Advances in Nucleic Acid Therapeutics

Sudhir Agrawal; Michael J Gait

BOOK

Advances in Nucleic Acid Therapeutics

Sudhir Agrawal | Michael J Gait

(2019)

Additional Information

Book Details

ISBN: 978-1-78801-571-4
Edition: 1
Language: English
Pages: 566
Subjects: Organic chemistry
Genetics (non-medical)
Computational biology / bioinformatics
Biochemistry
Chemical biology

Abstract

The sequencing of the human genome and subsequent elucidation of the molecular pathways that are important in the pathology of disease have provided unprecedented opportunities for the development of new therapeutics. Nucleic acid-based drugs have emerged in recent years to yield extremely promising candidates for drug therapy to a wide range of diseases. Advances in Nucleic Acid Therapeutics is a comprehensive review of the latest advances in the field, covering the background of the development of nucleic acids for therapeutic purposes to the array of drug development approaches currently being pursued using antisense, RNAi, aptamer, immune modulatory and other synthetic oligonucleotides. Nucleic acid therapeutics is a field that has been continually innovating to meet the challenges of drug discovery and development; bringing contributions together from leaders at the forefront of progress, this book depicts the many approaches currently being pursued in both academia and industry. A go-to volume for medicinal chemists, Advances in Nucleic Acid Therapeutics provides a broad overview of techniques of contemporary interest in drug discovery.

Section Title	Page	Action	Price
Cover	Cover
Advances in Nucleic Acid Therapeutics	i
Foreword	vii
Preface	ix
Contents	xiii
Chapter 1 - History and Development of Nucleotide Analogues in Nucleic Acids Drugs	1
1.1 Introduction	1
1.2 The Antisense Concept	2
1.3 Developments in Oligonucleotide Synthesis	2
1.4 Choices for Antisense Oligodeoxynucleotide Modifications	3
1.4.1 Backbone Modifications	3
1.4.1.1 Phosphorothioates	4
1.4.1.2 Non-anionic Internucleotide Linkages	7
1.4.1.2.1\rPhosphorodiamidate Morpholino Oligonucleotodes.Phosphorodiamidate morpholino oligonucleotides (PMO) are charge-neutral oligonucl...	8
1.4.1.2.2\rPeptide Nucleic Acids.Peptide nucleic acids (PNA) are charge-neutral synthetic mimics of DNA or RNA containing N-2 aminoethylgly...	9
1.4.2 Heterocyclic Bases	9
1.4.3 Sugar Modifications	10
1.4.3.1 Oligoribonucleotides	10
1.4.3.2 2ʹ-O-Alkyl Sugars	10
1.4.3.3 Bridged Nucleic Acids	11
1.5 Gapmers Using Combinations of Modified Oligodeoxy and/or Oligoribonucleotides	12
1.6 Antisense Conjugates	13
1.7 The Role of Innate Immune Receptors in Nucleic Acid Therapeutics	14
1.8 Future Directions	15
Acknowledgements	16
References	17
Chapter 2 - Mechanisms of Antisense Oligonucleotides	22
2.1 Introduction	22
2.2 RNase H and ASO Action	23
2.3 ASOs and Regulation of Splicing	25
2.4 ASOs and Activation of Frataxin, a Case Study for an Emerging Mechanism	27
2.5 Summary	29
Acknowledgements	29
References	29
Chapter 3 - The Medicinal Chemistry of RNase H-activating Antisense Oligonucleotides	32
3.1 Introduction to Gapmers	32
3.2 Human RNase H1	33
3.2.1 Biochemistry of Human RNase H1	33
3.2.2 Structural Biology of Human RNase H1	34
3.3 Structure–Activity Relationships of Gap Modifications	36
