Estimated number of off‐target candidate sites for antisense oligonucleotides in human <scp>mRNA</scp> sequences

Yoshida, Tokuyuki; Naito, Yukiko; Sasaki, Kiyomi; Uchida, Eiji; Sato, Yoji; Naito, Mikihiko; Kawanishi, Toru; Obika, Satoshi; Inoué, Takao

doi:10.1111/gtc.12587

Cited by 29 publications

(15 citation statements)

References 23 publications

(25 reference statements)

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“…Thus, when searching for off‐target candidate genes of gapmer ASOs, an in silico analysis of a database that fully includes human pre‐mRNA should be conducted. In a prior study, we showed the theoretical number of complementary regions of ASOs with mismatches in human mRNA (Yoshida et al, ). As the total size of human pre‐mRNA coding for proteins (1.17 Gb) is approximately 17‐fold higher than that of human mRNA (68.1 Mb) (see Section ), the number of complementary regions in human pre‐mRNA is also correspondingly greater (Table , see Section ).…”

Section: Resultsmentioning

confidence: 98%

“…We previously used mathematical calculations and in silico analysis to estimate the general number of complementary regions of ASOs with mismatches in human mRNA sequences using several thousand hypothetical ASOs. Our analysis showed that the number of complementary regions increases dramatically as the number of tolerated mismatches increases (Yoshida et al, ). However, to what extent the expression of genes with mismatches is affected by the mechanism of off‐target effects when ASOs actually act on human cells is unclear.…”

Section: Introductionmentioning

confidence: 83%

“…We mathematically calculated the theoretical number of complementary regions of ASOs with perfect matches or mismatches in the total size of human pre‐mRNA sequences in a similar manner as described previously (Yoshida et al, ). We hypothesized that the four bases (A, G, C and T) are used randomly in human pre‐mRNA sequences.…”

Section: Methodsmentioning

confidence: 99%

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Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

et al. 2019

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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.

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Section: Resultsmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 83%

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Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

et al. 2019

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“…Oligonucleotides are thought to be selective for fully complementary targets, however they can have off-targets (30). These off-targets include RNAs that are fully complementary and those that contain mismatches (30)(31)(32). Other factors that affect selectivity include the expression levels of the on-and the off-targets, with the more highly expressed target more likely to be occupied and affected (32).…”

Section: Discussionmentioning

confidence: 99%

Small molecules exploiting structural differences within microRNA-200 precursors family members reverse a type 2 diabetes phenotype

Haniff

Liu

Knerr

et al. 2020

Preprint

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MicroRNA families are pervasive in the human transcriptome, but specific targeting of individual members is a challenge because of sequence homology. Many of the secondary structures of the precursors to these miRs (pre-miRs), however, are quite different. Here, we demonstrate both in vitro and in cellulis that design of structure-specific small molecules can inhibit specific miR family members to modulate a disease pathway. In particular, the miR-200 family consists five miRs, miR-200a, -200b, -200c, -141, and -429, and is associated with Type II Diabetes (T2D). We designed a small molecule that potently and selectively targets pre-miR-200c's structure. The compound reverses a proapoptotic effect in a pancreatic β-cell model. In contrast, oligonucleotides targeting the RNA's sequence inhibit all family members. Global proteomics analysis further demonstrates selectivity for miR-200c. Collectively, these studies establish that miR-200c plays an important role in T2D and that small molecules targeting RNA structure can be an important complement to oligonucleotides targeting sequence.

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“…However, while this short ASO design lended itself to the greatest specificity, it does pose potential problems when considering off-target silencing. A recent paper by Yoshida et al estimated that 12-bp ASOs may have up to 3.4 potential off-target binding regions with zero-sequence mismatches, and up to 122 potential off-target binding regions if a single mismatched-base is tolerated [35]. While these numbers are large, they do not take into account potential secondary structure of the RNA, which is known to impact target-affinity in ASOs [36], or whether or not the transcripts containing those potential off-target sequences are expressed in the target cell-type.…”

Section: Discussionmentioning

confidence: 99%

Dissociation of disease phenotype and allele silencing in hypertrophic cardiomyopathy

Dainis

Zaleta-Rivera

Ribeiro

et al. 2019

Preprint

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Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy to date have focused on mouse models of disease. Here, we investigate two methods of allele-specific silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of disease. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. We find a dissociation between allelic-specificity and functional improvements between the two tested therapeutic strategies, suggesting a more complex method of allelic control underlying HCM-associated transcripts. Author summaryAllele-specific silencing, whereby a therapeutic molecule is used to lower the expression of just one of the two copies or alleles of a gene, may be a potential therapeutic strategy in diseases caused by a single mutation. In this paper, we examine two such strategies in hypertrophic cardiomyopathy, a disease characterized by an overgrowth of the left-ventricular heart muscle as well as contractile dysfunction. We used a human cell model of disease, creating induced pluripotent stem cell derived cardiomyocytes from a patient with HCM caused by a single base pair change in just one allele of the gene MYH7. We used two strategies to silence the disease-associated copy of MYH7, both focused on reducing RNA expression from the mutated allele, as well as state-of-the-art biophysical techniques for measuring contractility. We found that one silencing strategy, which reduced expression of both the disease-associated and the healthy alleles of MYH7, showed great improvements in contractility between treated and untreated cells. Our

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Estimated number of off‐target candidate sites for antisense oligonucleotides in human mRNA sequences

Cited by 29 publications

References 23 publications

Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

Evaluation of off‐target effects of gapmer antisense oligonucleotides using human cells

Small molecules exploiting structural differences within microRNA-200 precursors family members reverse a type 2 diabetes phenotype

Dissociation of disease phenotype and allele silencing in hypertrophic cardiomyopathy

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