Antisense Oligonucleotide Targeting of 3’-UTR of mRNA for Expression Knockdown

Part of the Methods in Molecular Biology book series (MIMB, volume 1828)


With the recent conditional approval of an antisense oligonucleotide (AON) that restores the reading frame of DMD transcript in a subset of Duchenne muscular dystrophy patients, it has been established that AONs sharing similar chemistry have clear clinical potential. Genetic diseases, such as facioscapulohumeral dystrophy (FSHD), can be the result of gain-of-function mutations. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3’UTR elements, it follows that targeting these elements with AONs have the potential to induce gene silencing. Aberrant expression of the Double homeobox 4 (DUX4) transcription factor and the downstream consequences of such expression is the hallmark of FSHD. Here we describe the bioinformatic strategies behind the design of AONs targeting polyadenylation signals and the methodologies relevant to their in vitro screening for efficacy and safety, including analysis of expression at the transcript and protein level of the specific target and downstream genes, and measurement of the effect on the fusion index of myotubes. The targeting of permissive DUX4 and MSTN are used as examples. MSTN encodes for myostatin, a negative regulator of myogenesis; the downregulation of MSTN expression has the potential to address the muscular atrophy associated with muscular dystrophies, sarcopenia, cancer and acquired immunodeficiency syndrome.

Key words

Antisense oligonucleotides Facioscapulohumeral dystrophy DUX4 Myostatin Polyadenylation signal 



The authors thank the Rosetrees Trust UK and Muscular Dystrophy UK, and by association French Association against Myopathies (AFM-Téléthon, France) for funding the work. A patent to protect PMOs targeting the DUX4 polyadenylation signal has been filed by University Pierre Marie Currie and Royal Holloway-University of London. The authors declare that they have no conflict of interest other than those listed above.


  1. 1.
    Colot HV, Stutz F, Rosbash M (1996) The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex. Genes Dev 10(13):1699–1708CrossRefGoogle Scholar
  2. 2.
    Danckwardt S, Hentze MW, Kulozik AE (2008) 3′ end mRNA processing: molecular mechanisms and implications for health and disease. EMBO J 27(3):482–498CrossRefGoogle Scholar
  3. 3.
    Proudfoot NJ, Furger A, Dye MJ (2002) Integrating mRNA processing with transcription. Cell 108(4):501–512CrossRefGoogle Scholar
  4. 4.
    Maniatis T, Kee SG, Efstratiadis A, Kafatos FC (1976) Amplification and characterization of a beta-globin gene synthesized in vitro. Cell 8(2):163–182CrossRefGoogle Scholar
  5. 5.
    Hewitt JE (2015) Loss of epigenetic silencing of the DUX4 transcription factor gene in facioscapulohumeral muscular dystrophy. Hum Mol Genet 24(R1):R17–R23CrossRefGoogle Scholar
  6. 6.
    Tawil R, van der Maarel SM, Tapscott SJ (2014) Facioscapulohumeral dystrophy: the path to consensus on pathophysiology. Skelet Muscle 4:12CrossRefGoogle Scholar
  7. 7.
    Marsollier AC, Ciszewski L, Mariot V et al (2016) Antisense targeting of 3'end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for Facioscapulohumeral dystrophy: a new gene silencing approach. Hum Mol Genet 25(8):1468–1478CrossRefGoogle Scholar
  8. 8.
    McPherron AC, Lawler AM, Lee SJ (1997) Regulation of skeletal muscle mass in mice by a new TGF-p superfamily member. Nature 387(6628):83–90CrossRefGoogle Scholar
  9. 9.
    Hübner C, Riebel T, Kömen W et al (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N Engl J Med 350(26):2682–2688CrossRefGoogle Scholar
  10. 10.
    McPherron AC, Lee S (1997) Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Scie USA 94:12457–12461CrossRefGoogle Scholar
  11. 11.
    Smith RC, Lin BK (2013) Myostatin inhibitors as therapies for muscle wasting associated with cancer and other disorders. Curr Opin Support Palliat Care 7(4):352–360CrossRefGoogle Scholar
  12. 12.
    Siva K, Covello G, Denti M (2014) Exon-skipping antisense oligonucleotides to correct missplicing in neurogenetic diseases. Nucleic Acid Therapeutics 24(1):69–86CrossRefGoogle Scholar
  13. 13.
    Ahmed F, Kunar M, Raghava GPS (2009) Prediction of polyadenylation signals in human DNA sequences using nucleotide frequencies. In Silico Biol 9:135–148PubMedGoogle Scholar
  14. 14.
    Popplewell LJ, Trollet C, Dickson G, Graham IR (2009) Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene. Mol Ther 17:554–561CrossRefGoogle Scholar
  15. 15.
    Gebski BL, Mann CJ, Fletcher S, Wilton SD (2003) Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle. Hum Mol Genet 12:1801–1811CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre of Biomedical Sciences, School of Biological Sciences, Royal HollowayUniversity of LondonEghamUK

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