3.3.1 Phosphorothioate (PS) DNA	38
3.3.2 Chiral PS DNA	39
3.3.3 Methyl Phosphonates, Phosphoramidates, Phosphotriesters and Boranophosphonate DNA	40
3.3.4 2ʹ-Fluoro Arabino Nucleic Acids (FANA)	40
3.3.5 DNA-like Modifications	41
3.4 SAR of Wing Modifications	41
3.4.1 2ʹ-O-Methoxyethyl RNA (MOE)	42
3.4.2 Locked Nucleic Acids (LNA) and Constrained Ethyl 2ʹ-4ʹ-Bridged Nucleic Acids (cEt)	42
3.4.3 α-l-LNA and Related Analogs	43
3.4.4 F-HNA and F-CeNA	43
3.4.5 Tricyclo DNA (tcDNA)	44
3.4.6 Phosphorodiamidate Linked Morpholinos (PMOs)	44
3.5 Design of Gapmer ASOs	45
3.5.1 MOE Gapmer ASOs	45
3.5.2 LNA Gapmers	46
3.5.3 cEt Gapmers	47
3.5.4 ASO Gapmer Duplexes	47
3.6 Control of Protein Binding	47
3.6.1 Interaction of Gapmer ASOs with Plasma Proteins	48
3.6.2 Interaction of Gapmer-ASOs with Cell-surface Proteins	48
3.6.3 Targeting Cell-surface Proteins for Cell-specific Delivery of Gapmers	49
3.6.4 Avoiding Interactions with TLR Receptors to Avoid Immune-stimulatory Toxicities	50
3.6.5 Optimizing Intracellular Distribution	51
3.7 Conclusions	51
References	52
Chapter 4 - Antisense Technology: Liver Targeting and Beyond for Drug Discovery	62
4.1 Introduction	62
4.2 Liver Targeting	64
4.2.1 The Beginning	64
4.2.2 Broadening the Liver-targeting Pipeline	66
4.3 Innovations in Liver Targeting	67
4.4 Beyond the Liver	70
4.5 Conclusions	72
References	73
Chapter 5 - Oligonucleotide-based Toll-like Receptor Antagonists and Therapeutic Applications	80
5.1 Introduction	80
5.2 Oligonucleotide-based TLR Antagonists	82
5.2.1 Structure–Activity Relationship Studies	83
5.2.2 TLR Antagonists	83
5.2.3 Inhibitory Activity of TLR Antagonists	85
5.2.4 Inhibitory Activity of Clinical Candidates	85
5.3 Studies of TLR Antagonists in Disease Models	87
5.3.1 Psoriasis	87
5.3.2 Systemic Lupus Erythematosus (SLE)	88
5.3.3 Rheumatoid Arthritis (RA)	89
5.3.4 Duchenne Muscular Dystrophy (DMD)	90
5.3.5 MyD88 L265P-positive B Cell Lymphoma (BCL)	90
5.3.6 Restenosis and Atherosclerosis	91
5.3.7 Inflammatory Bowel Disease (IBD)	92
5.3.8 HIV-1	92
5.4 Clinical Development of Lead TLR Antagonist Candidates	93
5.4.1 Moderate-to-severe Plaque Psoriasis	93
5.4.2 Waldenström's Macroglobulinemia	94
5.5 Conclusions	96
Acknowledgements	96
References	96
Chapter 6 - Splicing Modulation for Therapeutics	103
6.1 Introduction	103
6.1.1 RNA Splicing	103
6.1.1.1 Splicing Machinery and Sequence Motifs Required for RNA Splicing	104
6.1.1.2 Other Sequence Motifs that Influence RNA Splicing	104
6.1.2 Constitutive Splicing	106
6.1.3 Alternative Splicing	106
6.1.4 Cryptic Splicing	108
6.2 Therapeutic Exon Skipping Options	108
6.2.1 Antisense Oligonucleotides (ASOs) and Chemical Modifications	108
6.2.2 Restoring Cryptic Splicing	109
6.2.2.1 β-Thalassemia	109
6.2.2.2 Ataxia Telangiectasia	110
6.2.3 Reading Frame Restoration	111
6.2.3.1 Duchenne Muscular Dystrophy	111
6.2.3.2 Dystrophic Epidermolysis Bullosa	115
6.2.4 Exon Inclusion	116
6.2.4.1 Spinal Muscular Atrophy	116
6.2.4.2 Glycogen Storage Disease Type II or Pompe Disease	118
6.2.5 Generating Less Protein or Non-toxic Protein	118
6.2.5.1 Hutchinson–Gilford Progeria Syndrome	118
6.2.5.2 Huntington Disease	119
6.3 Future Perspectives, Towards Additional Approved Splice-modulating ASOs	120
6.3.1 The Challenge of Personalized Medicine Development	120
