Advances in oligonucleotide drug delivery

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.

Fig. 1. Chemistry of FDA-approved oligonucleotide drugs.

Chemical composition of the FDA-approved oligonucleotide drugs fomivirsen (part a), mipomersen (part b), inotersen (part c), eteplirsen (part d), golodirsen (part e), nusinersen (part f), patisiran (part g), givosiran (part h) and pegaptanib (part i). Drugs are ordered by mechanism of action. Drug names, trade names, principal developing company, modality and RNA target are described in Table 1 for each compound. The drug defibrotide consists of a mixture of single-stranded and double-stranded ribonucleotides of variable length and sequence composition harvested from pig intestine. It therefore cannot be easily represented in the same manner as the other oligonucleotide drugs and so is not shown here. GalNAc, N-acetylgalactosamine; PEG, polyethylene glycol; PMO, phosphorodiamidate morpholino oligonucleotide. Part i structure adapted from ref., Springer Nature Limited.

Fig. 2. Oligonucleotide-mediated gene regulatory mechanisms.

a | Gapmer antisense oligonucleotides (ASOs), consisting of a DNA-based internal ‘gap’ and RNA-like flanking regions (often consisting of 2ʹ-O-methyl (2ʹ-OMe) or locked nucleic acid (LNA) modified bases) bind to target transcripts with high affinity. The resulting RNA–DNA duplex acts as a substrate for RNASEH1, leading to the degradation of the target transcript. b | Steric block oligonucleotides targeted to pre-mRNA splicing signals modulate alternative splicing to either promote exon skipping or exon inclusion (depending on the type of splicing signal targeted). The resulting mature mRNA species can be spliced in a productive manner (for example, to restore the reading frame or to switch to an alternative isoform) or in a non-productive manner (for example, to remove an exon that is required for protein function and/or to disrupt the translation reading frame). c | Steric block antisense oligonucleotides can disrupt translation initiation by targeting the AUG start codon. d | Some transcripts contain upstream open reading frames (uORFs) that modulate the translational activity of the primary open reading frame (pORF). Targeting the uORF with steric block ASOs disrupts this regulation, leading to activation of pORF translation. e | Transcript stability can be modulated by shifting the usage of cleavage and polyadenylation signals. For example, a steric block ASO targeted to a distal polyadenylation signal results in the preferential usage of a weaker proximal polyadenylation signal. The resulting shorter transcript is more stable as it lacks RNA destabilization signals. f | Small interfering RNAs (siRNAs) enter the RNA-induced silencing complex (RISC), which consists of Argonaute 2 protein (AGO2), DICER1 and TARBP2, and the passenger strand is discarded. The guide strand directs the RISC to complementary target genes that are cleaved by the slicer activity of AGO2. g | Endogenous microRNAs (miRNAs) are loaded into miRISC. miRNA activity can be inhibited by steric block ASOs that either complex with the mature miRNA loaded in the RISC complex or by masking a target site through interactions with the targeted transcript. h | Natural antisense transcripts (NATs) recruit epigenetic silencing complexes, such as PRC2, to a sense gene locus. Interference of the epigenetic modifier protein association with the NAT using steric block ASOs or degradation of the NAT via siRNA or gapmer ASO results in ‘unsilencing’ of the sense gene. i | Small activating RNAs (saRNAs) can recruit the RNA-induced transcriptional activation (RITA) complex (consisting of AGO2, CTR9 and DDX5 (ref.)) to low-copy promoter-associated RNA, leading to transcriptional activation of the proximal gene. EZH2, Enhancer of zeste homolog 2; PRC2, polycomb repressive complex 2.

Fig. 3. Common chemical modifications used in oligonucleotide drugs.

Schematic of an RNA nucleotide and how it can be chemically modified at the backbone, nucleobase, ribose sugar and 2ʹ-ribose substitutions. B, nucleobase; cEt, constrained ethyl bridged nucleic acid; ENA, ethylene-bridged nucleic acid; 2ʹ-F, 2ʹ-fluoro; LNA, locked nucleic acid; 2ʹ-MOE, 2ʹ-O-methoxyethyl; 2ʹ-OMe, 2ʹ-O-methyl; PMO, phosphorodiamidate morpholino oligonucleotide; PNA, peptide nucleic acid; PS, phosphorothioate; tcDNA, tricyclo DNA.

Fig. 4. Oligonucleotide delivery strategies.

Schematics of various delivery strategies for small interfering RNAs (siRNAs) and antisense oligonucleotides (ASOs). a | Lipid–siRNA conjugate wherein cholesterol is conjugated to the 3ʹ terminus of the passenger strand. b | Triantennary N-acetylgalactosamine (GalNAc) moiety conjugated to an ASO. c | Antibody–siRNA conjugate. Oligonucleotides can be attached to the antibody or Fab fragment using click chemistry or thiol–maleimide linkages. d | Aptamer–siRNA conjugate. In vitro transcription can be used to generate a chimaeric aptamer–passenger strand as a single molecule. e | Peptide–ASO conjugate. The example is a PMO (phosphorodiamidate morpholino oligonucleotide) conjugated to a cell-penetrating peptide (Pip–9b2). f | Stable nucleic acid lipid particle encapsulating siRNAs. g | Engineered exosome with the brain-targeting rabies virus glycoprotein (RVG) peptide displayed on the outer surface. The exosome consists of a membrane containing lipids and proteins derived from the donor cell. The exosome also contains therapeutic cargo (for example, siRNA) and proteins and nucleic acids (for example, microRNA) derived from the donor cell. h | Spherical nucleic acid nanoparticle consisting of a gold core coated in densely packed ASOs attached by metal–thiol linkages. i | Self-assembled DNA cage tetrahedron nanostructure. Oligonucleotide therapeutics (for example, siRNAs and ASOs) can be incorporated into the design of the DNA cage itself. Additional targeting ligands and polyethylene glycol (PEG) can be further conjugated to the nanostructure. LAMP2, lysosome-associated membrane protein 2; Pip, PMO/peptide nucleic acid internalization peptide. Part d shows a schematic of the PSMA (prostate-specific membrane antigen) aptamer adapted from ref., Springer Nature Limited.