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.
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References
Liu MM, Zack DJ (2013) Alternative splicing and retinal degeneration. Clin Genet 84(2):142–149. https://doi.org/10.1111/cge.12181
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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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
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DOI: https://doi.org/10.1007/978-1-4939-8651-4_34
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