6.3.2 ASO Delivery	121
6.3.3 Future Perspective	121
References	122
Chapter 7 - Targeting Toxic Repeats	126
7.1 Introduction	126
7.2 Expanded Repetitive Sequences and Human Disease	127
7.2.1 Repeat Instability	127
7.2.2 Molecular Mechanisms of Disease	128
7.2.2.1 Loss-Of-Function	129
7.2.2.2 Gain-Of-Function	130
7.3 Why are Expanded Repeats so Special as Therapeutic Targets	130
7.3.1 Structures Formed by Expanded Repeats	131
7.3.1.1 R-Loop	131
7.3.1.2 Triple Helix	131
7.3.1.3 Imperfect Hairpin	131
7.3.1.4 Quadruplexes	133
7.3.2 Structures of Repeat-Expanded Transcripts	133
7.3.3 Structural Implications for Therapeutic Targeting	134
7.4 Therapeutics to Target Expanded Repeats	135
7.4.1 Therapeutic Strategies	135
7.4.2 Small Molecules Targeting Repeat Structures	136
7.4.3 Antisense Strategies	138
7.4.3.1 ASOs Directed Against DNA	138
7.4.3.2 ASOs Directed Against RNA	139
7.4.3.3 RNA Interference	140
7.4.3.4 Other Antisense Strategies	141
7.4.4 CRISPR/Cas9, TALEN, ZFN and Other Protein Effectors	141
7.5 Challenges for Multisystemic Repeat Diseases	142
Abbreviations	143
Acknowledgements	144
References	144
Chapter 8 - Research and Development of Oligonucleotides Targeting MicroRNAs (miRNAs)	151
8.1 Introduction: MicroRNA Biogenesis and Functions	151
8.2 miRNAs as Targets for Drugs	154
8.3 AntimiR Oligonucleotides as Drugs	154
8.3.1 The Development of AntimiR Medicinal Chemistry	154
8.3.1.1 2ʹ-O-Methyl-modified AntimiRs	155
8.3.1.2 Antagomirs	157
8.3.1.3 2ʹ-O-Methoxyethyl AntimiRs	158
8.3.1.4 2ʹ-Fluoro-modified AntimiRs	158
8.3.1.5 Locked Nucleic Acids and Constrained Ethyl Derivatives	159
8.3.1.6 Peptide Nucleic Acids	161
8.3.1.7 Morpholino Oligonucleotides	162
8.3.2 New Insights into Mechanisms of Oligonucleotide-based miRNA Targeting	162
8.3.2.1 AntimiR-mediated Tailing and Trimming of miRNAs	163
8.3.2.2 Targeting miRNAs in the RISC	163
8.3.2.3 AntimiR-mediated Unloading of miRNAs from RISC	164
8.3.3 New Chemistries and Alternative Approaches for Targeting miRNAs	165
8.4 AntimiRs in Clinical Trials	167
8.4.1 AntimiRs Targeting miR-122 for Treatment of HCV	167
8.4.2 AntimiR-targeting of Other miRNAs in Clinical Studies	169
8.5 Conclusions	170
Acknowledgement	171
References	171
Chapter 9 - Oligonucleotide Targeting of Long Non-coding RNAs	181
9.1 Introduction	181
9.2 History of lncRNAs	182
9.3 Biology and Functions of lncRNA	185
9.3.1 lncRNAs as Regulators of Transcription	186
9.3.2 lncRNA as Regulators of Post-transcriptional Processing	187
9.3.2.1 Splicing	188
9.3.2.2 Polyadenylation	188
9.3.2.3 mRNA Stability	188
9.3.2.4 Competing Endogenous RNAs	188
9.3.2.5 Interactions with miRs	189
9.3.2.6 Endogenous siRNA and miRNA Generation	189
9.3.3 lncRNA as Regulators of Translation	189
9.4 Classification of lncRNA	190
9.4.1 Functional Classification	191
9.4.2 Genomic Classification	191
9.5 Targeting of Long Non-coding RNA by Oligonucleotides	192
9.5.1 Antisense Oligonucleotides	192
9.5.2 siRNAs	192
9.5.3 CRISPR and Other Approaches	193
9.6 Therapeutic Applications	194
9.6.1 Neurology and Psychiatry	194
9.6.1.1 Dravet's Syndrome (DS)	194
9.6.1.2 Angelman's Syndrome (AS)	195
9.6.1.3 Spinal Muscular Atrophy (SMA)	195
9.6.1.4 Alzheimer's Disease (AD)	196
9.6.1.5 Fragile X Mental Retardation 1 (FMR1)	197
