Oligonucleotide-Based Drugs and Therapeutics (eBook, PDF)

Preclinical and Clinical Considerations for Development

• Format: PDF

Produktbeschreibung

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.

Produktdetails

• Verlag: John Wiley & Sons
• Erscheinungstermin: 6. Juni 2018
• Englisch
• ISBN-13: 9781119070290
• Artikelnr.: 53058833

Autorenporträt

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.

Inhaltsangabe

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 β‐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

---

List of Contributors xvii Preface xxi Acknowledgments xxii 1 Mechanisms of Oligonucleotide Actions 1 Annemieke AartsmäRus, 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 AartsmäRus 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