Cover
Preface
1. Acknowledgments
1 Mechanisms of Oligonucleotide Actions
1. 1.1 Introduction
2. 1.2 Antisense Oligonucleotide Therapeutics
3. 1.3 Oligonucleotides that Sterically Block Translation
4. 1.4 Oligonucleotides that Act Through the RNAi Pathway
5. 1.5 Chemical Modification of siRNAs and miRNAs
6. 1.6 Clinical Use of Oligonucleotides that Act through the RNAi Pathway
7. 1.7 Oligonucleotides that Modulate Splicing
8. 1.8 Conclusions
9. References
2 The Medicinal Chemistry of Antisense Oligonucleotides
1. 2.1 Introduction: The Antisense Approach and the Need for Chemical Modification
2. 2.2 Why Chemically Modify an Oligonucleotide?
3. 2.3 Chemical Modifications of Current Importance by Structural Class
4. 2.4 Conclusion
5. References
3 Cellular Pharmacology of Antisense Oligonucleotides
1. 3.1 Introduction
2. 3.2 Molecular Mechanisms of Antisense Oligonucleotides
3. 3.3 Cellular Pharmacology of Antisense Oligonucleotides
4. 3.4 Conclusion
5. References
4 Pharmacokinetics and Pharmacodynamics of Antisense Oligonucleotides
1. 4.1 Introduction
2. 4.2 Pharmacokinetic Properties of Antisense Oligonucleotides
3. 4.3 Pharmacodynamic Properties of Antisense Oligonucleotides
4. 4.4 PD and PK Results and Strategies of ASOs in Clinical Development
5. 4.5 Summary and Conclusions
6. References
5 Tissue Distribution, Metabolism, and Clearance
1. 5.1 Introduction
2. 5.2 Tissue Distribution
3. 5.3 Cellular Uptake
4. 5.4 Metabolism and Clearance
5. 5.5 Conclusion
6. References
6 Hybridization‐Independent Effects
1. 6.1 Background
2. 6.2 Mechanisms of Hybridization‐independent Toxicities
3. 6.3 Hybridization‐independent Effects Following Local Delivery of Oligonucleotides
4. 6.4 Conclusion
5. References
7 Hybridization‐Dependent Effects
1. 7.1 Introduction
2. 7.2 Specificity Studies with ASOs
3. 7.3 Implications of the Nuclear Site of Action of RNase H1
4. 7.4 Mechanism of OTE
5. 7.5 Determining the Extent that Accessibility, Affinity and, Mismatch Tolerance Contribute to Off‐target Activity
6. 7.6 Consequences of Unintended Transcript Knockdown: In Vivo and In Vitro Toxicity
7. 7.7 Identification and Evaluation of Putative OTEs
8. 7.8 Summary
9. Acknowledgments
10. References
8 Class‐Related Proinflammatory Effects
1. 8.1 Introduction
2. 8.2 Proinflammatory Effects of ASO for Consideration in Drug Development
3. 8.3 Conclusions
4. References
9 Exaggerated Pharmacology
1. 9.1 Introduction
2. 9.2 Regulatory Expectations
3. 9.3 Scope of EP Assessment
4. 9.4 EP Evaluation Strategies
5. 9.5 Conclusions
6. References
10 Genotoxicity Tests for Novel Oligonucleotide‐Based Therapeutics
1. 10.1 Introduction
2. 10.2 Experience with ONs in the Standard Battery
3. 10.3 OSWG Recommendation for Genotoxicity Testing of ONs
4. 10.4 Triplex Formation
5. 10.5 Impurities
6. 10.6 Conclusions
7. Acknowledgments
8. References
11 Reproductive and Developmental Toxicity Testing Strategies for Oligonucleotide‐Based Therapeutics
1. 11.1 Introduction
2. 11.2 General Design of Reproductive and Developmental Toxicity Studies
3. 11.3 Product Attributes of Oligonucleotide Drugs
4. 11.4 The Role of Intended Pharmacology in Reproductive and Developmental Effects
5. 11.5 Selection of Animal Species
6. 11.6 Justification of Dosing Regimen
7. 11.7 Exposure Assessment
8. 11.8 Subclass‐specific Considerations
9. 11.9 Conclusions
10. Acknowledgments
11. References
12 Specific Considerations for Preclinical Development of Inhaled Oligonucleotides
1. 12.1 Background
2. 12.2 Oligonucleotide Delivery Systems
3. 12.3 Repeat‐dose Toxicity
4. 12.4 Toxicokinetics
5. 12.5 Safety Pharmacology
6. 12.6 Additional Testing
7. 12.7 Conclusion
8. References
13 Lessons Learned in Oncology Programs
1. 13.1 Introduction
2. 13.2 Clinical Development of First‐generation ASOs
3. 13.3 Clinical Development of Second‐generation ASOs
4. 13.4 Regulatory Considerations
5. 13.5 Future Opportunities for ASOs as Therapeutic Agents for Cancer Treatment
6. References
14 Inhaled Antisense for Treatment of Respiratory Disease
1. 14.1 Introduction
2. 14.2 Atopic Asthma
3. 14.3 Antisense Oligonucleotides in Animal Models
4. 14.4 Clinical Data
5. 14.5 General Conclusion
6. References
15 Antisense Oligonucleotides for Treatment of Neurological Diseases
1. 15.1 Introduction
2. 15.2 Potential ASO Therapies in Neurodegenerative Diseases
3. 15.3 Conclusion
4. References
16 Nucleic Acids as Adjuvants
1. 16.1 Introduction
2. 16.2 Categories of Nucleic Acid Adjuvants
3. 16.3 Conclusion
4. Acknowledgments
5. References
17 Splice‐Switching Oligonucleotides
1. 17.1 Introduction of Splice Switching
2. 17.2 Preclinical and Clinical Development of Splice‐switching Oligos
3. 17.3 Future Directions
4. Conflict of Interest
5. Acknowledgments
6. References
18 CMC Aspects for the Clinical Development of Spiegelmers
1. 18.1 Introduction
2. 18.2 Technology (Mirror‐imaged SELEX Process) Selected Pharmaceutical Properties
3. 18.3 Preclinical Efficacy Data for Spiegelmers
4. 18.4 Clinical Development
5. 18.5 CMC Aspects for the Development of Spiegelmers
6. 18.6 Future Prospects for Spiegelmer Therapeutics
7. References
Index
End User License Agreement

