Targeting Splicing in the Treatment of Human Disease

Abstract

The tightly regulated process of precursor messenger RNA (pre-mRNA) alternative splicing (AS) is a key mechanism in the regulation of gene expression. Defects in this regulatory process affect cellular functions and are the cause of many human diseases. Recent advances in our understanding of splicing regulation have led to the development of new tools for manipulating splicing for therapeutic purposes. Several tools, including antisense oligonucleotides and trans-splicing, have been developed to target and alter splicing to correct misregulated gene expression or to modulate transcript isoform levels. At present, deregulated AS is recognized as an important area for therapeutic intervention. Here, we summarize the major hallmarks of the splicing process, the clinical implications that arise from alterations in this process, and the current tools that can be used to deliver, target, and correct deficiencies of this key pre-mRNA processing event.

Keywords: alternative splicing; genetic disease; precursor messenger RNA; therapy.

Figure 1

Schematic representation of the spliceosome assembly and pre-mRNA splicing. In the first step of the splicing process, the 5′ splice site (GU, 5′ SS) is bound by the U1 snRNP, and the splicing factors SF1/BBP and U2AF cooperatively recognize the branch point sequence (BPS), the polypyrimidine (Py) tract, and the 3′ splice site (AG, 3′ SS) to assemble complex E [11,12]. The binding of the U2 snRNP to the BPS results in the pre-spliceosomal complex A [13]. Subsequent steps lead to the binding of the U4/U5–U6 tri-snRNP and the formation of complex B [14]. Complex C is assembled after rearrangements that detach the U1 and U4 snRNPs [15] to generate complex B*. Complex C is responsible for the two trans-esterification reactions at the SS. Additional rearrangements result in the excision of the intron, which is removed as a lariat RNA, and ligation of the exons. The U2, U5, and U6 snRNPs are then released from the complex and recycled for subsequent rounds of splicing [16,17].

Figure 2

AS regulation by cis elements and trans-acting factors. The core cis sequence elements that define the exon/intron boundaries (5′ and 3′ splice sites (SS), GU-AG, polypyrimidine (Py) tract, and branch point sequence (BPS)) are poorly conserved. Additional enhancer and silencer elements in exons and in introns (ESE: exonic splicing enhancers; ESI: exonic splicing silencers; ISE: intronic splicing enhancers; ISI: intronic splicing silencers) contribute to the specificity of AS regulation. Trans-acting splicing factors, such as SR family proteins and heterogeneous nuclear ribonucleoprotein particles (hnRNPs), bind to enhancers and silencers and interact with spliceosomal components [18,19,20]. In general, SR proteins bound to enhancers facilitate exon definition, and hnRNPs inhibit this process.

Figure 3

Schematic representation of different types of alternative transcriptional or splicing events, with exons (boxes) and introns (lines). Constitutive exons are shown in green and alternatively spliced exons in purple. Dashed lines indicate the AS event. Exon skipping (a); alternative 3′ (b) and 5′ SS selection (c); intron retention (d); mutually exclusive exons (e); alternative promoter usage (f); and alternative polyadenylation (g) events are shown. Like alternative splicing (AS), usage of alternative promoter and polyadenylation sites allow a single gene to encode multiple mRNA transcripts.

Figure 4

Three different strategies to target splicing for gene modification. (a) The diagram depicts an antisense oligonucleotide (ASO)-based strategy to target an alternatively spliced exon (in orange). In the absence of the ASO, the spliceosome is assembled and the exon is included in the mRNA; in the presence of the ASO, the spliceosome is sterically blocked and the exon is skipped and not included in the mRNA. (b) SMaRT strategy for trans-splicing by 5′ exon replacement. Schematic representation of the gene-specific pre-trans-splicing molecule (PTM). The coding sequence of the PTM consists of an exon (in green), and the trans-splicing domain of the PTM comprises a binding-domain (BD) complementary to the 3′ end of the gene intron as well as highly conserved BPS and Py sequences. (c) Illustration depicting the mechanism by which siRNA can inhibit the expression of specific exon-containing target gene products by hybridizing to the mRNA and triggering RISC-mediated degradation or translational inhibition.

Figure 5

Schematic representation of different types of nanoparticles used to deliver biomolecules. (a) Solid lipid nanoparticles (SLNs); (b) Polymeric nanoparticles; (c) Inorganic core-shell nanoparticles (Au: gold, Fe₃O₄: iron oxide); (d) Lipid bilayer-based liposomes.