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Antisense technology: an overview and prospectus

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From Nature Reviews Drug Discovery

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Abstract

Antisense technology is now beginning to deliver on its promise to treat diseases by targeting RNA. Nine single-stranded antisense oligonucleotide (ASO) drugs representing four chemical classes, two mechanisms of action and four routes of administration have been approved for commercial use, including the first RNA-targeted drug to be a major commercial success, nusinersen. Although all the approved drugs are for use in patients with rare diseases, many of the ASOs in late- and middle-stage clinical development are intended to treat patients with very common diseases. ASOs in development are showing substantial improvements in potency and performance based on advances in medicinal chemistry, understanding of molecular mechanisms and targeted delivery. Moreover, the ASOs in development include additional mechanisms of action and routes of administration such as aerosol and oral formulations. Here, we describe the key technological advances that have enabled this progress and discuss recent clinical trials that illustrate the impact of these advances on the performance of ASOs in a wide range of therapeutic applications. We also consider strategic issues such as target selection and provide perspectives on the future of the field.

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Fig. 1: An overview of progress in antisense technology.
Fig. 2: Advances in the medicinal chemistry of antisense technology.
Fig. 3: Post-hybridization mechanisms of antisense oligonucleotides.
Fig. 4: A major molecular mechanism of toxicity of phosphorothioate antisense oligonucleotides.
Fig. 5: Local delivery of phosphorothioate antisense oligonucleotides.
Fig. 6: Schematic representation of RNA degradation mediated by RNase H1 or AGO2.

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Acknowledgements

The authors wish to thank K. Butler for assistance in manuscript preparation, and W. Sullivan for figure preparation. This work is supported by internal funding from Ionis Pharmaceuticals.

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  1. Ionis Pharmaceuticals, Inc., Carlsbad, CA, USA

    Stanley T. Crooke, Brenda F. Baker, Rosanne M. Crooke & Xue-hai Liang

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Correspondence to Stanley T. Crooke.

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Akcea and Ionis report positive topline phase 2 results of AKCEA-APOCIII-LRx: https://ir.ionispharma.com/news-releases/news-release-details/akcea-and-ionis-report-positive-topline-phase-2-results-akcea

BioMarin announces withdrawal of market authorization application for Kyndrisa (drisapersen) in Europe: https://www.globenewswire.com/news-release/2016/05/31/844933/0/en/BioMarin-Announces-Withdrawal-of-Market-Authorization-Application-for-Kyndrisa-drisapersen-in-Europe.html

Miragen announces internal review of preliminary topline data for the phase 2 solar clinical trial of cobomarsen in patients with cutaneous T-cell lymphoma (CTCL): http://investors.miragen.com/press-releases/press-release/2020/miRagen-Announces-Internal-Review-of-Preliminary-Topline-Data-for-the-Phase-2-SOLAR-Clinical-Trial-of-Cobomarsen-in-Patients-with-Cutaneous-T-Cell-Lymphoma-CTCL/default.aspx

OncoGenex announces results from the phase 3 AFFINITY trial of custirsen in men with metastatic castrate-resistant prostate cancer: https://www.prnewswire.com/news-releases/oncogenex-announces-results-from-the-phase-3-affinity-trial-of-custirsen-in-men-with-metastatic-castrate-resistant-prostate-cancer-300313767.html

ProQR announces positive findings from an interim analysis in the phase 1/2 trial of QR-421a for Usher syndrome and provides business update: https://www.globenewswire.com/news-release/2020/03/31/2008955/0/en/ProQR-Announces-Positive-Findings-From-an-Interim-Analysis-in-the-Phase-1-2-trial-of-QR-421a-for-Usher-Syndrome-and-Provides-Business-Update.html

Regulus announces pipeline updates and advancements: https://www.prnewswire.com/news-releases/regulus-announces-pipeline-updates-and-advancements-300472142.html

Sarepta Therapeutics announces FDA approval of AMONDYS 45 (casimersen) injection for the treatment of Duchenne muscular dystrophy (DMD) in patients amenable to skipping exon 45: https://www.globenewswire.com/news-release/2021/02/25/2182845/0/en/Sarepta-Therapeutics-Announces-FDA-Approval-of-AMONDYS-45-casimersen-Injection-for-the-Treatment-of-Duchenne-Muscular-Dystrophy-DMD-in-Patients-Amenable-to-Skipping-Exon-45.html

Wave Life Sciences announces discontinuation of suvodirsen development for Duchenne muscular dystrophy: https://www.globenewswire.com/news-release/2019/12/16/1960830/0/en/Wave-Life-Sciences-Announces-Discontinuation-of-Suvodirsen-Development-for-Duchenne-Muscular-Dystrophy.html

Wave Life Sciences announces topline data and addition of higher dose cohort in ongoing phase 1b/2a PRECISION-HD2 trial in Huntington’s disease: https://ir.wavelifesciences.com/news-releases/news-release-details/wave-life-sciences-announces-topline-data-and-addition-higher

Glossary

Antisense oligonucleotide

(ASO). A short (12–24 nucleotides) oligonucleotide that can base-pair with complementary RNA and trigger different post-hybridization mechanisms to modulate gene expression. It can be either single-stranded or double-stranded.

