Delivery of oligonucleotide‐based therapeutics: challenges and opportunities

Review6 April 2021Open Access Delivery of oligonucleotide-based therapeutics: challenges and opportunities Suzan M Hammond orcid.org/0000-0002-5640-7569 Department of Paediatrics, University of Oxford, Oxford, UKMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Annemieke Aartsma-Rus orcid.org/0000-0003-1565-654X Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Sandra Alves orcid.org/0000-0002-8881-9197 Department of Human Genetics, Research and Development Unit, National Health Institute Doutor Ricardo Jorge, Porto, Portugal Search for more papers by this author Sven E Borgos orcid.org/0000-0002-6222-9252 Department of Biotechnology and Nanomedicine, SINTEF AS, Trondheim, Norway Search for more papers by this author Ronald A M Buijsen orcid.org/0000-0002-9722-8110 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Rob W J Collin orcid.org/0000-0003-4347-6503 Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Giuseppina Covello orcid.org/0000-0001-5660-8283 Department of Biology, University of Padova, Padova, Italy Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Michela A Denti orcid.org/0000-0001-7203-7062 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Lourdes R Desviat orcid.org/0000-0002-2081-0815 Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, SpainMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Lucía Echevarría orcid.org/0000-0001-9307-6992 SQY Therapeutics, Montigny-le-Bretonneux, FranceMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Camilla Foged orcid.org/0000-0003-2812-5588 Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Ø, DenmarkMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Gisela Gaina orcid.org/0000-0001-7415-5281 Victor Babes National Institute of Pathology, Bucharest, Romania Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania Search for more papers by this author Alejandro Garanto orcid.org/0000-0001-5721-1560 Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands Department of Pediatrics, Radboud University Medical Center, Nijmegen, The NetherlandsMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Aurelie T Goyenvalle orcid.org/0000-0003-3938-1165 Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, FranceMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Magdalena Guzowska Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, PolandMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Irina Holodnuka orcid.org/0000-0002-7208-4499 Institute of Microbiology and Virology, Riga Stradins University, Riga, Latvia Search for more papers by this author David R Jones MHRA 10 South Colonnade, London, UK Search for more papers by this author Sabine Krause orcid.org/0000-0002-3141-886X Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University of Munich, Munich, Germany Search for more papers by this author Taavi Lehto orcid.org/0000-0002-7131-2998 Institute of Technology, University of Tartu, Tartu, Estonia Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, SwedenMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Marisol Montolio orcid.org/0000-0001-5494-5737 Duchenne Parent Project España, Madrid, Spain Department of Cell Biology, Fisiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain Search for more papers by this author Willeke Van Roon-Mom orcid.org/0000-0002-3035-0533 Department of Human Genetics, Leiden University Medical Center, Leiden, The NetherlandsMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Virginia Arechavala-Gomeza Corresponding Author [email protected] orcid.org/0000-0001-7703-3255 Neuromuscular Disorders Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain Ikerbasque, Basque Foundation for Science, Bilbao, SpainMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Suzan M Hammond orcid.org/0000-0002-5640-7569 Department of Paediatrics, University of Oxford, Oxford, UKMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Annemieke Aartsma-Rus orcid.org/0000-0003-1565-654X Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Sandra Alves orcid.org/0000-0002-8881-9197 Department of Human Genetics, Research and Development Unit, National Health Institute Doutor Ricardo Jorge, Porto, Portugal Search for more papers by this author Sven E Borgos orcid.org/0000-0002-6222-9252 Department of Biotechnology and Nanomedicine, SINTEF AS, Trondheim, Norway Search for more papers by this author Ronald A M Buijsen orcid.org/0000-0002-9722-8110 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands Search for more papers by this author Rob W J Collin orcid.org/0000-0003-4347-6503 Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands Search for more papers by this author Giuseppina Covello orcid.org/0000-0001-5660-8283 Department of Biology, University of