List of Tables

Chapter 01
1. Table 1.1 Therapeutics in clinical development that exploit the RISC mechanism.
2. Table 1.2 Oligonucleotides currently in clinical development that modulate splicing.
Chapter 04
1. Table 4.1 Overview of bioanalytical methods for oligonucleotide quantification in biological matrices such as plasma and tissue.
2. Table 4.2 ASOs recently under active development in phase II clinical trials or above demonstrating target alternations in patients.
Chapter 06
1. Table 6.1 List of antisense oligonucleotide sequences with mixed phosphorothioate/phosphodiester linkages.
Chapter 10
1. Table 10.1 Classes of ONs and their structures and mechanisms.
2. Table 10.2 Summary of oligonucleotide genotoxicity testing database.
3. Table 10.3 Genotoxicity testing recommendations for ONs.
Chapter 11
1. Table 11.1 Comparison of product attributes across product classes.
Chapter 12
1. Table 12.1 Summary of changes in bronchoalveolar lavage and lung tissue following intratracheal aerosol instillation of antisense oligonucleotides.
2. Table 12.2 Summary of delayed histopathologic changes in the liver and kidneys in recovery mice and monkeys following daily administration via inhalation exposure for 14 days.
3. Table 12.3 Summary of delayed histopathologic changes in the liver and kidneys in recovery mice treated with a phosphodiester 2F′‐ANA antisense oligonucleotide (s.c.).
4. Table 12.4 Summary of delayed histopathologic changes in the liver and kidneys in recovery rats treated with a phosphorothioate 2F′‐ANA gapmer antisense oligonucleotide (s.c.).
Chapter 13
1. Table 13.1 ASO products currently in clinical development for oncology indications.
2. Table 13.2 Clinical development of custirsen (OGX‐011): Phase 2 and phase 3 studies with >40 patients.
Chapter 14
1. Table 14.1 Antisense therapeutics and their specific targets in animal models of asthma and the effects on asthma outcomes.
Chapter 16
1. Table 16.1 Human experience with CpG ODN as vaccine adjuvants.
2. Table16.2 Animal experience with CpG ODN as adjuvants.
Chapter 17
1. Table17.1 List of disorders and related splice‐switching oligonucleotides discussed in the chapter.
Chapter 18
1. Table 18.1 Summary of preclinical efficacy data for various Spiegelmers.
2. Table 18.2 Clinical studies conducted with Spiegelmers.
3. Table 18.3 In‐process controls for the manufacturing of Spiegelmer drug substance.
4. Table 18.4 Analytical test methods for the quality control of Spiegelmer drug substance and drug product.

