RNA-Targeting Splicing Modifiers: Drug Development and Screening Assays

Abstract

RNA splicing is an essential step in producing mature messenger RNA (mRNA) and other RNA species. Harnessing RNA splicing modifiers as a new pharmacological modality is promising for the treatment of diseases caused by aberrant splicing. This drug modality can be used for infectious diseases by disrupting the splicing of essential pathogenic genes. Several antisense oligonucleotide splicing modifiers were approved by the U.S. Food and Drug Administration (FDA) for the treatment of spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD). Recently, a small-molecule splicing modifier, risdiplam, was also approved for the treatment of SMA, highlighting small molecules as important warheads in the arsenal for regulating RNA splicing. The cellular targets of these approved drugs are all mRNA precursors (pre-mRNAs) in human cells. The development of novel RNA-targeting splicing modifiers can not only expand the scope of drug targets to include many previously considered "undruggable" genes but also enrich the chemical-genetic toolbox for basic biomedical research. In this review, we summarized known splicing modifiers, screening methods for novel splicing modifiers, and the chemical space occupied by the small-molecule splicing modifiers.

Keywords: RNA-targeting; alternative splicing; antisense oligonucleotide; high-throughput screening; small molecule; splicing modifier.

Figure 1

Splicing reaction with (a) adenosine as the branch point, which occurs in spliceosome-dependent splicing and group II introns, and (b) exogenous guanosine binding in the group I introns.

Figure 2

Regulatory mechanism for spliceosome-dependent splicing. ESE = exonic splicing enhancer, ESS = exonic splicing silencer, ISE = intronic splicing enhancer, ISS = intronic splicing silencer. A stem-loop structure at the 5′ splice was shown as an illustration of functional structural elements in regulating RNA splicing. The figure is modified from ViralZone, SIB Swiss Institute of Bioinformatics.

Figure 3

Therapeutic strategy of Eteplirsen for the treatment of DMD.

Figure 4

Differential splicing patterns for SMN1 and SMN2. ~85% of the SMN2 transcript has exon 7 skipped due to the C-to-T transition, which mediates the loss of interaction of SRSF1 and gain of the interaction of hnRNPA1. Most type I SMA patients have deleted SMN1 but wildtype SMN2. Nusinersen, RG-7916, and LMI-070 are all interventional therapy to restore exon 7 inclusion in SMN2.

Figure 5

(a) Binding sites of the existing drugs for the treatment of SMA. Risdiplam has two binding sites on the SMN2 pre-mRNA exon7. (b) The structures of known RNA-targeting small-molecule splicing modifiers for SMN2 exon 7.

Figure 6

(a,b) Two genes in influenza A undergo splicing: M and NS1. The dotted box in M is the proposed region responsible for splicing switch (enlarged in (c)). The spliced isoform of NS1 is named nuclear export protein (NEP). (c) An equilibrium between a hairpin and a pseudoknot structure was proposed to control the splicing of M in influenza A [115].

Figure 7

RNA splicing in HIV-1 genes tat and rev. The figure represents only two sets of the potential splice sites out of four alternative 5′ splice sites and eight alternative 3′ splice sites [125]. The figure is modified from ViralZone, SIB Swiss Institute of Bioinformatics.

Figure 8

(a) A stem-loop controls the exon 10 splicing in the MAPT gene and the binding site of the known splicing modifiers. (b) The structures of known RNA-targeting small-molecule splicing modifiers for MAPT exon 10.

Figure 9

Binding sites for the ASOs used in the SMN2 exon 7 and intron 7 walk. The position of complementarity of each ASO along the sequence of interest is indicated by a horizontal line. +: promotion of the exon 7 inclusion, −: inhibition of the exon 7 inclusion, *: no effect on alternative splicing. The figure is modified from refs [101,153] (copyright © 2021 Hua et al. [153]; © 2021 The American Society of Human Genetics [101]). The “ASO walking” experiment determined the splicing silencer elements (red underline).

Figure 10

(a) Design of a minigene reporter assay for spliceosome-dependent splicing. By single-point addition, the firefly (FF) luciferase at the 3′-end will only be in-frame when exon x is included. (b) Design of in vitro FRET assay for group II intron splicing.

Figure 11

(a) RT-qPCR assay for the exon–exon junction sequence. (b) Fluorescent competition assay for MAPT exon 10 splicing. (c) Exon junction complex (EJC) detection assay (Copyright © 2021, American Society for Microbiology. All Rights Reserved [166]). HRP = horseradish peroxidase.

Figure 12

Chemical modifications of ASOs: (a) phosphorothioate and 2′-O-methoxyethyl (MOE), (b) locked nucleic acid (LNA), (c) peptide nucleic acid (PNA), and (d) phosphorodiamidate morpholino oligomer (PMO). Red = modifications to the native DNA or RNA structure.

Figure 13

Distribution of key physicochemical properties of the known splicing modifiers and the FDA approved drugs in Table 4.