REVIEWS Advances in oligonucleotide drug delivery Thomas C. Roberts 1,2 ✉ , Robert Langer 3 and Matthew J. A. Wood 1,2 ✉ Abstract | Oligonucleotides can be used to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing. As such, these molecules have potential therapeutic applications for myriad indications, with several oligonucleotide drugs recently gaining approval. However, despite recent technological advances, achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation. Here, we provide an overview of oligonucleotide-based drug platforms, focusing on key approaches — including chemical modification, bioconjugation and the use of nanocarriers — which aim to address the delivery challenge. Oligonucleotides are nucleic acid polymers with the In addition to their ability to recognize specific tar- potential to treat or manage a wide range of diseases. get sequences via complementary base pairing, nucleic Although the majority of oligonucleotide therapeutics acids can also interact with proteins through the for- have focused on gene silencing, other strategies are being mation of three-dimensional secondary structures — a pursued, including splice modulation and gene activa- property that is also being exploited therapeutically. For tion, expanding the range of possible targets beyond example, nucleic acid aptamers are structured nucleic what is generally accessible to conventional pharma- acid ligands that can act as antagonists or agonists for ceutical modalities. The majority of oligonucleotide specific proteins11 (BOx 1). Similarly, guide RNA mole- modalities interact with their cognate target molecules cules contain hairpin structures that bind to exogenously via complementary Watson–Crick base pairing, and introduced Cas9 protein and direct it to specific genomic so interrogation of the putative target sequence is rela- DNA loci for targeted gene editing12 (BOx 2). An in-depth tively straightforward. Highly specific lead compounds discussion of these modalities is beyond the scope of this can often be rationally designed based on knowledge of Review. the primary sequence of a target gene alone and lead As of January 2020, ten oligonucleotide drugs have candidates identified by rapid screening. By contrast, received regulatory approval from the FDA (Fig. 1; conventional small-molecule pharmaceuticals require TABle 1). However, a major obstacle preventing wide- much larger, and often iterative, screening efforts fol- spread usage of oligonucleotide therapeutics is the dif- lowed by extensive medicinal chemistry optimiza- ficultly in achieving efficient delivery to target organs 1Department of Paediatrics, tion. In addition, the use of oligonucleotides allows for and tissues other than the liver. In addition, off-target 13–17 University of Oxford, Oxford, precision and/or personalized medicine approaches interactions , sequence and chemistry-dependent UK. as they can theoretically be designed to selectively toxicity and saturation of endogenous RNA process- 2MDUK Oxford Neuromuscular target any gene with minimal, or at least predictable, ing pathways18 must also be carefully considered. The Centre, University of Oxford, off-target effects. Furthermore, it is possible to target most commonly used strategies employed to improve Oxford, UK. patient-specific sequences that are causative of rare nucleic acid drug delivery include chemical modifica- 3Department of Chemical disease1, specific alleles (for example, SNPs or expanded tion to improve ‘drug-likeness’, covalent conjugation to Engineering and Koch Institute for Integrative repeat-containing mutant transcripts can be preferen- cell-targeting or cell-penetrating moieties and nanopar- 2–5 Cancer Research, tially targeted without silencing the wild-type mRNA ), ticle formulation. More recently developed approaches Massachusetts Institute distinct transcript isoforms6, pathogenic fusion tran- such as endogenous vesicle (that is, exosome) loading, of Technology, Cambridge, scripts (for example, Bcr–Abl7), traditionally ‘undrug- spherical nucleic acids (SNAs), nanotechnology appli- MA, USA. gable’ targets (for example, proteins that may lack cations (for example, DNA cages) and ‘smart’ materials ✉e-mail: thomas.roberts@ hydrophobic pockets that may accommodate a small are also being pursued. paediatrics.ox.ac.uk; 8,9 matthew.wood@ molecule that also inhibits protein activity) and viral This Review will provide an overview of paediatrics.ox.ac.uk sequences that evolve resistance to an oligonucleotide oligonucleotide-based drug platforms and focus on https://doi.org/10.1038/ therapy (whereby the oligonucleotide design is modified recent advances in improving