9.6.1.6 Psychiatric Diseases	197
9.6.2 Oncology	198
9.6.3 Cardiology	201
9.6.4 Gastroenterology	202
9.7 Perspectives	202
References	203
Chapter 10 - Conjugate-mediated Delivery of RNAi-based Therapeutics: Enhancing Pharmacokinetics–Pharmacodynamics Relationships of Medicinal Oligonucleotides	206
10.1 Introduction	206
10.2 Chemical Stabilization as a Prerequisite for Conjugate-mediated Delivery of siRNAs: Effects on Clearance, Distribution and S...	207
10.3 Modulating Biodistribution of Therapeutic Oligonucleotides Using Conjugated Modalities: Targeted versus Broad Functional Del...	213
10.3.1 Broad Functional Delivery of Conjugated siRNAs	213
10.3.2 Targeted Delivery of Conjugated siRNAs	216
10.4 Productive Delivery of Therapeutic Oligonucleotides: Overcoming the Endosomal Barrier	218
10.5 The Effects of the Route of Administration: Local versus Systemic Delivery	219
10.5.1 Local Delivery of Conjugated siRNAs	219
10.5.2 Systemic Delivery of Conjugated siRNAs	221
10.6 Enhancing PK Properties of Conjugated siRNAs: Reducing Clearance Kinetics and Accelerating Target Tissue Uptake	223
10.7 Conjugation Chemistry for RNAi-based Therapeutics: Future Perspectives	224
Acknowledgements	226
References	226
Chapter 11 - Liver-targeted RNAi Therapeutics: Principles and Applications	233
11.1 Introduction	233
11.2 The Role of Chemistry	234
11.3 Liver-specific Delivery of siRNA	237
11.3.1 Ionizable Lipid Nanoparticles (iLNPs)	238
11.3.2 Lipid-conjugated siRNA provided Proof of Concept for RNAi Therapeutics	239
11.3.3 Discovery of GalNAc Conjugates	240
11.4 Clinical Candidates	240
11.4.1 ONPATTROTM (Patisiran)	240
11.4.2 Inclisiran	254
11.4.3 Givosiran	255
11.4.4 Fitusiran	256
11.4.5 TTRsc02	256
11.4.6 Revusiran	256
11.5 Conclusions and Outlook	257
References	257
Chapter 12 - Advances and Challenges of RNAi-Based Anti-HIV Therapeutics	266
12.1 Introduction	266
12.2 Potential Targets for Anti-HIV-1 RNAi Therapeutics	268
12.2.1 Targeting the HIV-1 Viral Genome	268
12.2.2 Targeting Host Factors	270
12.3 Challenges in Obtaining Effective Anti-HIV-1 RNAi Activity	272
12.3.1 Key Barriers to In Vivo RNAi Efficacy	272
12.3.2 The Need for Rational Design and for Chemical Modifications	274
12.3.3 The Need for Combinatorial RNAi	275
12.4 Recent Progress and Clinical Development of Anti-HIV-1 RNAi	276
12.4.1 In Vivo Delivery of Anti-HIV-1 RNAi Effectors	276
12.4.2 Ex vivo Delivery of Anti-HIV-1 shRNAs	281
12.4.3 Clinical Development of Anti-HIV-1 RNAi	282
12.5 Conclusions and Perspective	283
Conflict of Interest Declaration	284
Acknowledgements	285
References	285
Chapter 13 - Nucleic Acid Innate Immune Receptors	292
13.1 Introduction	292
13.2 Toll-like Receptors	293
13.2.1 TLR3 Recognizes dsRNA	293
13.2.2 TLR7 and TLR8 Recognize ssRNA and Guanosine or Uridine	293
13.2.3 TLR9 Recognizes CpG-DNA	294
13.2.4 Chaperones Regulate the Maturation of NA-sensing TLRs	295
13.2.5 Unc93B1 Regulates the Balance of TLR7 and TLR9 Responses	296
13.2.6 Proteolytic Cleavage of NA-sensing TLRs is Essential for Their Function	296
13.2.7 Trafficking of TLR7 and TLR9 is Essential for Type I Interferon Production in pDCs	297
13.3 Nucleic Acids Sensing in the Cytoplasm	298
13.3.1 Cytosolic DNA Sensors Recognize dsDNA	298
13.3.2 RIG-I and MDA5 Recognize dsRNA and Activate MAVS to Induce Immune Responses	299
