Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides

Scharner, J., Ma, W. K., Zhang, Q., Lin, K. T., Rigo, F., Bennett, C. F., Krainer, A. R. (December 2019) Hybridization-mediated off-target effects of splice-switching antisense oligonucleotides. Nucleic Acids Res, 48 (2). ISSN 0305-1048 (Public Dataset)

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URL: https://www.ncbi.nlm.nih.gov/pubmed/31802121

DOI: 10.1093/nar/gkz1132

Abstract

Splice-switching antisense oligonucleotides (ASOs), which bind specific RNA-target sequences and modulate pre-mRNA splicing by sterically blocking the binding of splicing factors to the pre-mRNA, are a promising therapeutic modality to treat a range of genetic diseases. ASOs are typically 15-25 nt long and considered to be highly specific towards their intended target sequence, typically elements that control exon definition and/or splice-site recognition. However, whether or not splice-modulating ASOs also induce hybridization-dependent mis-splicing of unintended targets has not been systematically studied. Here, we tested the in vitro effects of splice-modulating ASOs on 108 potential off-targets predicted on the basis of sequence complementarity, and identified 17 mis-splicing events for one of the ASOs tested. Based on analysis of data from two overlapping ASO sequences, we conclude that off-target effects are difficult to predict, and the choice of ASO chemistry influences the extent of off-target activity. The off-target events caused by the uniformly modified ASOs tested in this study were significantly reduced with mixed-chemistry ASOs of the same sequence. Furthermore, using shorter ASOs, combining two ASOs, and delivering ASOs by free uptake also reduced off-target activity. Finally, ASOs with strategically placed mismatches can be used to reduce unwanted off-target splicing events.

Item Type:	Paper
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics Investigative techniques and equipment Investigative techniques and equipment > cloning > PCR Investigative techniques and equipment > assays > cloning > PCR bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > antisense Investigative techniques and equipment > assays organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types > cell line organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions > cell types organs, tissues, organelles, cell types and functions > cell types and functions Investigative techniques and equipment > cloning Investigative techniques and equipment > assays > cloning bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > mRNA dynamics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > oligonucleotide organs, tissues, organelles, cell types and functions bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNA splicing
CSHL Authors:	Krainer, Adrian R. Scharner, Juergen Lin, Kuan-Ting Ma, Wai Kit Zhang, Qian
Communities:	CSHL Cancer Center Program > Gene Regulation and Inheritance Program CSHL labs > Krainer lab CSHL Cancer Center Shared Resources > Animal Shared Resource
Depositing User:	Adrian Gomez
Date:	5 December 2019
Date Deposited:	11 Dec 2019 15:38
Last Modified:	02 Feb 2024 20:34
PMCID:	PMC6954394
Related URLs:	Publisher
Dataset ID:	Supplement: https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkz1132/5658446#supplementary-data
URI:	https://repository.cshl.edu/id/eprint/38735

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