List of Illustrations

Chapter 02
1. Figure 2.1 Some oligonucleotides based on 2′‐modified sugars.
2. Figure 2.2 Oligonucleotides based on constrained sugars.
3. Figure 2.3 Oligonucleotides based on sugars with expanded ring size.
4. Figure 2.4 The R _p and S _p stereoisomers of the phosphorothioate linkage.
5. Figure 2.5 Charged mimics of the internucleotide phosphate linkage.
6. Figure 2.6 Neutral mimics of the phosphate linkage based on amides (top), triazoles (bottom), and cyclic phosphotriesters (right).
7. Figure 2.7 Terminal phosphonates used as phosphatase‐resistant 5′‐phosphate analogues.
8. Figure 2.8 Phosphorodiamidate morpholino oligomers and a recent derivative termed PMO plus.
9. Figure 2.9 Peptide nucleic acid and recent derivatives. The stereochemistry of the α‐modified analogues developed by Ugichem is not disclosed [226], but older literature [227] suggests that the oligomer is likely to bind with higher affinity when the stereocenters marked * are configured as drawn. Note that for both α and γ modification of PNA, favorable properties are obtained with partial modification – there is no need to modify every unit of the PNA backbone.
10. Figure 2.10 Simple nucleobase modifications include S‐for‐O substitutions and a large family of C5‐modified pyrimidines.
11. Figure 2.11 Nucleobase analogues with extended hydrogen bonding.
Chapter 03
1. Figure 3.1 Oligonucleotide mechanisms of action. Four mechanisms are illustrated. Starting from the top: (i) classical ASO‐mediated mRNA degradation via ribonuclease H; (ii) siRNA‐mediated mRNA degradation via the Ago 2/RISC complex; (iii) removal of a mutant extron using splice switching oligonucleotides; and (iv) miRNA's modulation of mRNA function.
2. Figure 3.2 Pathways of endocytosis and intracellular trafficking. There are multiple pathways for cellular entry of macromolecules including oligonucleotides. In all cases the initial step of endocytosis involves initial binding to the plasma membrane and engulfment of cargo into intracellular vesicles. The second step often involves sorting of the cargo through endosomes, which is followed by the final stage during which the cargo is delivered to its final destination or recycled to extracellular milieu.
Chapter 06
1. Figure 6.1 Morphological changes in macrophages following ASO exposure in vitro. Representative light micrographs (63×) of RAW 264.7 cells treated with vehicle (a) or a phosphorothioate ASO at 5 mM (b), 15 mM (c), or 30 mM (d) for 48 h. Incubation of cells with ASO resulted in minimal to mild vacuolation at the highest ASO concentration.
2. Figure 6.2 Comparison of target mRNA inhibition between selected mixed phosphorothioate/phosphodiester ASO sequences with (referred to as POX‐DAP) or without (referred to as POX) substitution of adenosines with 2‐amino‐2′‐deoxyadenosine (DAP). FIp‐In T‐REx 293 cells remained either untransfected (Ctrl NT) or transfected for 24 h with 236 nM of each ASO or a sense control sequence (sense). ASO sequences are listed in Table 6.1. The mRNA expression (mean±SEM) of PDE7A was quantified using the QuantiGene 2.0 assay with normalization to the expression levels of β2M.
3. Figure 6.3 Comparison between a selected mixed phosphorothioate/phosphodiester ASO sequence with DAP (referred to as PO7‐DAP) and (a) the same ASO sequence as a phosphorothioate 2′F‐ANA gapmer (PS‐FANA) (ASO concentrations up to 1250 nM), and (b) a phosphorothioate LNA gapmer (PS‐LNA) (ASO concentrations up to 713 nM). ASO sequences are listed in Table 6.1. Flp‐In T‐REx 293 cells were transfected for 24 h. The mRNA expression (mean±SEM) of PDE7A was quantified using the QuantiGene 2.0 assay with normalization to the expression levels of β2M.
4. Figure 6.4 Comparison between a selected mixed phosphorothioate/phosphodiester ASO sequence (referred to as PO7‐DAP) with the same sequence fully phosphorothioated (referred to as PS‐DNA) on the immune response in vitro. (a) Mouse macrophage cells (RAW 264.7) or (b) human PBMC was exposed to different concentrations of ASO or a known immunostimulatory sequence CpG as positive control and levels of TNF‐α and IFN‐α in culture supernatants measured 24 h later (n = 3–5). ASO sequences are listed in Table 6.1.
