
8.2.2 Cytokine Release 229
8.2.3 Mononuclear Cellular Infiltrate 232
8.2.4 Hematological Changes 236
8.2.5 Immunogenicity 237
8.3 Conclusions 238
References 239
9 Exaggerated Pharmacology 243
Alain Guimond and Doug Kornbrust
9.1 Introduction 243
9.2 Regulatory Expectations 244
9.3 Scope of EP Assessment 245
9.3.1 Species Selection 245
9.3.2 Determination of Pharmacologic Relevance 247
9.4 EP Evaluation Strategies 248
9.4.1 Concerns About the Use of Animal-active Analogues 248
9.4.2 Animal-active Analogues in Reproductive and/or Carcinogenicity Studies 250
9.4.3 Other Considerations for Use of Animal Analogues 250
9.4.4 The Use of Inactive Analogues as Control Articles 250
9.4.5 The Role of Formulations 251
9.4.6 Aptamer Oligonucleotides 251
9.4.7 Immunostimulatory Oligonucleotides 252
9.4.8 MicroRNA 253
9.5 Conclusions 254
References 255
10 Genotoxicity Tests for Novel Oligonucleotide-Based Therapeutics 257
Cindy L. Berman, Scott A. Barros, Sheila M. Galloway, Peter Kasper, Frederick B. Oleson, Catherine C. Priestley, Kevin S. Sweder, Michael J. Schlosser, and Zhanna Sobol
10.1 Introduction 257
10.1.1 History of Regulatory Guidance on Genotoxicity Testing 259
10.1.2 Relevance of the Standard Genotoxicity Test Battery to ONs 260
10.2 Experience with ONs in the Standard Battery 262
10.2.1 ON Chemical Classes Tested for Genotoxicity 264
10.2.2 Conclusions Based on the Database 265
10.3 OSWG Recommendation for Genotoxicity Testing of ONs 266
10.3.1 Recommended Test Battery 266
10.3.2 Requirement for Evidence for Uptake 270
10.3.3 Need for Testing of ONs 271
10.3.3.1 Nonconjugated ONs in Simple Aqueous Formulations 271
10.3.3.2 ONs in Complex Formulations or Conjugates 272
10.3.4 Recommended Test Conditions 273
10.3.4.1 Top Concentration for In Vitro Tests 273
10.3.4.2 Use of S-9 in In Vitro Tests 273
10.3.4.3 In Vivo Tests 274
10.4 Triplex Formation 275
10.4.1 Biochemical Requirements for Triplex Formation 275
10.4.2 Assessment of New ONs for Triplex Formation 277
10.5 Impurities 278
10.5.1 ON-Related Impurities 278
10.5.2 Potentially Mutagenic Impurities 278
10.6 Conclusions 279
Acknowledgments 280
References 280
11 Reproductive and Developmental Toxicity Testing Strategies for Oligonucleotide-Based Therapeutics 287
Tacey E.K. White and Joy Cavagnaro
11.1 Introduction 287
11.2 General Design of Reproductive and Developmental Toxicity Studies 289
11.3 Product Attributes of Oligonucleotide Drugs 291
11.4 The Role of Intended Pharmacology in Reproductive and Developmental Effects 293
11.5 Selection of Animal Species 294
11.5.1 Design and Use of Animal-active Analogues 294
11.6 Justification of Dosing Regimen 296
11.7 Exposure Assessment 297
11.8 Subclass- specific Considerations 298
11.8.1 Single-stranded DNA Antisense Oligonucleotides 299
11.8.2 CpG and Immunostimulatory (IS) Oligonucleotides 300
11.8.3 microRNA Mimetics/Antagonists and siRNAs 301
11.8.4 Aptamer Oligonucleotides 303
11.9 Conclusions 304
Acknowledgments 305
References 305
12 Specific Considerations for Preclinical Development of Inhaled Oligonucleotides 311
Nicolay Ferrar
12.1 Background 311
12.2 Oligonucleotide Delivery Systems 312
12.2.1 Inhalation Exposure Systems 312
12.2.2 Intratracheal Aerosol Instillation 313
