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Targeting RNA splicing for disease therapy

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Abstract Splicing of pre‐messenger RNA into mature messenger RNA is an essential step for the expression of most genes in higher eukaryotes. Defects in this process typically affect cellular function and can have pathological consequences. Many human genetic diseases are caused by mutations that cause splicing defects. Furthermore, a number of diseases are associated with splicing defects that are not attributed to overt mutations. Targeting splicing directly to correct disease‐associated aberrant splicing is a logical approach to therapy. Splicing is a favorable intervention point for disease therapeutics, because it is an early step in gene expression and does not alter the genome. Significant advances have been made in the development of approaches to manipulate splicing for therapy. Splicing can be manipulated with a number of tools including antisense oligonucleotides, modified small nuclear RNAs (snRNAs), trans‐splicing, and small molecule compounds, all of which have been used to increase specific alternatively spliced isoforms or to correct aberrant gene expression resulting from gene mutations that alter splicing. Here we describe clinically relevant splicing defects in disease states, the current tools used to target and alter splicing, specific mutations and diseases that are being targeted using splice‐modulating approaches, and emerging therapeutics. WIREs RNA 2013, 4:247–266. doi: 10.1002/wrna.1158 The authors have declared no conflicts of interest for this article. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing RNA in Disease and Development > RNA in Disease

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Splicing, alternative splicing, and pathogenic mutations that affect splicing outcomes. (a) A model of the splicing sequences and the components involved in their initial recognition during splicing by the major spliceosome. Exons are depicted as boxes and introns are lines. The canonical 5′ splice site (5′ss), branch point sequence (BPS), polypyrimidine tact (py tract), and 3′ splice site (3′ss) sequences are shown along with their interactions with the U1 and U2 snRNPs. The gray‐lined snRNA and the major protein components of the snRNPs are labeled. Intronic and exonic splicing silencers (orange: ISS and red: ESS) and enhancers (dark green: ESE and light green: ISE) are depicted either with or without their trans‐acting proteins bound. Alternative splicing of the middle exon produces mRNA isoforms 1 and 2 and results in two distinct protein isoforms, 1 and 2. (b) Common types of disease‐causing mutations that disrupt splicing are labeled in red along with their possible outcomes and the aberrant splicing pathway (bottom). De novo cryptic splice site mutations are represented by the terminal dinucleotides, GU (5′ss) and AG (3′ss).

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Tools used to induce aberrant splicing for disease therapy. (a) Modulating alternative splicing for disease therapy using ASOs, bifunctional ASOs (e.g., 2′F ASO), or small molecules (SM). These therapeutics can be used to either promote exon inclusion or skipping in the presence or absence of a mutation. (b) Targeting splicing to disrupt gene expression by the use of ASOs to promote exon skipping to disrupt the reading frame (green versus yellow exons) and promote nonsense‐mediated decay (NMD). This process is known as forced‐splicing‐dependent nonsense‐mediated decay (FSD‐NMD). (c) Splicing‐mediated rescue of gene expression disrupted by nonsense mutations. Top panel: The C to T point mutation that introduces a PTC either resulting in NMD, or a truncated, nonfunctional protein. Middle panel: The use of ASOs (blue) to block the 5′ and 3′ splice sites promotes exon skipping. The reading frame remains intact (green exons) resulting in a truncated but functional protein. Bottom panel: The use of a PTM to replace the 3′ portion of the mRNA because the exons flanking the exon containing the PTC mutation are not in the same reading frame (green versus yellow exons). The use of the PTM results in a full‐length and functional protein.

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Tools to correct aberrant splicing caused by mutations. (a) ASO‐based correction of mutation‐induced aberrant splicing depicting ways in which ASOs (blue) can be used to correct for mutations (red) to promote proper splicing and exon inclusion. (b) Trans‐splicing rescue of mutation‐induced aberrant splicing depicting the replacement of the 3′ or 5′ portion of an RNA with mutated 3′ or 5′ splice sites, respectively, by a pre‐trans‐splicing molecule (PTM). Core splicing sequence mutations are depicted in red. (c) Modified U1 snRNA compensation for a 5′ splice site mutation (red). Exogenous U1 snRNA with a compensatory mutation allows for base‐pairing with the 5′ splice site and the restoration of exon recognition and inclusion. (d) Small molecule compounds that modulate alternative splicing. Small molecules act in trans by binding spliceosome components to promote alternative exon inclusion to compensate for a mutation (examples noted in red lettering and represented with an X) or to alter mRNA isoforms for therapeutic benefit. Depicted are examples of small molecules that inhibit splicing (spliceostation A, pladienolide B, and sudemycins) and two small molecules that promote exon inclusion (PTK‐SMA1 and digitoxin).

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RNA in Disease and Development > RNA in Disease
RNA Processing > Splicing Mechanisms
RNA Processing > Splicing Regulation/Alternative Splicing

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