Opportunities and challenges for antisense oligonucleotide therapies

Abstract

Antisense oligonucleotide (AON) therapies involve short strands of modified nucleotides that target RNA in a sequence-specific manner, inducing targeted protein knockdown or restoration. Currently, 10 AON therapies have been approved in the United States and Europe. Nucleotides are chemically modified to protect AONs from degradation, enhance bioavailability and increase RNA affinity. Whereas single stranded AONs can efficiently be delivered systemically, delivery of double stranded AONs requires capsulation in lipid nanoparticles or binding to a conjugate as the uptake enhancing backbone is hidden in this conformation. With improved chemistry, delivery vehicles and conjugates, doses can be lowered, thereby reducing the risk and occurrence of side effects. AONs can be used to knockdown or restore levels of protein. Knockdown can be achieved by single stranded or double stranded AONs binding the RNA transcript and activating RNaseH-mediated and RISC-mediated degradation respectively. Transcript binding by AONs can also prevent translation, hence reducing protein levels. For protein restoration, single stranded AONs are used to modulate pre-mRNA splicing and either include or skip an exon to restore protein production. Intervening at a genetic level, AONs provide therapeutic options for inherited metabolic diseases as well. This review provides an overview of the different AON approaches, with a focus on AONs developed for inborn errors of metabolism.

Keywords: RNA therapeutics; antisense oligonucleotides; personalized medicine; splicing modulation; targeted gene knockdown; therapies.

FIGURE 1

AON‐mediated protein knockdown. A, The pre‐mRNA transcribed from the DNA is spliced and capped to obtain the final mRNA, which is exported to the cytoplasm and translated into protein at the ribosomes. B, As the single stranded AON binds to the mRNA, this complex is recognized and degraded by RNaseH, an endonuclease present in nucleus and cytoplasm, blocking protein production. C, From the double stranded siRNA, the guide strand is incorporated into the RNA‐induced silencing complex (RISC). This complex specifically binds the targeted mRNA, inducing its degradation and inhibiting protein production. D, The AON binds the mRNA, thereby changing its conformation, preventing the formation of the ribosome and blocking the process of protein translation

FIGURE 2

Schematic depiction of AON‐mediated protein restoration strategies using on disease examples. A, Exon inclusion. Normally, the majority of SMN2 transcripts does not include exon 7 which prevents the production of functional protein. By blocking an intronic splice silencer that prevents recognition of exon 7, AON treatment stimulates the inclusion of exon 7 and thereby production of functional protein. B, Exon skipping. The deletion of exons 49 and 50 disrupts the DMD open reading frame leading to a premature stop codon. AON‐mediated skipping of exon 51 restores the reading frame and allows production of partially functional protein. C, Restoring pseudoexon inclusion. When a variant creates a novel intronic cryptic splice site, a pseudoexon can be included in the CLN7 transcript. Blocking of this cryptic splice site by AONs restores normal splicing. D, Restoring partial intron retention. A variant‐induced intronic cryptic splice site leads to partial intron retention of FECH as this splice site is now used as splice acceptor site. Blocking of the cryptic splice acceptor site restores normal splicing. E, Restoring cryptic splicing. In case of a variant‐induced exonic cryptic splice site as in IDS, part of the exon is excluded as the cryptic splice site is used. AON treatment prevents the used of the cryptic splice site and stimulates use of the canonical splice site. F, Modulating alternative splicing. A variant in GAA silences the normal splice acceptor site of exon 2, thereby enabling the inclusion of a natural pseudoexon. Blocking of the pseudoexon splice sites with AONs restores normal splicing. G, Another way to prevent the inclusion of a pseudoexon in GAA is by strengthening the canonical splice sites of exon 2 by blocking of exonic splice silencers in exon 2 with AONs. Note this figure shows several but limited examples of protein restoring strategies. Star, variant; triangle, cryptic splice site