Small interfering RNAs

(siRNAs). A short (~21 base pairs) RNA duplex that can trigger degradation of RNAs containing homologous sequences through endonucleolytic cleavage by AGO proteins.

RNase H1

An endoribonuclease that specifically cleaves the RNA strand present in a DNA–RNA heteroduplex and is the cellular enzyme used by antisense oligonucleotides to trigger specific degradation of complementary RNA. Its primary role in the cell is to remove R-loops.

Phosphorothioate

(PS). A backbone modification in a nucleic acid in which one non-bridging oxygen in the phosphodiester is replaced by a sulfur atom. PS modifications dramatically improve antisense oligonucleotide stability and pharmacological performance.

2′-O-methoxyethyl

(2′-MOE). A modification in which the 2′-OH in the ribose is replaced with a 2′-O-methoxyethyl group, which increases the binding affinity for RNA and improves antisense oligonucleotide drug performance.

2′-constrained ethyl

(2′-cEt). A modification that links the 2′ and 4′ positions in the ribose, which substantially increases the binding affinity for RNA and improves antisense oligonucleotide drug performance.

N-acetylgalactosamine

(GalNAc). A sugar derivative that binds with high affinity to the asialoglycoprotein receptor, which is selectively expressed on hepatocytes. Conjugation to triantennary GalNAc ligands is used to target antisense oligonucleotides to the liver.

Gapmer

A term that describes the design of a phosphorothioate antisense oligonucleotide with several 2ʹ-modified nucleotides at both the 5′ and 3′ ends (or ‘wings’) to enhance its affinity for target RNAs and its nuclease resistance, with a central portion that consists of PS 2′-H nucleotides to support RNase-H1-mediated cleavage of the target RNA.

AGO2

An endoribonuclease component of the RNA-induced silencing complex (RISC), which mediates the RNA-degrading activity of small interfering RNAs.

Nonsense-mediated mRNA decay

(NMD). An mRNA quality control mechanism in which mRNAs containing premature stop codons are selectively degraded.

No-go decay

An mRNA quality control mechanism in which mRNAs containing stacks of stalled ribosomes are degraded.

Cap structures

These contain N7-methylated guanosine linked through 5′–5′ triphosphate to the first nucleotide of mRNAs. The cap structure is required for mRNA processing, nuclear export, stability and cap-dependent translation. Antisense oligonucleotides can be designed to alter the cap structure and thus inhibit translation.

Polyadenylation sites

Cleavage sites of mRNA precursors where the polyadenine tail is added. Polyadenylation site use determines the length of the 3′ untranslated region (UTR), mRNA stability and translation. Antisense oligonucleotides can alter polyadenylation site utilization, thus modulating the fate of mRNAs.

Exon skipping

A process by which an exon that is normally present in mature mRNA is omitted during splicing, and thus is not present in mature mRNA. Antisense oligonucleotides can be designed to promote exon skipping with the goal of producing a shorter, but still functional, protein.

Upstream open reading frames

(uORFs). A uORF in an mRNA is an ORF with an AUG start codon upstream of the start codon for the ORF encoding a particular protein. The presence of the uORF can inhibit the translation of the primary ORF and thereby reduce the level of protein expression.

Translation inhibitory elements

(TIEs). mRNA structural elements present in the 5′ untranslated region of many mRNAs that inhibit translation in a position- and structural-strength-dependent manner.

Exon junction complex

(EJC). A protein complex loaded to mRNA exons near the exon–exon junction positions during splicing. An EJC present downstream of a stop codon is recognized by nonsense-mediated mRNA decay (NMD) factors to trigger mRNA degradation. Antisense oligonucleotides can be designed to displace the EJC complex, inhibiting NMD.

Off-target

In the context of oligonucleotide therapeutics, off-target refers to an RNA that is not designed to be targeted by an antisense oligonucleotide (ASO) or small interfering RNA (siRNA), but is unintentionally affected owing to complementarity to the ASO or siRNA sequence.

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Crooke, S.T., Baker, B.F., Crooke, R.M. et al. Antisense technology: an overview and prospectus. Nat Rev Drug Discov 20, 427–453 (2021). https://doi.org/10.1038/s41573-021-00162-z

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