Padova, Padova, Italy Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Michela A Denti orcid.org/0000-0001-7203-7062 Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy Search for more papers by this author Lourdes R Desviat orcid.org/0000-0002-2081-0815 Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, SpainMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Lucía Echevarría orcid.org/0000-0001-9307-6992 SQY Therapeutics, Montigny-le-Bretonneux, FranceMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Camilla Foged orcid.org/0000-0003-2812-5588 Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Ø, DenmarkMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Gisela Gaina orcid.org/0000-0001-7415-5281 Victor Babes National Institute of Pathology, Bucharest, Romania Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania Search for more papers by this author Alejandro Garanto orcid.org/0000-0001-5721-1560 Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands Department of Pediatrics, Radboud University Medical Center, Nijmegen, The NetherlandsMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Aurelie T Goyenvalle orcid.org/0000-0003-3938-1165 Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, FranceMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Magdalena Guzowska Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, PolandMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Irina Holodnuka orcid.org/0000-0002-7208-4499 Institute of Microbiology and Virology, Riga Stradins University, Riga, Latvia Search for more papers by this author David R Jones MHRA 10 South Colonnade, London, UK Search for more papers by this author Sabine Krause orcid.org/0000-0002-3141-886X Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University of Munich, Munich, Germany Search for more papers by this author Taavi Lehto orcid.org/0000-0002-7131-2998 Institute of Technology, University of Tartu, Tartu, Estonia Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, SwedenMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Marisol Montolio orcid.org/0000-0001-5494-5737 Duchenne Parent Project España, Madrid, Spain Department of Cell Biology, Fisiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain Search for more papers by this author Willeke Van Roon-Mom orcid.org/0000-0002-3035-0533 Department of Human Genetics, Leiden University Medical Center, Leiden, The NetherlandsMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Virginia Arechavala-Gomeza Corresponding Author [email protected] orcid.org/0000-0001-7703-3255 Neuromuscular Disorders Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain Ikerbasque, Basque Foundation for Science, Bilbao, SpainMiddle authors are listed in alphabetical order, particularly engaged authors are highlighted with the symbol. Search for more papers by this author Author Information Suzan M Hammond1, Annemieke Aartsma-Rus2, Sandra Alves3, Sven E Borgos4, Ronald A M Buijsen2, Rob W J Collin5, Giuseppina Covello6,7, Michela A Denti7, Lourdes R Desviat8, Lucía Echevarría9, Camilla Foged10, Gisela Gaina11,12, Alejandro Garanto5,13, Aurelie T Goyenvalle14, Magdalena Guzowska15, Irina Holodnuka16, David R Jones17, Sabine Krause18, Taavi Lehto19,20, Marisol Montolio21,22, Willeke Van Roon-Mom2 and Virginia Arechavala-Gomeza *,23,24 1Department of Paediatrics, University of Oxford, Oxford, UK 2Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 3Department of Human Genetics, Research and Development Unit, National Health Institute Doutor Ricardo Jorge, Porto, Portugal 4Department of Biotechnology and Nanomedicine, SINTEF AS, Trondheim, Norway 5Department of Human Genetics and Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands 6Department of Biology, University of Padova, Padova, Italy 7Department of Cellular, Computational and Integrative Biology - CIBIO, University of Trento, Trento, Italy 8Centro de Biología Molecular Severo Ochoa UAM-CSIC, CIBERER, IdiPaz, Universidad Autónoma de Madrid, Madrid, Spain 9SQY Therapeutics, Montigny-le-Bretonneux, France 10Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen Ø, Denmark 11Victor Babes National Institute of Pathology, Bucharest, Romania 12Department of Biochemistry and Molecular Biology, University of Bucharest, Bucharest, Romania 13Department of Pediatrics, Radboud University Medical Center, Nijmegen, The Netherlands 14Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France 15Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life Sciences – SGGW, Warsaw, Poland 16Institute of Microbiology and Virology, Riga Stradins University, Riga, Latvia 17MHRA 10 South Colonnade, London, UK 18Department of Neurology, Friedrich-Baur-Institute, Ludwig-Maximilians-University of Munich, Munich, Germany 19Institute of Technology, University of Tartu, Tartu, Estonia 