RNA therapeutics: beyond RNA interference and antisense oligonucleotides

Abstract

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.

Figure 1. Mechanisms of oligonucleotide-induced downregulation of gene expression

A. SiRNA. Synthetic double-stranded short interfering RNA (siRNA) is complexed with components of RNA interference (RNAi) pathway, dicer, AGO2 and other proteins, forming RNA-interference-silencing-complex, RISC. RISC binds to a target mRNA via the unwound guide strand of siRNA, allowing AGO 2 to degrade the RNA. RISC-bound siRNA can also bind with mismatches to unintended mRNAs, leading to significant off-target effects (see main text). B. Antisense gapmer oligonucleotides. These usually have a PS backbone with flanks additionally modified with 2′MOE or 2′OMe residues (Red in figure. flank modifications increase resistance of the ASO to degradation and enhance binding to target mRNA. The unmodified “gap” in a gapmer/mRNA duplex is recognized by RNase H, a ribonuclease that degrades duplexed mRNA. C. Translation suppressing oligomers (TSO). PMO and their derivatives or oligonucleotides fully substituted with 2′MOE or 2′OMe residues are not recognized by RISC or RNase H and do not lead to RNA degradation. Nevertheless, they lead to downregulation of gene expression by steric blockade of ribosome access to mRNA and suppression of protein translation. D. External Guide Sequence (EGS) and RNase P. A PPMO is designed to hybridize to targeted bacterial mRNA and form stem-loop structures such that the resulting duplex resembles tRNA. In bacteria, a tRNA processing ribozyme RNase P recognizes this structure and cleaves mRNA.

Figure 2

Oligonucleotide Chemistries. Top. All oligonucleotides are negatively charged. Phosphorothioate (PS) backbone and 2′MOE and 2′OMe substituents increase resistance to degradation and promote protein binding. In addition, LNA modification markedly increases binding of the oligonucleotide to the target mRNA. Bottom. In Phosphorodiamidate morpholino oligomers (PMO) ribose (RNA) or deoxyribose (DNA) are replaced with morpholine rings and the phosphorothioate or phosphodiester (RNA) groups are replaced with uncharged phosphorodiamidate groups, resulting in a compound that is neutral and very resistant to degradation. Positively charged piperazine residues in PMOplus or positively charged, arginine rich peptides in PPMO dramatically improve intracellular uptake of the oligomers.

Figure 3

Mechanisms of oligonucleotide induced modulation of gene expression. A. Exon skipping and splice switching oligonucleotides (SSO). A chemically modified, RNA blocking oligonucleotide targeted to a splice site or an exon-internal exon splicing enhancer (ESE) in pre-mRNA prevents proper assembly of the spliceosome on the exon and redirects splicing to another pathway, effecting skipping of the targeted exon. Such alternatively spliced mRNA may code for a novel protein with favorable properties, may restore translation, if exon skipping restores a reading frame, as is the case in DMD, or may change the balance of alternative splice variants These outcomes cannot be accomplished by ASO or siRNA, which degrade target mRNA. B. Exon retention by SSO. Some exons are poorly spliced into mRNA because they contain exon splicing silencer (ESS) elements. An SSO designed to block an ESS interferes with this element’s role in splicing and promotes exon inclusion, as has been demonstrated in the case of SMA, a genetic disorder. C. Restoration of correct splicing and RNA repair by SSO. Top. An intron mutation may create and/or activate aberrant splice sites, which leads to inclusion of an intronic fragment into the spliced mRNA, in essence creating a pseudo exon and interfering with the translational reading frame. Bottom. An SSO targeted to the aberrant splicing elements restores correct splicing and allows translation of the correct, fully functional protein. D. Displacement of splicing factors from triplet repeats. An extended triplet repeat CUGCUGCUG… in DMPK pre-mRNA attracts a splicing factor, muscleblind (MBNL) and titrates it out from the nucleoplasm. As a result, DMPK and several other pre-mRNAs are not properly processed, preventing translation of a number of proteins and causing myotonic dystrophy, a neuromuscular disorder. A modified, steric-blocking oligonucleotide displaces MBNL, allowing it to participate in splicing of appropriate mRNAs and restoring function the affected muscle.