13.4 Conclusions	299
References	300
Chapter 14 - Synthetic Agonists of Toll-like Receptors and Therapeutic Applications	306
14.1 Introduction	306
14.1.1 RIG-I-like Receptors	307
14.1.2 AIM2-like Receptors (ALRs)	308
14.1.3 NOD-like Receptors, NLRP3 Inflammasome	308
14.1.4 Cyclic GMP–AMP Synthase (cGAS) and the STING Pathway	309
14.1.5 Toll-like Receptors (TLR)	309
14.2 Agonists of TLR3	310
14.2.1 Synthetic Agonists of TLR3	310
14.3 Agonists of TLR 7 and TLR 8	311
14.3.1 Synthetic Agonists of TLR7 and TLR8	312
14.3.2 Preclinical Studies of Agonists of TLR7 and TLR8	314
14.4 Agonists of TLR9	315
14.4.1 Synthetic Agonists of TLR9	316
14.4.1.1 Role of the 5ʹ-end	317
14.4.1.2 Synthetic Immune-stimulatory Motifs	319
14.4.1.3 Secondary Structures	320
14.5 Therapeutic Applications of Synthetic Agonists of TLR9	322
14.5.1 Preclinical Studies in Cancer	322
14.5.2 Treatment for Asthma and Allergies	324
14.5.3 Use as Vaccine Adjuvants	325
14.6 Clinical Development of Synthetic Agonists of TLR9	325
14.6.1 Clinical Trials in Hepatitis C Patients	325
14.6.2 Clinical Trials in Cancer	326
14.7 Conclusions	327
References	328
Chapter 15 - Prostate-specific Membrane Antigen (PSMA) Aptamers for Prostate Cancer Imaging and Therapy	339
15.1 Introduction	339
15.1.1 Aptamers and SELEX	339
15.1.2 DNA vs. RNA Aptamers	342
15.2 Prostate Specific Membrane Antigen (PSMA) Aptamers	343
15.2.1 PSMA	343
15.2.2 DNA and RNA PSMA Aptamers	345
15.2.2.1 PSMA RNA Aptamers	345
15.2.2.2 PSMA DNA Aptamers	347
15.3 PSMA Aptamers Applications	348
15.3.1 Imaging and Diagnostic Applications	348
15.3.1.1 Radio-imaging Applications	348
15.3.1.2 Computed Tomography (CT) Applications	349
15.3.1.3 Magnetic Resonance Imaging (MRI) Applications	349
15.3.1.4 Optical Imaging Applications	350
15.3.2 PSMA Aptamers as Therapeutic Inhibitors	351
15.3.3 Targeted Delivery Applications	351
15.3.3.1 PSMA Aptamer RNAi Conjugates	351
15.3.3.2 PSMA Aptamer–CRISPR Conjugates	355
15.3.3.3 Bispecific PSMA Aptamer Conjugates	356
15.3.3.4 PSMA Aptamer Protein Toxin Conjugates	356
15.3.3.5 PSMA Aptamer Small-molecule Conjugates	357
15.3.3.6 PSMA Aptamer Functionalized Nanoparticles	357
15.4 Conclusions	359
15.5 Future Perspectives	359
Acknowledgements	361
References	361
Chapter 16 - Aptamers and Clinical Applications	367
16.1 Introduction	367
16.2 Recent Preclinical Studies of Aptamer Drugs	369
16.2.1 Aptamer Structures	369
16.2.1.1 SOMAmers and X-Aptamers	369
16.2.1.2 Thioaptamers	373
16.2.1.3 Photo-regulated Aptamers	374
16.2.2 Non-ocular Diseases	376
16.2.2.1 Cardiovascular and Cerebrovascular Diseases	376
16.2.2.2 Alzheimer's Disease	377
16.2.2.3 Antiviral Applications	378
16.2.2.4 Antibacterial Applications	380
16.2.2.5 Antiparasitic Applications	381
16.3 Recent Studies of Aptamer-based Targeting of Drugs	381
16.4 Clinical Studies of Aptamer Drugs Registered in ClinicalTrials.gov	384
16.4.1 Completed Clinical Studies	385
16.4.2 Terminated or Withdrawn Studies	390
16.4.3 Active Studies	391
16.5 Conclusions and Prospects	392
References	394
Chapter 17 - CRISPR-based Technologies for Genome Engineering: Properties, Current Improvements and Applications in Medicine	400
17.1 Introduction	400
17.2 Sequence-specific CRISPR Nucleases and Improved Variants	401