5. Figure 6.5 Comparison between a selected mixed phosphorothioate/phosphodiester ASO sequence (referred to as PO7‐DAP) with the same sequence fully phosphorothioated (referred to as PS‐DNA) on the inflammatory response in the lung of mice following repeated dosing. Mice (n = 4 per group) received 10 doses (2.5 mg/kg) of the ASO over a 2‐week period (intratracheal aerosol dosing using a MicroSprayer from PennCentury) and inflammatory response assessed by (a) lung tissue histopathology using a severity grade scoring (grade 1 = minimal; grade 2 = mild; grade 3 = moderate to severe) and in (b) bronchoalveolar lavage (total cells and neutrophils are shown). ASO sequences are listed in Table 6.1.
Chapter 07
1. Figure 7.1 A summary of unintended interactions predicted for six BACH1‐targeting ASOs. As a part of the design process, ASOs with potential interactions with mature mRNA at the perfect or near‐perfect level were screened out. “PM” stands for perfect match, “1MM” for one mismatch, and “2MM” for two mismatches.
2. Figure 7.2 Graphical representation of ASO gapmer‐mediated on‐ and off‐target events. The ASO can bind to accessible regions within the intended target or off‐target transcript and recruit RNase H1. Some regions of the transcripts are inaccessible to the ASO due to secondary or tertiary RNA structure or because they are masked by RNA‐binding proteins.
3. Figure 7.3 List of ASO‐, transcript‐, and RNase H1‐based features that affect the potency of off‐target knockdown.
4. Figure 7.4 Comparison between the change of binding affinity (T_m) and activity for nine off‐target hits. Top: Off‐target activity measured 48 h following unassisted transfection of human NHBE cells with ASO GSK2910546A. Below: T_m measured for nine off‐target RNA oligos duplexed with 16‐mer ASO GSK2910546A. Change in T_m from the perfect matched duplex GSK2910546A/Bach1 RNA oligo is shown. Each off‐target had two mismatches in the following positions: FBXW11 and ZNF674 1+16, RIC8B 13+14, SPATS2L 1+15, TBCK 13+16, PRICKLE1 12+16, RBMS3 2+16, NRF1 9+16, and VPS13C 12+14. Correlation between activity and change in T_m Pearson r = 0.84, P‐value = 0.0025.
5. Figure 7.5 The effect of mismatches in different positions in a 16‐mer ASO on affinity and activity. Two series of ASOs (GSK2910546 and GSK2910613) with a single mismatch in each position were used to determine the effect of mismatch position of activity and affinity. Top – Activity: Bach1 mRNA knockdown was measured 24 h following Lipofectamine 2000 transfection of 10 nM ASOs in primary NHBE cells. Mismatch position is indicated with number from 1 to 16 with 1 being the first monomer from the 5′ end, and 0 indicates the perfectly matched ASO. The position of LNA residues is not shown due to a confidentiality agreement. Below – Affinity: An RNA oligo of the appropriate Bach1 target sequences was annealed with the mismatch ASO and the T_m values of the duplexes were measured using the assay described in Ref. [5]. Graph is change in T_m from the perfectly matched duplex value.
6. Figure 7.6 Apoptosis induced by an ASO in vitro is RNase H1 dependent and can be ablated by changing the LNA content and spiking pattern. (a) A549 cells were transfected with 0.01–50 nM of three different BACH1 targeting or control ASOs using Lipofectamine 2000. Forty‐eight hours after transfection, apoptosis was measured using Promega ApoTox‐Glo assay. Decline in signal at higher concentrations of ASO BACH1 C is due to cell loss at this time point. (b) A549 cells were transfected with 20 nM control siRNA or siRNA‐targeting RNase H1 for 24 h, followed by transfection of ASO BACH1 C for 24 h. Apoptosis was measured using Promega ApoTox‐Glo assay. (c) Variants of BACH1 C with modified LNA content and position: LNA residues are indicated in red, and “*” represents PS residue. The LNA spiking pattern cannot be shown for “BACH1 C” due to a confidentiality agreement. (d) BACH1 target mRNA knockdown following Lipofectamine 2000 transfection of A549 cells with 5 nM of ASO. (e) Induction of apoptosis following Lipofectamine 2000 transfection of A549 cells with 5 nM of ASO. Apoptosis was measured using Promega Apo‐One Assay.