12.3 Repeat-dose Toxicity 314
12.3.1 General Principles 314
12.3.2 Recovery Phase 317
12.4 Toxicokinetics 319
12.5 Safety Pharmacology 322
12.5.1 Respiratory System 323
12.5.2 Cardiovascular and Central Nervous Systems 324
12.6 Additional Testing 326
12.6.1 Complement Activation 326
12.6.2 Proinflammatory Effects 327
12.7 Conclusion 328
References 328
13 Lessons Learned in Oncology Programs 331
Cindy Jacobs, Monica Krieger, Patricia S. Stewart, Karen D. Wisont,and Scott Cormack
13.1 Introduction 331
13.2 Clinical Development of First-generation ASOs 332
13.2.1 Aprinocarsen 332
13.2.2 Oblimersen 334
13.2.3 Challenges Associated with First-generation ASOs 335
13.3 Clinical Development of Second-generation ASOs 336
13.3.1 Custirsen 337
13.3.2 Lessons Learned from Custirsen Clinical Development 343
13.3.3 Apatorsen 344
13.3.4 Bladder Cancer 346
13.3.5 Lung Cancer 346
13.3.6 Pancreatic Cancer 347
13.3.7 Prostate Cancer 347
13.4 Regulatory Considerations 348
13.5 Future Opportunities for ASOs as Therapeutic Agents for Cancer Treatment 349
References 349
14 Inhaled Antisense for Treatment of Respiratory Disease 355
Gail M. Gauvreau, Beth E. Davis, and John Paul Oliveria
14.1 Introduction 355
14.2 Atopic Asthma 355
14.2.1 Pharmacotherapy of Asthma 356
14.2.2 Anti-IL-5 Monoclonal Antibodies 357
14.2.3 Anti-IL-4/13 Monoclonal Antibodies 359
14.3 Antisense Oligonucleotides in Animal Models 361
14.3.1 CpG Immunostimulatory Sequences 361
14.3.2 Antisense to Receptors on Eosinophils 366
14.3.3 Antisense to IL-4 and IL-13 Receptors 368
14.3.4 Summary of Antisense Oligonucleotides in Animal Models 368
14.4 Clinical Data 369
14.4.1 Allergen Challenge: A Model of Asthma Exacerbation 369
14.4.2 Allergen Challenge for Evaluation of Efficacy 369
14.4.3 1018 Immunostimulatory Sequence 370
14.4.3.1 Study Design for 1018 ISS 370
14.4.3.2 Results for 1018 ISS 371
14.4.4 AIR645 372
14.4.4.1 Study Design for AIR645 373
14.4.4.2 Results for AIR645 373
14.4.5 TPI ASM8 374
14.4.5.1 Mechanism of TPI ASM8 374
14.4.5.2 Study #1 for TPI ASM8 375
14.4.5.3 Study #2 for TPI ASM8 377
14.5 General
Conclusion 378
References 378
15 Antisense Oligonucleotides for Treatment of Neurological Diseases 389
Rosanne Seguin
15.1 Introduction 389
15.1.1 Delivery of ASO to Central Nervous System 389
15.2 Potential ASO Therapies in Neurodegenerative Diseases 390
15.2.1 Spinal Muscular Atrophy (SMA) 390
15.2.2 Amyotrophic Lateral Sclerosis (ALS) 393
15.2.3 Huntington's Disease (HD) 396
15.2.4 Muscular Sclerosis (MS) 399
15.2.5 Alzheimer's Disease (AD) 401
15.3 Conclusion 403
References 403
16 Nucleic Acids as Adjuvants 411
Kevin Brown, Montserrat Puig, Lydia Haile, Derek Ireland, John Martucci, and Daniela Verthelyi
16.1 Introduction 411
16.1.1 TLR as Nucleic Acid-Sensing Pathogen Recognition Receptors (PRR) 412
16.2 Categories of Nucleic Acid Adjuvants 413
16.2.1 DNA-Based Adjuvants and Vaccine Studies in Mice 417
16.2.2 Classes of CpG ODN that Activate Human TLR9 421
16.2.3 Preclinical Studies with Human CpG ODN 422
16.2.4 Safety Issues Raised in Animal Models 424
16.2.5 Clinical Trial Experience 425
16.2.6 Safety Issues from Human Clinical Trials 427
16.2.7 Novel Delivery Systems for CpG ODN as Adjuvants 427
16.3 Conclusion 429
Acknowledgments 429
References 430