17.2.1 Cas 9 from Streptococcus pyogenes, Orthologues and Variants	401
17.2.1.1 CRISPR Systems from the Bacterial Immune Response	401
17.2.1.2 Variation Around the SpCas9–sgRNA Complex to Improve Activity	403
17.2.1.3 Expanding the Target Repertoire	404
17.2.2 Expanding Targeted Functions with “CRISPR Fusions”	404
17.2.2.1 Catalytically Dead CRISPR Systems	404
17.2.2.2 Multiplexing Strategies	405
17.2.3 Specificity of CRISPR Systems	405
17.2.3.1 Approaches to Determine Specificity	406
17.2.3.2 Computational Tools for Guide RNA Selection	406
17.2.3.3 Approaches to Improve Specificity	407
17.3 Genome Editing Mechanisms and Current Improvements	408
17.3.1 Principles: Genome Editing Takes Place During Repair of DSB Breaks Induced by Cas9	408
17.3.1.1 End-joining Pathways	409
17.3.1.2 Homology-directed Repair	409
17.3.1.3 Regulation of DNA Repair Pathways During the Cell Cycle	410
17.3.2 Improvement of HDR-based CRISPR Strategies for Programmable Genome Modification	410
17.3.2.1 Strategies to Repress the NHEJ-mediated Repair	410
17.3.2.2 Strategies to Enhance HDR	412
17.3.2.2.1\rMain Pathways in HDR-mediated DSB Repair.In HDR-mediated repair, the DSB ends are resected to expose 3ʹ ssDNA tails. Resection s...	412
17.3.2.2.2\rDNA Homology Template Donors for HDR-mediated Genome Editing.In HDR-mediated genome editing, cells accept different types of DNA...	412
17.3.2.2.3\rFusion of Cas9 to HDR Partners.Several approaches to enhance HDR include the fusion to Cas9 of another protein thereby combined ...	413
17.3.2.2.4\rModifications of the Cell Cycle During Gene Editing and Other Approaches.Orthwein et al. showed that the replacement of partner ...	413
17.4 Epigenome Editing and Base Editing with the CRISPR Systems	414
17.4.1 Modulation of Transcription, CRISPRa and CRISPRi Systems	414
17.4.2 Modulation of Chromatin Status	416
17.4.3 Base Editors	417
17.5 Applications in Medicine and Challenges	419
17.5.1 Applications in Biomedical Research	419
17.5.2 The Delivery Challenge	420
17.5.3 The Genome Editing Precision Challenge	422
17.5.4 Clinical Trials Based on Genome Editing	422
17.6 Conclusions	423
References	424
Chapter 18 - Therapeutic Potential of Ribozymes	434
18.1 Introduction	434
18.2 Trans-cleaving Ribozymes	435
18.3 Ribozyme-mediated Genetic Repair	439
18.3.1 Spliceosome-mediated RNA Trans-splicing (SMaRT)	440
18.3.2 Group I Intron Ribozyme-mediated Trans-splicing	441
18.3.3 Twin Ribozyme-mediated RNA Repair	443
18.3.4 Correction of Genetic Disorders by Retro-homing Group II Introns	445
18.4 Conclusions	445
References	447
Chapter 19 - Large-scale Automated Synthesis of Therapeutic Oligonucleotides: A Status Update	453
19.1 Introduction	453
19.2 Chemical Modifications in Clinical and Commercial Products	454
19.2.1 First-generation Backbone Modifications – the Phosphorothioate (PS) Internucleotide Linkage	454
19.2.2 Second-generation Sugar Modifications	455
19.3 The Oligonucleotide Manufacturing Process	457
19.3.1 Use of an Automated Synthesizer	457
19.3.2 Starting Materials	458
19.3.2.1 Nucleoside-loaded Solid Support	458
19.3.2.2 Nucleoside Phosphoramidites	460
19.3.2.3 Sulfur-transfer Reagents	462
19.3.3 Reagent-related Impurities	462
19.3.4 The Four-step Synthesis Cycle	464
19.3.5 Cleavage and Deprotection	465