7. Figure 7.7 In vivo toxicity can be reproduced in cell culture and is RNase H1 dependent. (a) Mouse Hepa1‐6 cells were transfected with different doses of NTS‐1, TS‐2 & HTS‐3 [32] using Lipofectamine2000. Twenty‐four hours after transfection, apoptosis was measured using Promega ApoTox‐Glo assay. (b) Apoptosis induced by HTS‐3 is partially mitigated by pretreatment with 25 nM siRNA designed against murine RNase H1. Cells were transfected with siRNA for 24 h, and then transfected with ASO for 24 h. (c) Myo1E knockdown is RNase H1 dependent. Murine Hepa1–6 cells were transfected with 25 nM control or mouse RNase H1‐specific siRNA for 24 h followed by transfection with 100 nM of TS‐2 ASO for 24 h. Zfp191 was previously reported as exon off‐target [32].
8. Figure 7.8 Graphical summary of computational and experimental steps used in ASO gapmer specificity evaluations. While RNA structure prediction and thermodynamic calculations are valuable and can bring substantial improvements to the prediction process in the future, they currently lack parameters and features that are necessary for comprehensive assessment of modified ASO‐mediated interaction likelihood.
Chapter 11
1. Figure 11.1 Overview of reproduction toxicity evaluations described in ICH guideline S5(R2): Detection of Toxicity to Reproduction for Medicinal Products and Toxicity to Male Fertility. A–F denote the stages in the complete reproductive life cycle from conception in one generation through conception in the following generation, as described in the guideline. The study types shown are standard study types described in the guideline as “the most probable option” for ensuring that all phases of the life cycle are evaluated and include studies for fertility and early embryonic development, embryo–fetal development, and pre‐ and postnatal development, including maternal function. Species listed are the typical species for each study type. NHP is shown in parentheses as one of several acceptable alternative species.
Chapter 13
1. Figure 13.1 Randomized phase 2 study in mCRPC: Survival results for custirsen/docetaxel/prednisone (investigational, dotted line) versus docetaxel/prednisone (control, solid line). Adapted from Chi et al. [40].
Chapter 14
1. Figure 14.1 Parallel group design to study the effects of 1018 on allergen‐induced responses. 1018 ISS or placebo was administered on days 1, 8, 15, and 22, and allergen challenges were conducted on days 9 and 23.
2. Figure 14.2 Parallel group design to study the effects of AIR645 on allergen‐induced responses. AIR645 or placebo was administered on days 1, 3, 5, 8, 15, and 22. Allergen challenge was conducted on day 24.
3. Figure 14.3 Crossover design to study the effects of TPI ASM8 on allergen‐induced responses. TPI ASM8 or placebo was administered on days 1–4. Allergen challenge was conducted on day 3.
4. Figure 14.4 Dose‐escalating design to study the effects of TPI ASM8 on allergen‐induced responses.
Chapter 17
1. Figure 17.1 Alternative splicing. Exon exclusion (upper panel) can be regulated by splicing repressors proteins (such as heterogeneous nuclear ribonucleoproteins [hnRNPs]) that bind to silencing motifs (black boxes) in the exons (exonic splicing silencers [ESS]) or in the introns (intron splicing silencers [ISS]). Splicing activator proteins (such as serine–arginine [SR]‐rich domain proteins) can influence the recognition and the splice of the exons by binding exonic or intronic splicing enhancers (ESE or ISE, respectively) (grey boxes), allowing the inclusion of the exon in the transcript (exon inclusion, lower panel). Mutations in these repressor or activator motifs, variation in levels of activating or repressing factors between tissues, or developmental stages can influence exon exclusion or inclusion. Exon inclusion can also be modulated by targeting the silencing or enhancing motifs with antisense oligonucleotides (AONs) to induce exon inclusion or exclusion, respectively.
2. Figure 17.2 Antisense‐mediated splicing modulation. (a) Correct cryptic splicing. A mutation (mut, a T>C transition at position 705 in intron 2) in the β‐globin gene generates a new donor splice site and activates an acceptor cryptic splice site (c.s.s). Rather than splicing out intron 2, part of the intronic sequence (the cryptic exon [dashed grey box]) is restored in the mRNA (route −AON). Antisense oligonucleotides (AONs) targeting the cryptic exon restore normal splicing (route +AON). (b) Isoform switching. AONs can modulate alternative splicing by binding to the donor (start of intron) or the acceptor (end of intron) splice site of an alternatively spliced exon (dashed box), resulting in primarily