17 Splice-Switching Oligonucleotides 445
Isabella Gazzoli and Annemieke Aartsma-Rus
17.1 Introduction of Splice Switching 445
17.1.1 Correct Cryptic Splicing 446
17.1.1.1 ß-Thalassemia 446
17.1.1.2 Cystic Fibrosis 450
17.1.2 Isoform Switching 451
17.1.2.1 Anticancer 451
17.1.2.2 Tauopathies 452
17.1.3 Induce Exon Inclusion 452
17.1.3.1 Tumorigenesis 452
17.1.3.2 Spinal Muscular Atrophy (SMA) 453
17.1.4 Reading Frame Correction 454
17.1.4.1 Duchenne Muscular Dystrophy 454
17.1.4.2 Dysferlinopathies 455
17.1.5 Knockdown 456
17.1.5.1 Atherosclerosis 456
17.1.5.2 Myostatin-Related Muscle Hypertrophy 457
17.2 Preclinical and Clinical Development of Splice-switching Oligos 457
17.2.1 Introduction to Different Chemistries to be Used for Splice Switching 457
17.2.2 AON Targets 459
17.2.3 AON Development for DMD 460
17.2.4 2'-O-Methyl Phosphorothioate AONs 461
17.2.4.1 Animal Studies 461
17.2.4.2 Human Studies 463
17.2.5 Phosphorodiamidate Morpholino Oligos 466
17.2.5.1 Animal Studies 466
17.2.5.2 Human Studies 467
17.2.6 Other Chemistries 468
17.2.6.1 Peptide-Conjugated PMOs 468
17.2.7 Preclinical and Clinical Studies for Other Diseases 470
17.2.7.1 Spinal Muscular Atrophy (SMA) 470
17.2.8 Biomarkers 472
17.3 Future Directions 474
Conflictof Interest 475
Acknowledgments 475
References 475
18 CMC Aspects for the Clinical Development of Spiegelmers 491
Stefan Vonhoff
18.1 Introduction 491
18.2 Technology (Mirror-imaged SELEX Process) Selected Pharmaceutical Properties 492
18.3 Preclinical Efficacy Data for Spiegelmers 494
18.4 Clinical Development 504
18.4.1 Emapticap Pegol: NOX-E36 504
18.4.2 Olaptesed Pegol: NOX-A12 506
18.4.3 Lexaptepid Pegol: NOX-H94 507
18.5 CMC Aspects for the Development of Spiegelmers 508
18.5.1 Discovery and Early Preclinical Stage 508
18.5.2 Generic Manufacturing Process 509
18.5.2.1 Solid-phase Synthesis 510
18.5.2.2 Deprotection 510
18.5.2.3 Purification of the Intermediate Spiegelmer Prior to Pegylation 510
18.5.2.4 Pegylation 510
18.5.2.5 Purification of the Pegylated Spiegelmer 510
18.5.3 CMC Aspects for the Selection of Development Candidates 511
18.5.4 GMP Production of Spiegelmers 514
18.5.4.1 Starting Materials 514
18.5.4.2 Drug Substance 516
18.5.4.3 Drug Product 516
18.5.5 Analytical Methods for the Quality Control of Spiegelmers 517
18.6 Future Prospects for Spiegelmer Therapeutics 521
References 521
Index 527
Nicolay Ferrari, PhD, is the Executive Director of the Canadian Critical Care Trials Group, a Canadian investigator-lead research network, Quebec, Canada. A former Director of Research in Pharmacology at Topigen Pharmaceuticals, Inc, over twenty years of research experience, Dr. Ferrari is the co-inventor of six patents.
Rosanne Seguin, PhD, is an Academic Associate at the Montreal Neurological Institute of McGill University, Montreal, Quebec, Canada. A former Director of Immunology and Development Support at Topigen Pharmaceuticals, Inc. Dr. Seguin has 20 years of research experience.
Rosanne Seguin, PhD, is an Academic Associate at the Montreal Neurological Institute of McGill University, Montreal, Quebec, Canada. A former Director of Immunology and Development Support at Topigen Pharmaceuticals, Inc. Dr. Seguin has 20 years of research experience.