20Division of Biomolecular and Cellular Medicine, Department of Laboratory Medicine, Karolinska Institutet, Huddinge, Sweden 21Duchenne Parent Project España, Madrid, Spain 22Department of Cell Biology, Fisiology and Immunology, Faculty of Biology, University of Barcelona, Barcelona, Spain 23Neuromuscular Disorders Group, Biocruces Bizkaia Health Research Institute, Barakaldo, Spain 24Ikerbasque, Basque Foundation for Science, Bilbao, Spain *Corresponding author. Tel: +34 946007967; E-mail: [email protected] EMBO Mol Med (2021)13:e13243https://doi.org/10.15252/emmm.202013243 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics. Glossary Anti-drug antibodies (ADAs) Antibody-mediated immunogenicity elicited in vivo to a given drug. Drug-specific antibodies can reduce the efficacy of the treatment and even fully inactivate the drug, and/or they can induce adverse effects. Antisense oligonucleotides (ASOs) Single-stranded oligonucleotides complementary to RNA target sequences. Aptamers Single-stranded oligonucleotides (20-100 nucleotides) which adopt three-dimensional structures that allow them to bind very specifically to protein target sites. Blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) Selectively permeable membranes of the central nervous system (CNS) vasculature. Only small molecules (molecular weight below 400-500 Da) and high lipid solubility (logP value of approximately 2.1) can cross these vascular barriers. Generally, oligonucleotides display a molecular weight of approximately 10 kDa and are hydrophilic; hence, they are too large and hydrophilic to cross biological barriers by passive diffusion. Cell-penetrating peptides (CPPs) Short cationic and/or amphipathic peptides (usually less than 30 amino acids) capable of translocating different types of cargoes across biological barriers and cell membranes. CPPs can be directly conjugated to oligonucleotides (ONs) or used to encapsulate ONs into nanoparticles. European Medicines Agency (EMA) Agency of the European Union in charge of the evaluation and supervision of medicinal products. The EMA facilitates development and access to medicines, evaluates applications for marketing authorisation and monitors the safety of human and veterinary medicines. Food and Drug Administration (FDA) The federal agency of the United States Department of Health and Human Services, responsible for protecting public health by ensuring the safety, efficacy and security of human and veterinary drugs. Gapmer Chimeric antisense oligonucleotides (ASOs) that contain a central block of DNA nucleotides, flanked by modified sequences, usually containing 2′-O-modified or locked nucleic acid (LNA) chemistries. Gapmers are used for gene silencing by stimulating RNA cleavage through the recruitment of RNase H. Lipid nanoparticles (LNPs) Delivery systems based on LNPs are composed of one or several lipid components, often an ionisable cationic lipid used for complexation of polyanionic DNA/RNA and stabilising helper lipids such as distearoylphosphatidylcholine (DSPC) and cholesterol. In addition, LNPs may be stabilised sterically by surface coating with polyethylene glycol (PEG). LNPs have a complex internal lipid architecture that is well suited for stable and efficient encapsulation of DNA/RNA cargoes. MicroRNAs (miRNAs) Small noncoding RNAs (∼22 nt), which regulate gene expression at the post-transcriptional level by degrading target mRNAs, when complementary to the sequence, or inhibiting their translation when not fully complementary. Each miRNA can influence the expression of hundreds of mRNAs. Pharmacodynamics (PD) The relationship between the drug concentration at the site of action and the observed biochemical response and its efficacy. Pharmacokinetics (PK) The time course of drug absorption, distribution, metabolism, excretion and toxicity (ADMET), as well as the liberation of a drug from its formulation. Phosphorodiamidate morpholino oligonucleotides (PMOs) Oligonucleotides containing uncharged chemistry. The nucleic acid backbone has been replaced with 6-membered morpholino rings and phosphorodiamidate linkages, while retaining standard nucleobases. Peptide nucleic acid (PNA) Uncharged oligonucleotide chemistry with amide bond linkages between the nucleobases. PNAs are manufactured by peptide synthesis. RNAse H cleavage RNAse H hydrolyses the phosphodiester bonds of RNA when hybridised to DNA. Small interfering RNA (siRNA) Double-stranded RNA (~21 nt) composed of a guide strand complementary to the target mRNA and a passenger strand. siRNAs act within the endogenous RNA-induced silencing complex (RISC) to degrade mRNA. Toll-like receptors (TLRs) Pattern-recognition receptors usually found on the plasma or endosomal membranes of sentinel cells such as macrophages and dendritic cells (DCs). Activation