transcripts where the exon is excluded. This mechanism switches from one isoform (route −AON) to another one (+AON). (c) Induce exon inclusion. (i) In the SMN2 gene, exon 7 is generally skipped (route –AON) and is due to a transition (C to T) that has changed an ESE site into an ESS site ( light and dark grey boxes). Exon exclusion is further enhanced by an ISS present in intron 7. AON targeting ISS (black box) or the acceptor splice site of intron 7 can enhance the inclusion of exon 7 (route +AON). (ii) The use of bifunctional AONs, targeting the ESS (mutated T allele) and carrying a tail mimicking an ESE (ESE‐AON, upper part of figure) or 10 RS repeats (RS10‐AON, bottom part) can recruit splicing factors and correction of the spliced transcript. Regulatory sequences in exon 7 (ESS and ESE) and introns 6 and 7 (ISS and ISE [dark gray box]) can silence or enhance the exclusion or inclusion of an exon, respectively. (d) Reading frame correction. (i) In this example, a deletion of exon 50 (dashed exon) in the DMD gene disrupts the open reading frame, resulting in a premature stop codon in exon 51 (route –AON). AON can be used to hide exon 51 from the splicing machinery, leading to the skip of the exon and restoration of the reading frame allowing the production of a shorter, partially functional protein. (ii) Double exon skipping with a combination of two AONs (route +AONs ex45 and 51), targeting exons 45 and 51, restores the DMD reading frame in patient cell line carrying a 46–50 exon deletion (dashed exons). The use of single AON (route +AON ex45, route +AON ex51) provides out‐of‐frame transcripts. (e) Knockdown. An opposite scenario results in downregulation of a protein. Using AON targeting exon 2, one of the three exons of myostatin gene (MSTN), results in exon 2 exclusion and a transcript from which no functional protein can be produced (route +AON).
3. Figure 17.3 Antisense oligonucleotide backbone structures of the most in use compounds (2OMe‐RNA, 2F‐RNA, 2OMOE‐RNA, PNA, LNA, ENA, PMO, vivo‐PMO, and phosphorothioate (PS)‐RNA).
Chapter 18
1. Figure 18.1 Illustration of the mirror‐imaged SELEX process using the selection against the chemokine MCP‐1 as example. Using a randomly synthesized RNA library of 10¹⁵ different sequences, or three‐dimensional shapes, the selection is conducted against the chemically synthesized enantiomer of the natural target, i.e. D‐MCP‐1. Binding sequences are isolated and amplified for a new round of selection. After several rounds of selection during which the stringency is gradually increased, the sequences of the binding aptamers are identified and synthesized as L‐enantiomers or Spiegelmers. The Spiegelmer sequences are optimized and characterized for their targeted therapeutic application by truncation and point mutations. At the end of the optimization process, the MCP‐1 binding Spiegelmer NOX‐E36 is identified.
2. Figure 18.2 Manufacturing process flow diagram for Spiegelmers using NOX‐E36 as example.
3. Figure 18.3 Sequences and AX‐HPLC chromatograms of the two hepcidin‐binding Spiegelmers NOX‐H91 (top chromatogram) and NOX‐H94 (bottom chromatogram).
4. Figure 18.4 μDSC scans of 0.2 mM NOX‐A12 in 200 mM glucose. Top row left: first scan with Tm 27.1°C, calorimetric enthalpy ΔH 27.4 kcal/mol; top row right: second scan with Tm 27.0°C, ΔH 27.3 kcal/mol, and in 20 mM sodium phosphate, 180 mM glucose, 1 mM EDTA; bottom row left: first scan with Tm 32.6 and 43.8°C, ΔH 120.6 kcal/mol; bottom row right: second scan with Tm 25.5, 32.2, and 43.9°C, ΔH 71.8 kcal/mol).
5. Figure 18.5 Synthesis scheme for N‐benzoyl‐2′‐tert‐butyldimethylsilyl‐5′‐(4,4′‐dimethoxytrityl)‐L‐adenosine‐3′‐(2‐cyanoethyl‐N,N‐diisopropyl) phosphoramidite. (a) Bis‐N,O‐trimethylsilyl‐acetamide (BSA); (b) trimethylsilyl triflate (TMSOTf); (c) methylamine; (d) di‐tert‐butyl‐silyl bis‐triflate, DMF; (e) tert‐butyl‐dimethylsilyl chloride, imidazole; (f) benzoic anhydride; (g) HF‐pyridine, THF; (h) dimethoxytrityl chloride (DMT‐Cl), pyridine; (i) cyanoethyl N,N,N′,N′‐tetra‐isopropyl phosphane, benzylthiotetrazole (BTT). CEP, cyanoethyl N,N‐di‐isopropyl phosphoramidite.
6. Figure 18.6 Surrogate pegylation of NOX‐A12 pre‐PEG with a cleavable biotin affinity label.
7. Figure 18.7 Integration of the same AX‐HPLC of a 40 kDa pegylated Spiegelmer by Gaussian skim, drop‐down, and Tangential skim.

