Splicing Modulation as Therapy for Human Genetic Disease


The sequencing of the human genome has led to a greater appreciation of the contribution that genetic abnormalities and splicing errors make to the burden of human disease. The modulation of splicing using antisense oligonucleotides provides an attractive therapeutic option for tailored interventions in rare genetic disease. In this review, we outline the various approaches to splicing modulation and provide examples of each strategy. Finally, we address some of the practical issues facing the field in trying to bring this technology towards clinical application using the treatment of Duchenne muscular dystrophy and spinal muscular atrophy as examples.

Key Concepts

  • Ninety‐five percent of human genes undergo splicing.
  • An estimated 50% of all human genetic diseases result from splicing abnormalities.
  • Splicing modulation using antisense oligonucleotide (ASO) technology offers the future prospect of personalised medicine tailored to specific mutations.
  • Strategies to modulate splicing include suppression of an aberrant splice isoform, switching between known splicing variants or generating a novel variant with a potential therapeutic attribute.
  • ASO chemistries are varied and have been optimised to improve target binding and resist nuclease degradation.
  • ASO targets include all pre‐mRNA sites which influence the splicing process.
  • Splicing events can be complex and unpredictable. Therefore an iterative method of design and test is required to optimise ASO efficacy.
  • ASO side‐effects are likely to stem from the ASO chemistry, off‐target effects as the result of nonspecific binding, and ASO distribution to organs when given in vivo.
  • Optimising tissue delivery of ASO remains a significant challenge.

Keywords: splicing; therapeutics; antisense oligonucleotide; genetic disease

Figure 1. Commonest ASOs in use for splicing therapeutics. (a) 2′‐O‐methyl RNA with a phosphorothioate substitution (2′‐Me‐PS). (b) 2′‐O‐methyloxyethyl RNA with a phosphorothioate substitution (2′‐MOE‐PS). (c) Phosphoroamidate morpholino oligonucleotides (PMO). (d) Locked nucleic acids (LNA).
Figure 2. Cryptic splice site blockade – ASO‐mediated splicing modulation of GHR. A mutation in intron 6 (A to G) generates a cryptic 5′ spice site leading to the splicing and inclusion of pseudoexon 6Ψ in the growth hormone receptor. The abnormal growth hormone receptor fails to localise at the cell membrane leading to growth hormone insensitivity (left panel). ASOs targeting the cryptic splice sites of pseudoexon 6Ψ cause pseudoexon skipping and restore the wild type receptor (right panel). GHR – growth hormone receptor.
Figure 3. Reading frame correction – ASO‐mediated splicing modulation of the DMD. Figure shows three exons from DMD pre‐mRNA. Exons 45–50 are absent as the result of a deletion in this example. Left panel demonstrates open reading frame disruption which leads to a prematurely terminated nonfunctional protein. The right panel demonstrates the effect of an ASO targeting exon 51. Exon 51 skipping produces a shortened mRNA but with a corrected open reading frame which produces a partially functional protein.
Figure 4. RNA engineering of novel isoforms – ASO‐mediated splicing modulation of . Figure shows three exons of . Left panel demonstrates normal splicing and the generation of the full length transcript, APOB100. Right panel demonstrates the generation of APOB87 as the result of ASO induced exon 27 skipping. APOB87 leads to a reduction in LDL secretion.


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Further Reading

Aartsma‐Rus AM (2012a) Exon skipping: methods and protocols. In: Methods in Molecular Biology. New York: Humana Press.

Aartsma‐Rus A (2012b) Overview on AON design. Methods in Molecular Biology 867: 117–129.

Cohn RD and Campbell KP (2000) Molecular basis of muscular dystrophies. Muscle and Nerve 23 (10): 1456–1471.

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Srirangalingam, Umasuthan, and Khoo, Bernard(Jul 2015) Splicing Modulation as Therapy for Human Genetic Disease. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0024458]