of TLRs can promote an inflammatory response. For example, TLR9 is activated by unmethylated cytidine-phosphate-guanosine (CpG) dinucleotides present in bacterial and viral DNA. Introduction Synthetic oligonucleotides (ONs) are small, single- or double-stranded pieces of modified nucleic acids that have been exploited as therapeutic modalities in different ways (Table 1). The unique characteristic of ONs is that they bind to their target via Watson–Crick base pairing, enabling intervention at a genetic level by targeting RNA in a specific manner (Zamecnik & Stephenson, 1978). ONs encompass many types of nucleic acid-based therapeutics, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), anti-miRNA (antagomirs), miRNA mimics (agomirs), aptamers and unmethylated CpG-containing ONs. Depending on their mechanism of action, treatment with therapeutic nucleic acids may cause decreased, increased or restored protein expression. Currently, 11 ON-based drugs across many disease areas have received regulatory approval by the US Food and Drug Administration (FDA), the European Medicines Agency (EMA) and/or the Japanese Ministry of Health, Labour and Welfare. However, further therapeutic development is challenged by unfavourable absorption, distribution, metabolism, excretion and toxicity (ADMET) properties for most clinical applications (Godfrey et al, 2017). This review mainly focuses on the development of single-stranded ONs and covers (i) the numerous methods developed to date to deliver ONs across biological barriers, (ii) the model systems used to test ONs and (iii) the hurdles existing for translating laboratory breakthroughs to the clinic. The content represents the joint efforts of members of the EU Cooperation of Science and Technology (COST) network Delivery of RNA Therapeutics (DARTER, COST Action 17103, www.antisenserna.eu), which aims to facilitate RNA-targeting nucleic acid-based drugs to reach their full potential. Table 1. Mechanisms of action of therapeutic oligonucleotides. Modality Mechanism Example(s) RNase H RNase H-mediated cleavage of target transcript Gapmers Steric Blockage Interference with post-transcriptional RNA-binding elements, e.g. splicing modulation and blocking endogenous miRNA 2nd and 3rd generation ASOs and antagomirs Protein Binding Bind target proteins in a structure-specific manner Aptamer Innate Immunity Inhibits protein expression via target-specific mRNA degradation Unmethylated CpG-containing ONs RNAi Inhibition of gene expression via target-specific mRNA degradation siRNAs, microRNAs Chemistry dictates the drug properties of oligonucleotides Therapeutic nucleic acids are chemically modified in several ways to endow them with properties such as increased resistance to nucleases and improved target binding affinity (Jarver et al, 2014) (Fig 1). Each modification confers the ON different properties, and some may be combined, but other modifications are not compatible or may modify the ON in ways that complicate their synthesis or interfere with the mechanisms by which they exert their effect. First-generation chemistries include the widely used phosphate backbone modifications, e.g. phosphorothioate (PS), which imparts resistance to endonucleases and improves bioavailability by reducing renal clearance due to increased affinity for serum proteins (Eckstein, 2014). However, this modification also reduces the affinity for the target RNA. Second-generation chemistries include ribose modifications at the 2′-O position of RNA and 2′ position of DNA, of which the 2ʹ-O-methyl (2ʹ-OMe), 2ʹ-O-methoxy-ethyl (2ʹ-MOE) and 2ʹ-fluoro (2ʹ-F) modifications are the most commonly used types. These modifications increase the binding affinity to RNA and further improve the nuclease resistance. An even greater binding affinity chemistry is the conformationally constrained DNA analogues locked nucleic acid (LNA) and tricyclo-DNA (tcDNA). LNA contains a methyl bridge between the 2′-O and 4′ position of the ribose ring (Koshkin et al, 1998; Obika et al, 1998). The backbone considerably changed for tcDNA via introduction of an ethylene bridge with a cyclopropane ring between the ribose 3' and 5' carbon positions (Renneberg & Leumann, 2002). The bridge imposes a locked conformation on the ribose ring, which is ideal for binding to RNA. All first- and second-generation chemistries are compatible with nucleic acid synthesis and can easily be mixed with DNA and RNA in ON chimeras. Third-generation chemistries include changes in the nucleobase, e.g. phosphorodiamidate morpholino oligomers (PMO) (Summerton & Weller, 1997) and peptide nucleic acid (PNA) (Nielsen et al, 1991; Hanvey et al, 1992). For PMOs, the nucleic acid backbone has been replaced with a 6-membered morpholino ring and phosphorodiamidate linkages, while retaining standard