List of Contributors

Annemieke Aartsma‐Rus
Department of Human Genetics
Leiden University Medical Center
Leiden
The Netherlands

Scott A. Barros
Sage Therapeutics
Cambridge, MA
USA

Cindy L. Berman
Berman Consulting
Wayland, MA
USA

Kevin Brown
Fluidigm Corporation
South San Francisco, CA
USA

Joy Cavagnaro
Access BIO
Boyce, VA
USA

Scott Cormack
OncoGenex Pharmaceuticals
Bothell, WA
USA

Beth E. Davis
Department of Medicine
University of Saskatchewan
Saskatoon, Saskatchewan
Canada

Mehrdad Dirin
Department of Pharmaceutical Chemistry
University of Vienna
Vienna, Austria

Sheila M. Galloway
MRL, Merck & Co., Inc.
West Point, PA
USA

Gail M. Gauvreau
Department of Medicine
McMaster University
Hamilton, Ontario
Canada

Isabella Gazzoli
Department of Human Genetics
Leiden University Medical Center
Leiden
The Netherlands

Alain Guimond
InSymbiosis
Montreal
Quebec
Canada

Lydia Haile
Centre for Drug Evaluation and Research, Office of New Drugs
US FDA Silver Spring, MD
USA

Jonathan Hall
ETH‐Zurich
Zurich
Switzerland

Derek Ireland
Centre for Drug Evaluation and Research, Office of Biotechnology
Products, US FDA
Silver Spring, MD
USA

Aimee L. Jackson
miRagen Therapeutics
Boulder, CO
USA

Cindy Jacobs
OncoGenex Pharmaceuticals
Bothell, WA
USA

Piotr J. Kamola
GlaxoSmithKline R&D
Ware
Hertfordshire
UK
Current address
RIKEN Center for Integrative Medical Sciences
Yokohama
Japan

Lauren Kane
GlaxoSmithKline R&D
Stevenage
Hertfordshire
UK
Current address
MRC Human Genetics Unit
Institute of Genetics and Molecular Medicine
University of Edinburgh
Scotland

Peter Kasper
Federal Institute for Drugs and Medical Devices (BfArM)
Bonn
Germany

Jeremy D. A. Kitson
GlaxoSmithKline R&D
Stevenage
Hertfordshire
UK

Doug Kornbrust
Preclinsight
Reno, NV
USA

Monica Krieger
OncoGenex Pharmaceuticals
Bothell, WA
USA

Arthur A. Levin
Avidity Biosciences
La Jolla, CA
USA

Helen Lightfoot
ETH‐Zurich
Zurich
Switzerland

John Martucci
Centre for Drug Evaluation and Research, Office of Biotechnology
Products, US FDA
Silver Spring, MD
USA

Xin Ming
Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy
University of North Carolina
Chapel Hill, NC
USA

Nicolay Ferrari
Centre de recherche du CHUM – Tour Viger
Montreal, Quebec
Canada

Frederick B. Oleson
Independent Consultant
Concord, MA
USA

John Paul Oliveria
Department of Medicine
McMaster University
Hamilton, Ontario
Canada

Catherine C. Priestley
Innovative Medicines & Early Development
AstraZeneca
Cambridge
UK

Montserrat Puig
Centre for Drug Evaluation and Research Office of Biotechnology
Products, US FDA
Silver Spring, MD
USA

Michael J. Schlosser
MSR Pharma Services, Inc.
Lincolnshire, IL
USA

Anneliese Schneider
Preclinical Services & Consulting
Munich
Germany

Rosanne Seguin
Montreal Neurological Institute
McGill University
Montreal
Quebec
Canada

Zhanna Sobol
Pfizer Inc.
Groton, CT
USA

Patricia S. Stewart
OncoGenex Pharmaceuticals
Bothell, MA
USA

Kevin S. Sweder
Forensic and National Security Sciences Institute
Syracuse University
Syracuse, NY
USA

Daniela Verthelyi
Centre for Drug Evaluation and Research, Office of Biotechnology
Products, US FDA
Silver Spring, MD
USA

Stefan Vonhoff
NOXXON Pharma AG
Berlin
Germany

Jonathan K. Watts
RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology
University of Massachusetts Medical School
Worcester, MAUSA

