Antisense Oligonucleotides: An Emerging Area in Drug Discovery and Development

Abstract

Antisense oligonucleotides (ASOs) bind sequence specifically to the target RNA and modulate protein expression through several different mechanisms. The ASO field is an emerging area of drug development that targets the disease source at the RNA level and offers a promising alternative to therapies targeting downstream processes. To translate ASO-based therapies into a clinical success, it is crucial to overcome the challenges associated with off-target side effects and insufficient biological activity. In this regard, several chemical modifications and diverse delivery strategies have been explored. In this review, we systematically discuss the chemical modifications, mechanism of action, and optimized delivery strategies of several different classes of ASOs. Further, we highlight the recent advances made in development of ASO-based drugs with a focus on drugs that are approved by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for clinical applications. We also discuss various promising ASO-based drug candidates in the clinical trials, and the outstanding opportunity of emerging microRNA as a viable therapeutic target for future ASO-based therapies.

Keywords: RNA; antisense oligonucleotides; chemical modifications; clinical trials.

Figure 1

Antisense oligonucleotide (ASO) design. Chemically modified ASOs provide nuclease resistance and improved binding affinity to their target. Full length modified design represents chemical modifications throughout the sequence. Gapmer design includes a central region consist of DNA nucleotides and a stretch of LNA or 2′ modifications or PS nucleotides flanking both terminals of the sequence. Mixmer design contains LNA (or 2′ modifications) and DNA nucleotides present sequentially.

Figure 2

Mechanism of action of antisense oligonucleotides (ASOs): ASOs act by either causing (1) RNA cleavage or (2) RNA blockage. (1a) RNase H1 mediated cleavage, (1b) RNA interference (RNAi), (2a) Steric hindrance, and (2b) Splice modulation. (1a) ASO-mRNA heteroduplex recruits RNase H1 enzyme and this enzyme cleaves the target mRNA. (1b) mRNA degradation by siRNA associated with RNA inducing silencing complex (RISC). (2a) ASO-mRNA complex sterically blocks and prevents the interaction of mRNA with ribosomes for protein translation. (2b) is an example of splice switching oligonucleotides (SSO). Rectangles depict the coding exon regions separated by a curve depicting the non-coding intron region of the pre-mRNA. The red square represents the mutated region of the exon. The dashed line represents the splicing pattern of pre-mRNA. RNase H1 mediated cleavage, RNA interference, and steric hindrance mechanisms produce less protein, while splice modulation produce the correct form of protein. Phosphorothioate (PS) and 5′methylcytosine base modification induces mRNA cleavage. Peptide nucleic acids (PNA), 2′-O-methyl (2′-O-Me) and 2′-O-methoxyethyl (2′-O-MOE) modifications, phosphorodiamidate morpholino (PMO), locked nucleic acid (LNA) act on mRNA to sterically block its translation or these ASOs can act as SSO to modulate splicing pattern.

Figure 3

miRNA biogenesis and mechanism of action. miRNA is transcribed by RNA polymerase II (RNAP II) to form double stranded hairpin loop structure called pri-miRNA, which gets cleaved by nuclease Drosha to form pre-miRNA. Exportin transports the pre-miRNA to the cytoplasm where it is further processed by Dicer to form a single stranded mature miRNA. The mature miRNA is uploaded in the RNA induced silencing complex (RISC) where it associates with Argonaute 2 protein. This miRNA-RISC complex interacts with the seed region of the mRNA and regulates the mRNA translation by either mRNA cleavage or by steric hindrance.