nucleobases. The nucleobases of PNAs are linked by amide bonds, which are synthesised similarly to peptides. Both PMO and PNA are uncharged, very resistant to nucleases, and display variable affinity for the target RNA (Smulevitch et al, 1996; Summerton & Weller, 1997). The choice of chemical modifications is largely dictated by the modality and the target tissue. Figure 1. Oligonucleotide chemistries Commonly used nucleic acid chemistries. The often used phophorothioate (PS) backbone replaces the natural phosphodiester (PO). Modifications to the ribose at the 2ʹ-O position of RNA and 2ʹ-position of DNA include the 2ʹ-O-methyl (2ʹ-OMe), 2ʹ-O-methoxy-ethyl (2ʹ-MOE) and 2ʹ-fluoro (2ʹ-F) are the most commonly used. Conformationally constrained DNA analogues, locked nucleic acid (LNA), constrained 2′-O-ethyl (cEt) and tricyclo-DNA (tcDNA), provide greater binding affinity. LNA and cEt are constrained by a methyl bridged from the 2′-O and 4′ position of the ribose. tcDNA introduces of an ethylene bridge with a cyclopropane ring between the 3′ and 5′ carbon positions of ribose. Alternative chemistries include changes in the nucleobase, e.g. phosphorodiamidate morpholino oligomers (PMO) and peptide nucleic acid (PNA). Download figure Download PowerPoint Single-stranded ASOs complementary to target RNA were first utilised therapeutically by exploiting RNase H cleavage of DNA/RNA hybrids (Stein & Hausen, 1969; Wu et al, 2004) (Fig 2). RNase H-inducible ASOs are designed as gapmers, where central DNA nucleotides are flanked by RNase H-resistant modified nucleotides (Wahlestedt et al, 2000). The modified sequences improve target affinity while the central DNA sequence forms the DNA/RNA hybrid for RNase H recognition and cleavage (Monia et al, 1993). Fully modified second- and third-generation ASO chemistries act through RNase H-independent mechanisms (Fig 2) (Jarver et al, 2014). Steric blocking ASOs can inhibit or activate translation through the binding to regulatory elements, e.g. upstream open reading frames (Liang et al, 2016b; Liang et al, 2017). A common therapeutic modality is the modulation of pre-mRNA splicing (Arechavala-Gomeza et al, 2014), which is used to induce or suppress exon inclusion. In Duchenne muscular dystrophy (DMD) patients, ASO-induced exon skipping of mutated dystrophin pre-mRNA restores the reading frame and allows for the production of partially functional, rather than non-functional, dystrophin protein (Mitrpant et al, 2009). In contrast, for spinal muscular atrophy (SMA) patients, ASOs increase the level of exon 7 inclusion in survival motor neuron 2 (SMN2) mRNA, leading to increased levels of SMN protein (Singh et al, 2006). Similarly, ASOs can also induce the skipping of pseudoexons (Collin et al, 2012) or block RNA-splicing factors from recognising cryptic splice sites (Rivera-Barahona et al, 2015). ASOs can also sterically block the union of RNA-binding factors to repeat expansion regions of pathogenic mRNAs (Fig 2). In myotonic dystrophy 1, expanded microsatellite repeats sequester RNA-binding factors within nuclear expansion RNA foci (Miller et al, 2000). ASOs targeting the CUG repeat expansion mRNA release the sequestered RNA-binding factors and reverse the phenotype (Klein et al, 2019). RNA interference (RNAi)-based therapies, i.e. double-stranded siRNA and single-stranded microRNA (miRNA), exploit the endogenous RNAi pathway in the cytosol (Fire et al, 1998) to silence or modulate the expression of specific proteins (Fig 2). Commonly used chemical modifications for siRNA, including 2ʹ-OMe and 2ʹ-F modifications, decrease RNase recognition and are well tolerated throughout the entire siRNA duplex (Watts et al, 2008). In addition, these modifications are widely used to decrease immune stimulation (Judge et al, 2006). ASOs can influence miRNA function, either by sequestering a miRNA (antagomir) or by generating a miRNA mimic (agomir). Notably, a single miRNA generally regulates the expression of multiple genes in a given pathway; hence, antagomirs and agomirs have the potential to mediate increased or decreased expression of multiple genes, respectively (Friedman et al, 2009). Finally, two types of ONs which do not work through Watson–Crick base pairing are aptamers and unmethylated CpG-containing ONs. Aptamers are single-stranded ONs (20–100 nucleotides) selected from randomised libraries based on their high-avidity binding to specific targets (Ellington & Szostak, 1990; Tuerk & Gold, 1990). They adopt three-dimensional structures that bind to protein target sites through attractive electrostatic interactions and pocket-like structures (Ellington & Szostak, 1990), and they display binding affinities to their receptor targets which are comparable to those of monoclonal antibodies (Jayasena, 1999). Unmethylated CpG-cont