oligonucleotide drug s41573-020-0075-7 to compensate for acquired escape mutations)10. delivery. NATURE REVIEWS | DRUG DISCOVERY VOLUME 19 | OCTOBER 2020 | 673 REVIEWS Box 1 | Aptamers — evolved nucleic acid ligands they lack RNase H competence. Such oligonucleotides therefore comprise either nucleotides that do not form Aptamers are structured, single-stranded nucleic acid molecules (typically ~20–100 RNase H substrates when paired with RNA or a mixture nucleotides) that fold into defined secondary structures and act as ligands that interact of nucleotide chemistries (that is, ‘mixmers’) such that with target proteins by way of their three-dimensional structure and adaptive fit11. runs of consecutive DNA-like bases are avoided. In contrast with other kinds of nucleic acid therapeutics, aptamers are not rationally designed. Instead, they are generated by an in vitro evolution methodology called Steric block oligonucleotides can mask specific SELEX (systematic evolution of ligands by exponential enrichment)286–288. Pegaptanib sequences within a target transcript and thereby inter- (originally developed by NeXstar Pharmaceuticals and Eyetech Pharmaceuticals) fere with transcript RNA–RNA and/or RNA–protein (Fig. 1i; TABle 1), an RNA-based aptamer that targets the VEGF-165 vascular endothelial interactions. The most widely used application of steric growth factor isoform as an anti-angiogenic therapy for neovascular age-related block ASOs is in the modulation of alternative splicing macular degeneration, is currently the only aptamer approved for clinical use. in order to selectively exclude or retain a specific exon(s) Aptamers have primarily been used to target extracellular targets (for example, (that is, exon skipping and exon inclusion, respectively). receptors), which somewhat simplifies the delivery problem for this class of In these cases, the oligonucleotide ‘masks’ a splicing oligonucleotide. However, as with other RNA species, RNA aptamers are rapidly signal such that it becomes invisible to the spliceosome, degraded in most extracellular environments, meaning that chemical modification leading to alterations in splicing decisions24,25. Typically, of aptamers is essential for in vivo activity. SELEX can be performed with libraries of chemically modified RNAs to a limited extent, as some nucleotide analogues, such as such splice correction approaches have been used to 2 -fluoro and 2 -O-methyl, are tolerated in both reverse transcriptase and T7 RNA restore the translational reading frame in order to res- ʹ ʹ 26,27 polymerase enzymatic steps289,290. The introduction of post-SELEX chemical cue production of a therapeutic protein . However, modifications is an alternative approach to further enhance aptamer drug-like the same technology can also be used for splice cor- properties. ruption, whereby an exon is skipped in order to disrupt The inherent chirality of amino acids in nature, in turn, enforces chirality in the translation of the target gene28 (Fig. 2b). Given that enzymatically produced nucleic acids (that is, l-amino acids and d-nucleotides). alternative splicing is responsible for much proteomic However, SELEX can be performed using enantiomeric protein analogues of target diversity, it is possible that steric block oligonucleotides proteins synthesized with unnatural d-amino acids. The resulting aptamers are may also be utilized to promote isoform switching, necessarily composed of d-RNA as a consequence of the restrictions of the enzymatic thereby diminishing the expression of harmful protein SELEX steps. However, the l-RNA versions of these identified aptamers can now be generated by chemical synthesis, which will thereby recognize the natural l-protein. isoforms and/or promoting the expression of beneficial These highly stable ‘mirror image’ aptamers are called spiegelmers (or l-RNA aptamers) ones. To date, three splice-switching ASOs have been and are not substrates for natural nucleases291. FDA-approved; eteplirsen, golodirsen and nusinersen (Fig. 1d–f). Notably, steric block ASOs have also been demon- Oligonucleotide-based platforms strated to inhibit translation inhibition29,30 (Fig. 2c), Antisense oligonucleotides. Antisense oligonucleotides interfere with upstream open reading frames that neg- (ASOs) are small (~18–30 nucleotides), synthetic, atively regulate translation31 in order to activate protein single-stranded nucleic acid polymers of diverse chem- expression32 (Fig. 2d), inhibit nonsense-mediated decay in istries, which can be employed to modulate gene expres- a gene-specific manner by preventing assembly of exon sion via various mechanisms. ASOs can be subdivided junction