Tacey E.K. White
Aclairo Pharmaceutical Development Group, Inc., Vienna, VA
USA

Johannes Winkler
Department of Cardiology
Medical University of Vienna
Vienna, Austria
Department of Pharmaceutical Chemistry
University of Vienna
Vienna, Austria

Karen D. Wisont
OncoGenex Pharmaceuticals
Bothell, WA,
USA

Preface

Development of oligonucleotide (ODN)‐based therapeutics is being progressed for a wide range of indications and using various routes of administration. There is a diversity of structures, chemistries, and mechanisms of actions for ODN therapeutics, but most of the members of this class of drug candidates can be categorized on the basis of whether they target either mRNA or proteins. ODN‐based therapy is distinct from gene therapy as it does not involve the modification of genes. Antisense ODN (ASO), short interfering RNA (siRNA), antagomirs, microRNA mimetics, and DNAzymes are part of the RNA‐targeting group, while immunostimulatory sequences (ISS), aptamers, and decoys are members of the protein‐targeting group.

Currently, six ODN‐based pharmaceuticals, including four ASO, have achieved marketing authorization in Europe and/or United States, and many more are undergoing late‐stage clinical testing. The first ASO drug, VITRAVENE (fomivirsen, Ionis Pharmaceuticals – formerly Isis), was approved in 1998 to treat CMV eye infections in HIV patients but within a few years was rendered obsolete by advances in antiretroviral cocktails for HIV therapy. The field waited 15 years for another approval. In 2013, the second ASO drug, KYNAMRO (mipomersen, Ionis Pharmaceuticals), was approved by the Food and Drug Administration (FDA) for the treatment of familial hypercholesterolemia. In 2016, out of 22 new drugs approved by FDA, 3 were for ODN therapeutics: DEFITELIO (defibrotide, Jazz Pharmaceuticals), a treatment for veno‐occlusive disease of the liver in individuals who have undergone bone marrow transplants granted in March; EXONDYS 51 (eteplirsen, Sarepta Therapeutics), a treatment for Duchenne muscular dystrophy granted in August; and SPINRAZA (nusinersen, Biogen), a treatment for spinal muscular atrophy granted in December. In addition, Atlantic Pharmaceuticals is developing alicaforsen, an ASO targeting ICAM‐1 for the treatment of pouchitis, and currently supplies alicaforsen in response to physicians’ requests under international named patient supply regulations for patients with inflammatory bowel disease. In January 2017, Atlantic announced it received agreement from the FDA to initiate a rolling submission of its New Drug Application for alicaforsen to treat pouchitis ahead of data from an ongoing phase III study, which is expected at the end of 2018.

The recent ODN approvals are indicative of the enthusiasm, vigor, and vitality of the field observed in recent years. There are currently over 100 companies combining hundreds of ODN programs. In 2015 alone, there were more than 35 Investigational New Drug submissions for ODN candidates. More than 145 ODN clinical trials are listed on ClinicalTrials.gov, 31 of which are active/recruiting. The diverse types of indications for which ODN therapies have been approved and for those currently in clinical development demonstrate that these therapies are not a “one‐off” development but rather are poised to claim their space in the apothecary of pharmaceuticals.

The advancement of a growing number of ODN programs, in particular ASO, in late stage of clinical development and the rapid pipeline expansion by various companies are testament of the progress, much of which was made in the 15 years between first and second drug approvals, in understanding the pharmacologic, pharmacokinetic, and toxicologic properties, as well as improving the delivery of ODN. There are now numerous examples of pharmacologic activity in animal models, and evidence of antisense activity in patients has been demonstrated in clinical trials.

The discovery of novel therapeutics is an inherently complex and interdisciplinary process, requiring close integration of scientists from several disciplines in an environment in which lessons are shared and taught across an organization.

The purpose of this book is to review the current state of knowledge of ODN and to examine the scientific principles and the tools utilized by scientists in preclinical and clinical settings as applied to ODN therapeutics.

Acknowledgments

We have embarked on this endeavor without anticipating the long twisting road that was ahead of us in putting this book together. We would like to give our heartfelt thanks to all authors. As editors, we were depending on their goodwill, commitment, and patience. We hope that their contribution will offer a useful review of the current understanding and recent advances in the field. In light of the challenges we are facing with this technology, we also hope the knowledge summarized in this book will provide guidance and will support those readers currently working in the field as well as the future developers that will further advance oligonucleotide therapeutics.

Nicolay Ferrari and Rosanne Seguin

Oligonucleotide‐Based Drugs and Therapeutics

Preclinical and Clinical Considerations for Development

List of Contributors

Preface

Acknowledgments