19.3.6 The Purification Process	466
19.3.7 The Lyophilization Process	468
19.4 Analytical Protocols	468
19.5 Synthesis Yield and Product Purity	470
19.6 Conclusions and Future Outlook	470
References	471
Chapter 20 - Preclinical and Clinical Drug-metabolism, Pharmacokinetics and Safety of Therapeutic Oligonucleotides	474
20.1 Introduction	474
20.2 Oligonucleotide Chemistries and Mode of Action (MOA)	475
20.2.1 Chemistry and Design Considerations of Therapeutic ONDs	475
20.2.1.1 OND Chemistry	476
20.2.1.2 OND Design	476
20.2.2 Delivery Approaches	478
20.3 Distribution, Metabolism and Pharmacokinetics (DMPK)	479
20.3.1 DMPK Properties of ONDs	479
20.3.1.1 Plasma and Tissue Pharmacokinetics	479
Anti-drug Antibodies.Increasing experience with longer-term treatment duration of PS gapmer and non-gapmer ASOs in animal studie...	481
20.3.1.2 Tissue Distribution	481
20.3.1.3 Productive Uptake	482
20.3.1.4 Metabolism	484
20.3.1.5 Excretion	485
20.3.2 Delivery Strategies	485
20.3.2.1 Local Delivery	486
20.3.2.2 Advanced Formulation Approaches	486
20.3.2.3 Targeted Delivery	487
20.4 Class Profile of Toxicity	488
20.5 Hybridization-dependent Toxicities	490
20.5.1 On-target Safety and Exaggerated Pharmacology	490
20.5.2 Off-target Pharmacology	491
20.6 Hybridization-independent Toxicities	493
20.6.1 Effects Related to Transient Protein Binding	494
20.6.1.1 Inhibition of the Coagulation Cascade	494
20.6.1.2 Complement System Activation	494
20.6.2 Immune-mediated Effects	496
20.6.2.1 Pro-inflammatory Effects	496
20.6.2.1.1\rManifestations of Immune Stimulation.The manifestations of immune stimulation by DNA- and RNA-based ONDs have been described in ...	496
20.6.2.1.2\rPro-inflammatory Mechanisms.The pro-inflammatory properties of non-formulated ONDs stem from the activation of the innate immune...	498
20.6.2.1.3\rMitigating Proinflammatory Effects.Chemical modifications can modify the immune-stimulatory potential of ONDs of a given sequenc...	499
20.6.2.1.4\rImmunogenicity – Anti-drug Antibodies (ADAs).PS backbone ONDs were long considered to be devoid of antigenic properties based on...	500
20.6.2.2 Thrombocytopenia	500
20.6.3 Toxicity in High-exposure Organs	505
20.6.3.1 Liver Toxicity	505
20.6.3.1.1\rGalNAc-conjugation.At a given dose, GalNAc conjugation significantly increases hepatocyte uptake of both single-stranded and dou...	506
20.6.3.1.2\rToxic Sequence Motifs.Important efforts have been made to better understand the sequence component and define particular sequenc...	507
20.6.3.1.3\rMechanistic Hypotheses.The understanding of the mechanism involved in the high-affinity gapmer hepatotoxicity has greatly evolve...	507
20.6.3.2 Kidney Toxicity	509
20.6.3.2.1\rTubular Effects.The morphological and functional tubular changes in animal studies follow a predictable course, whereby initial ...	509
20.6.3.2.2\rGlomerular Effects.Glomerular lesions are not commonly observed with PS ONDs and 2ʹMOE gapmers,234 but glomerulopathies were a r...	511
20.7 Getting to the Clinic	511
20.7.1 Regulatory Considerations for Preclinical Development	511
20.7.2 Precision Medicine and Opportunity for Accelerated Timelines	515
20.8 Conclusions	515
Acknowledgements	516
References	517
Subject Index	532

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