Design of Exon Skipping Oligonucleotides
Presenter: Annemieke Aartsma-Rus, PhD, Leiden University Medical Center
Date: 20 February 2018
Description: Antisense-mediated splicing modulation utilizes antisense oligonucleotides (ASOs) to manipulate the splicing by sterically blocking the binding of splicing factors to pre-mRNA transcripts. While in industry often oligo walks are used to identify the most optimal ASO, generally academic groups do not have the funds to do this and can design only a limited number of ASOs for testing. While ASO design for splicing modulation is partially a trial and error approach, some lessons can be learnt from previous efforts.
Annemieke Aartsma-Rus has designed over 300 ASOs for exon skipping and retrospective analysis has provided her some insights in dos and don’ts with regards to ASO design. In this webinar Annemieke will share this expertise and will outline what to consider when selecting candidate ASOs for splice modulation (e.g. target site selection, thermodynamic properties, checking for off-target effects etc). This presentation will be followed by a Q&A session where you can ask Annemieke your questions during the broadcast.
Recording of the webinar:
Annemieke kindly provided a list of recommended references:
Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites.
Aartsma-Rus A, De Winter CL, Janson AA, Kaman WE, Van Ommen GJ, Den Dunnen JT, Van Deutekom JC.
Oligonucleotides. 2005 Dec;15(4):284-97.
Guidelines for antisense oligonucleotide design and insight into splice-modulating mechanisms.
Aartsma-Rus A, van Vliet L, Hirschi M, Janson AA, Heemskerk H, de Winter CL, de Kimpe S, van Deutekom JC, ‘t Hoen PA, van Ommen GJ.
Mol Ther. 2009 Mar;17(3):548-53
Overview on AON design.
Methods Mol Biol. 2012;867:117-29.
Exonic sequences provide better targets for antisense oligonucleotides than splice site sequences in the modulation of Duchenne muscular dystrophy splicing.
Aartsma-Rus A, Houlleberghs H, van Deutekom JC, van Ommen GJ, ‘t Hoen PA.
Oligonucleotides. 2010 Apr;20(2):69-77.
Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides.
Aartsma-Rus A, Janson AA, Heemskerk JA, De Winter CL, Van Ommen GJ, Van Deutekom JC.
Ann N Y Acad Sci. 2006 Oct;1082:74-6.
This is the paper discussed during the Q&A session:
In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy.
Echigoya Y, Mouly V, Garcia L, Yokota T, Duddy W.
PLoS One. 2015 Mar 27;10(3):e0120058.
Annemieke added the following comment about this paper:
“The paper Art referred to generates a secondary prediction based on windows of short pieces of the pre-mRNA – it is so to speak a more dynamic model than m-fold (which is static). I have never checked whether this model is useful in establishing whether an oligonucleotide for exon skipping is effective or not, so I cannot make recommendations. What I can recommend is that people using the model also take the length, GC-content, predicted self and self-self structuring and predicted ESE sites into account.”
Further reading on this subject:
A prospective study in the rational design of efficient antisense oligonucleotides for exon skipping in the DMD gene.
Pramono ZA, Wee KB, Wang JL, Chen YJ, Xiong QB, Lai PS, Yee WC.
Hum Gene Ther. 2012 Jul;23(7):781-90.
Design of phosphorodiamidate morpholino oligomers (PMOs) for the induction of exon skipping of the human DMD gene.
Popplewell LJ, Trollet C, Dickson G, Graham IR.
Mol Ther. 2009 Mar;17(3):554-61.
Antisense oligonucleotide-induced exon skipping across the human dystrophin gene transcript.
Wilton SD, Fall AM, Harding PL, McClorey G, Coleman C, Fletcher S.
Mol Ther. 2007 Jul;15(7):1288-96.
Dr. Annemieke Aartsma-Rus is a professor of translational genetics at the Department of Human Genetics of the Leiden University Medical Center, Leiden the Netherlands. She played an important role in the development of the antisense mediated exon skipping therapy for Duchenne muscular dystrophy and has designed and tested hundreds of exon skipping antisense oligonucleotides.
In 2016 and 2017 she was selected as the most influential scientist in Duchenne muscular dystrophy in the past 10 years (2006-2016) by Expertscape based on contributions to the understanding and treatment of Duchenne muscular dystrophy. Thus far, she has published over 140 peer-reviewed papers and 9 book chapters, as well as 13 patents and has edited one book. She has created and maintains a website on exon skipping (www.exonskipping.nl) and tweets under @oligogirl. In 2011 she received the Duchenne Award from the Dutch Duchenne Parent Project in recognition of this work and her dedication to the Duchenne field. She is currently the president elect of the oligonucleotide therapeutics society.