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Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts
Published on: May 11, 2018
Evaluation of off-target effects of gapmer antisense oligonucleotides using human cells
Tokuyuki Yoshida1,2, Yuki Naito3,4, Hidenori Yasuhara2
1Division of Molecular Target and Gene Therapy Products, National Institute of Health Sciences, Kawasaki, Kanagawa, Japan.
Abstract:
Antisense oligonucleotide (ASO) has the potential to induce off-target effects due to complementary binding between the ASO and unintended RNA with a sequence similar to the target RNA. Conventional animal studies cannot be used to assess toxicity induced by off-target effects because of differences in the genome sequence between humans and other animals. Consequently, the assessment of off-target effects with in silico analysis using a human RNA database and/or in vitro expression analysis using human cells has been proposed. Our previous study showed that the number of complementary regions of ASOs with mismatches in the human RNA sequences increases dramatically as the number of tolerated mismatches increases. However, to what extent the expression of genes with mismatches is affected by off-target effects at the cellular level is not clear. In this study, we evaluated off-target effects of gapmer ASOs, which cleave the target RNA in an RNase H-dependent manner, by introducing the ASO into human cells and performing microarray analysis. Our data indicate that gapmer ASOs induce off-target effects depending on the degree of complementarity between the ASO and off-target candidate genes. Based on our results, we also propose a scheme for the assessment of off-target effects of gapmer ASOs.
Insights
Antisense oligonucleotides (ASOs) can cause unintended effects by binding to similar RNA sequences. This study shows that gapmer ASOs induce off-target effects in human cells based on complementarity, informing safety assessments.
Area of Science:
- Pharmacogenomics
- Molecular Biology
- Biotechnology
Background:
- Antisense oligonucleotides (ASOs) offer therapeutic potential but can cause off-target effects via unintended RNA binding.
- Existing animal models are insufficient for assessing ASO off-target toxicity due to genomic differences.
- In silico and in vitro human cell-based methods are proposed for evaluating these effects.
Purpose of the Study:
- To evaluate the cellular-level off-target effects of gapmer antisense oligonucleotides (ASOs).
- To determine the relationship between ASO complementarity and gene expression changes in human cells.
- To propose a framework for assessing gapmer ASO off-target effects.
Main Methods:
- Introduction of gapmer ASOs into human cells.
- Microarray analysis to assess global gene expression changes.
- Evaluation of ASO complementarity to off-target candidate RNA sequences.
Main Results:
- Gapmer ASOs induce off-target effects in human cells.
- The extent of off-target effects is dependent on the degree of complementarity between the ASO and unintended RNA targets.
- A correlation exists between the number of mismatches and potential off-target binding sites.
Conclusions:
- Off-target effects of gapmer ASOs are driven by sequence complementarity.
- A scheme for assessing gapmer ASO off-target effects in vitro is proposed.
- This research contributes to the safe development of ASO therapeutics.
