Skip to main content
SpringerLink
Log in

Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays

  • Protocol
  • First Online:
Exon Skipping and Inclusion Therapies

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

Abstract

Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Liu MM, Zack DJ (2013) Alternative splicing and retinal degeneration. Clin Genet 84(2):142–149. https://doi.org/10.1111/cge.12181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Slijkerman RW, Vache C, Dona M et al (2016) Antisense oligonucleotide-based splice correction for USH2A-associated retinal degeneration caused by a frequent deep-intronic mutation. Mol Ther Nucleic Acids 5(10):e381. https://doi.org/10.1038/mtna.2016.89

    Article  CAS  PubMed  Google Scholar 

  3. Liquori A, Vache C, Baux D et al (2016) Whole USH2A gene sequencing identifies several new deep intronic mutations. Hum Mutat 37(2):184–193. https://doi.org/10.1002/humu.22926

    Article  CAS  PubMed  Google Scholar 

  4. Collin RW, den Hollander AI, van der Velde-Visser SD et al (2012) Antisense oligonucleotide (AON)-based therapy for leber congenital amaurosis caused by a frequent mutation in CEP290. Mol Ther Nucleic Acids e14:1. https://doi.org/10.1038/mtna.2012.3

    Article  CAS  Google Scholar 

  5. Gerard X, Perrault I, Hanein S et al (2012) AON-mediated exon skipping restores ciliation in fibroblasts harboring the common leber congenital amaurosis CEP290 mutation. Mol Ther Nucleic Acids e29:1. https://doi.org/10.1038/mtna.2012.21

    Article  CAS  Google Scholar 

  6. Skordis LA, Dunckley MG, Yue B et al (2003) Bifunctional antisense oligonucleotides provide a trans-acting splicing enhancer that stimulates SMN2 gene expression in patient fibroblasts. Proc Natl Acad Sci U S A 100(7):4114–4119. https://doi.org/10.1073/pnas.0633863100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Rutten JW, Dauwerse HG, Peters DJ et al (2016) Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept. Brain 139(Pt 4):1123–1135. https://doi.org/10.1093/brain/aww011

    Article  PubMed  Google Scholar 

  8. van Wijk E, Dona M, Slijkerman R et al (2017) Antisense oligonucleotide-induced skipping of USH2A exon13 restores visual function in Zebrafish. Invest Ophth Vis Sci 58(8):2490–2490

    Google Scholar 

  9. Aoki Y, Yokota T, Nagata T et al (2012) Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery. Proc Natl Acad Sci U S A 109(34):13763–13768. https://doi.org/10.1073/pnas.1204638109

    Article  PubMed  PubMed Central  Google Scholar 

  10. Allikmets R, Singh N, Sun H et al (1997) A photoreceptor cell-specific ATP-binding transporter gene (ABCR) is mutated in recessive Stargardt macular dystrophy. Nat Genet 15(3):236–246. https://doi.org/10.1038/ng0397-236

    Article  CAS  PubMed  Google Scholar 

  11. Sangermano R, Bax NM, Bauwens M et al (2016) Photoreceptor progenitor mRNA analysis reveals exon skipping resulting from the ABCA4 c.5461-10T-->C mutation in stargardt disease. Ophthalmology 123(6):1375–1385. https://doi.org/10.1016/j.ophtha.2016.01.053

    Article  PubMed  Google Scholar 

  12. Aartsma-Rus A, van Vliet L, Hirschi M et al (2009) Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms. Mol Ther 17(3):548–553. https://doi.org/10.1038/mt.2008.205

    Article  CAS  PubMed  Google Scholar 

  13. Aartsma-Rus A (2012) Overview on AON design. Methods Mol Biol 867:117–129. https://doi.org/10.1007/978-1-61779-767-5_8

    Article  CAS  PubMed  Google Scholar 

  14. Khvorova A, Watts JK (2017) The chemical evolution of oligonucleotide therapies of clinical utility. Nat Biotechnol 35(3):238–248. https://doi.org/10.1038/nbt.3765

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Singh NN, Seo J, Rahn SJ et al (2012) A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes. PLoS One 7(11):e49595. https://doi.org/10.1371/journal.pone.0049595

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors received funding from “Stichting Ushersyndroom” (HK and EvW); the Foundation Fighting Blindness USA (grant PPA-0517-0717-RAD) and “Stichting Wetenschappelijk Onderzoek Doof-Blindheid” (EvW) for the research that led to the protocol.

Conflict of Interest

EvW is employed by Radboudumc and inventor on a patent (PCT/EP2015/065736) for antisense oligonucleotide-based exon skipping. Radboudumc has licensed the rights to the patent exclusively to ProQR Therapeutics. As the inventor, EvW is entitled to a share of any future royalties paid to Radboudumc, should the therapy eventually be brought to the market.

Author information

Authors and Affiliations

  1. Hearing and Genes Division, Department of Otorhinolaryngology, Radboud University Medical Center, Nijmegen, The Netherlands

    Ralph Slijkerman, Hannie Kremer & Erwin van Wijk

  2. The Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands

    Ralph Slijkerman

  3. Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Center, Nijmegen, The Netherlands

    Hannie Kremer & Erwin van Wijk

  4. Department of Human Genetics, Radboud University Medical Center, Nijmegen, The Netherlands

    Hannie Kremer

Authors
  1. Ralph Slijkerman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hannie Kremer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Erwin van Wijk
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Erwin van Wijk .

Editor information

Editors and Affiliations

  1. Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Toshifumi Yokota

  2. Medical Genetics, University of Alberta, Edmonton, AB, Canada

    Rika Maruyama

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Slijkerman, R., Kremer, H., van Wijk, E. (2018). Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays. In: Yokota, T., Maruyama, R. (eds) Exon Skipping and Inclusion Therapies. Methods in Molecular Biology, vol 1828. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8651-4_34

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-8651-4_34

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8650-7

  • Online ISBN: 978-1-4939-8651-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation