All RNA-targeted therapeutic technologies exploit oligonucleotides that bind to target RNA, but they differ in their mechanism of action and produce different effects.
Small interfering RNAs, antisense oligonucleotides and external guide sequences lead to enzyme-dependent degradation of targeted mRNA. Drugs involving these approaches are designed to reduce the level of harmful gene products such as viral or bacterial proteins or disease-promoting cellular proteins. They could be useful against cancer as well as viral and bacterial infections, or used to prevent the accumulation of high levels of cholesterol in the bloodstream.
Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading RNA. Splice-switching oligonucleotides are discussed in detail in this Review; these oligonucleotides redirect alternative splicing, repair defective RNA or restore the production of proteins that are missing because of genetic defects. Splice-switching oligonucleotide-based drugs should be useful for the treatment of genetic diseases such as Duchenne muscular dystrophy, spinal muscular atrophy and β-thalassaemia.
Compared to classical small-molecule drugs, it is much more difficult to achieve intracellular delivery with oligonucleotides; this is still a major issue for this class of drugs. The advantage of oligonucleotides is their high specificity, which results from sequence-specific base pairing to target RNA.
The oligonucleotide-based drug fomivirsen was approved by the US Food and Drug Administration in 1998 for the treatment of viral retinitis in patients with AIDS. Oligonucleotide-based drugs are now in advanced clinical trials for the treatment of cancer and Duchenne muscular dystrophy as well as for lowering high cholesterol levels.
Here, we discuss three RNA-based therapeutic technologies exploiting various oligonucleotides that bind to RNA by base pairing in a sequence-specific manner yet have different mechanisms of action and effects. RNA interference and antisense oligonucleotides downregulate gene expression by inducing enzyme-dependent degradation of targeted mRNA. Steric-blocking oligonucleotides block the access of cellular machinery to pre-mRNA and mRNA without degrading the RNA. Through this mechanism, steric-blocking oligonucleotides can redirect alternative splicing, repair defective RNA, restore protein production or downregulate gene expression. Moreover, they can be extensively chemically modified to acquire more drug-like properties. The ability of RNA-blocking oligonucleotides to restore gene function makes them best suited for the treatment of genetic disorders. Positive results from clinical trials for the treatment of Duchenne muscular dystrophy show that this technology is close to achieving its clinical potential.
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R.K. would like to thank his past and present colleagues at AVI BioPharma for helpful comments on this article. A.R.K. thanks his collaborators at Cold Spring Harbor Laboratory and Isis Pharmaceuticals for helpful discussions.
Ryszard Kole is an employee and shareholder of AVI BioPharma.
Adrian R. Krainer is a consultant for, and has ongoing collaborations with, Isis Pharmaceuticals.
Sidney Altman declares no competing financial interests.
- RNA interference
(RNAi). A form of post-transcriptional gene silencing in which the expression or transfection of double-stranded RNA induces degradation by nucleases of the homologous endogenous transcripts, resulting in the reduction or loss of gene activity.
- Antisense oligonucleotides
Oligonucleotides that bind to complementary mRNA by base pairing and induce cleavage of targeted mRNA by ribonuclease H, an enzyme that degrades RNA in RNA–DNA duplexes.
- Small interfering RNAs
(siRNAs). Synthetic, short, 21–22-nucleotide-long double-stranded RNAs with chemical modifications designed to increase their stability and cellular uptake. One strand of siRNA hybridizes to targeted mRNA and allows mRNA degradation.
- RNA-induced silencing complex
(RISC). A multiprotein complex that, when combined with small interfering RNA (siRNA), affects mRNA degradation. A key component of RISC is an endonuclease, argonaute 2, which cleaves the targeted mRNA within the siRNA–mRNA duplex.
- Translation-suppressing oligonucleotides
(TSOs). Modified oligonucleotides that block mRNA sequences near the initiation of the translation codon (AUG), interfere with the binding of ribosomes to mRNA and inhibit the translation of undesirable proteins. TSO–mRNA duplexes are not recognized by ribonuclease H or RNA-induced silencing complex, and the mRNA is therefore not cleaved.
- External guide sequence
(EGS). A short RNA sequence designed to bind to targeted mRNA and form a structure that is recognized by a tRNA-processing ribozyme, ribonuclease P. Ribonuclease P cleaves mRNA and thereby downregulates the function of a targeted gene.
- Splice-switching oligonucleotides
(SSOs). Chemically modified oligonucleotides that block sequences in pre-mRNA that are involved in pre-mRNA splicing, and redirect mRNA splicing pathways. SSO–pre-mRNA duplexes are not recognized by ribonuclease H or the RNA-induced silencing complex, and the pre-mRNA is thus not cleaved.
- Alternative splicing
Splicing of pre-mRNA to yield more than one kind of mRNA — that is, different splice variants — by frequently including or excluding an exon.
- Translational reading frame
Arrangement of mRNA nucleotides into triplets (codons) that, when read by the ribosome, are translated into one amino acid per codon. The reading frame usually starts with a translation initiation codon, AUG — for example, AUG UUU ACA GCA. Deletion of a nucleotide, such as the third uridylic in the second codon, changes the reading frame to AUG 'UUA CAG CA', thus preventing the translation of the desired protein.
- Exonic splicing silencers
Sequences present in exons in pre-mRNA that contribute to the modulation of splicing. They can inhibit the inclusion of a given exon into mature mRNA during splicing. Exonic splicing enhancers also exist.
- Intronic splicing silencers
Sequences present in introns in pre-mRNA that contribute to the modulation of splicing. They can inhibit the inclusion of a given intron into mature mRNA during splicing. Intronic splicing enhancers also exist.
- Cryptic splice site
A splice site that is not functional under normal conditions but becomes activated if a mutation modifies its sequence to resemble a functional splice site or if an adjacent normal splice site is inactivated by a mutation.
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Kole, R., Krainer, A. & Altman, S. RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nat Rev Drug Discov 11, 125–140 (2012). https://doi.org/10.1038/nrd3625
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