Antisense oligonucleotides capable of promoting specific target mRNA reduction via competing RNase H1-dependent and independent mechanisms.
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ABSTRACT:
SUBMITTER: Vickers TA
PROVIDER: S-EPMC4191969 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation.
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Project description:RNase H1-dependent antisense oligonucleotides (ASOs) can degrade complementary RNAs in both the nucleus and the cytoplasm. Since cytoplasmic mRNAs are actively engaged in translation, ASO activity may thus be affected by translating ribosomes that scan the mRNAs. Here we show that mRNAs associated with ribosomes can be cleaved using ASOs and that translation can alter ASO activity. Translation inhibition tends to increase ASO activity when targeting the coding regions of efficiently translated mRNAs, but not nuclear non-coding RNAs or less efficiently translated mRNAs. Increasing the level of RNase H1 protein eliminated the enhancing effects of translation inhibition on ASO activity, suggesting that RNase H1 recruitment to ASO/mRNA heteroduplexes is a rate limiting step and that translating ribosomes can inhibit RNase H1 recruitment. Consistently, ASO activity was not increased by translation inhibition when targeting the 3' UTRs, independent of the translation efficiency of the mRNAs. Contrarily, the activity of 3' UTR-targeting ASOs tended to be reduced upon translation inhibition, likely due to decreased accessibility. These results indicate that ASO activity can be affected by the translation process, and the findings also provide important information toward helping better ASO drug design.
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Project description:RNase H1-dependent antisense oligonucleotides (ASOs) are active in reducing levels of both cytoplasmic mRNAs and nuclear retained RNAs. Although ASO activity in the nucleus has been well demonstrated, the cytoplasmic activity of ASOs is less clear. Using kinetic and subcellular fractionation studies, we evaluated ASO activity in the cytoplasm. Upon transfection, ASOs targeting exonic regions rapidly reduced cytoplasmically enriched mRNAs, whereas an intron-targeting ASO that only degrades the nuclear pre-mRNA reduced mRNA levels at a slower rate, similar to normal mRNA decay. Importantly, some exon-targeting ASOs can rapidly and vigorously reduce mRNA levels without decreasing pre-mRNA levels, suggesting that pre-existing cytoplasmic mRNAs can be cleaved by RNase H1-ASO treatment. In addition, we expressed a cytoplasm-localized mutant 7SL RNA that contains a partial U16 small nucleolar RNA (snoRNA) sequence. Treatment with an ASO simultaneously reduced both the nuclear U16 snoRNA and the cytoplasmic 7SL mutant RNA as early as 30 min after transfection in an RNase H1-dependent manner. Both the 5' and 3' cleavage products of the 7SL mutant RNA were accumulated in the cytoplasm. Together, these results demonstrate that RNase H1-dependent ASOs are robustly active in both the cytoplasm and nucleus.
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Project description:Pre-mRNA splicing, a dynamic process of intron removal and exon joining, is governed by a combinatorial control exerted by overlapping cis-elements that are unique to each exon and its flanking intronic sequences. Splicing cis-elements are usually 4-to-8-nucleotide-long linear motifs that provide binding sites for specific proteins. Pre-mRNA splicing is also influenced by secondary and higher order RNA structures that affect accessibility of splicing cis-elements. Antisense oligonucleotides (ASOs) that block splicing cis-elements and/or affect RNA structure have been shown to modulate splicing in vivo. Therefore, ASO-based strategies have emerged as a powerful tool for therapeutic manipulation of splicing in pathological conditions. Here we describe an ASO-based approach to increase the production of the full-length SMN2 mRNA in spinal muscular atrophy patient cells.
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Project description:Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.
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Project description:MYC is a prolific proto-oncogene driving the malignant behaviors of numerous common cancers, yet potent and selective cell-permeable inhibitors of MYC remain elusive. In order to ultimately realize the goal of therapeutic MYC inhibition in cancer, we have initiated discovery chemistry efforts aimed at inhibiting MYC translation. Here we describe a series of conformationally stabilized synthetic antisense oligonucleotides designed to target MYC mRNA (MYCASOs). To support bioactivity, we designed and synthesized this focused library of MYCASOs incorporating locked nucleic acid (LNA) bases at the 5'- and 3'-ends, a phosphorothioate backbone, and internal DNA bases. Treatment of MYC-expressing cancer cells with MYCASOs leads to a potent decrease in MYC mRNA and protein levels. Cleaved MYC mRNA in MYCASO-treated cells is detected with a sensitive 5' Rapid Amplification of cDNA Ends (RACE) assay. MYCASO treatment of cancer cell lines leads to significant inhibition of cellular proliferation while specifically perturbing MYC-driven gene expression signatures. In a MYC-induced model of hepatocellular carcinoma, MYCASO treatment decreases MYC protein levels within tumors, decreases tumor burden, and improves overall survival. MYCASOs represent a new chemical tool for in vitro and in vivo modulation of MYC activity, and promising therapeutic agents for MYC-addicted tumors.
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Project description:RNase H-dependent gapmer antisense oligonucleotides (ASOs) are a promising therapeutic approach via sequence-specific binding to and degrading target RNAs. However, the efficacy and mechanism of antiviral gapmer ASOs have remained unclear. Here, we investigated the inhibitory effects of gapmer ASOs containing locked nucleic acids (LNA gapmers) on proliferating a mosquito-borne flavivirus, Japanese encephalitis virus (JEV), with high mortality. We designed several LNA gapmers targeting the 3' untranslated region of JEV genomic RNAs. In vitro screening by plaque assay using Vero cells revealed that LNA gapmers targeting a stem-loop region effectively inhibit JEV proliferation. Cell-based and RNA cleavage assays using mismatched LNA gapmers exhibited an underlying mechanism where the inhibition of viral production results from JEV RNA degradation by LNA gapmers in a sequence- and modification-dependent manner. Encouragingly, LNA gapmers potently inhibited the proliferation of five JEV strains of predominant genotypes I and III in human neuroblastoma cells without apparent cytotoxicity. Database searching showed a low possibility of off-target binding of our LNA gapmers to human RNAs. The target viral RNA sequence conservation observed here highlighted their broad-spectrum antiviral potential against different JEV genotypes/strains. This work will facilitate the development of an antiviral LNA gapmer therapy for JEV and other flavivirus infections.
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Project description:RNase H1-dependent phosphorothioate oligonucleotides (PS-ASOs) have been developed to treat various diseases through specific degradation of target RNAs. Although many factors or features of RNA and PS-ASOs have been demonstrated to affect antisense activity of PS-ASOs, little is known regarding the roles of RNase H1-associated proteins in PS-ASO performance. In this study, we report that two nucleolar proteins, NAT10 and DDX21, interact with RNase H1 and affect the potency and safety of PS-ASOs. The interactions of these two proteins with RNase H1 were determined using BioID proximity labeling in cells and confirmed biochemically. Reduction of NAT10 and DDX21 decreased PS-ASO activity in cells, and purified NAT10 and DDX21 proteins enhanced RNase H1 cleavage rates, indicating that these two proteins facilitate RNase H1 endoribonuclease activity. Consistently, reduction of these proteins increased the levels of R-loops, and impaired pre-rRNA processing. In addition, reduction of the two proteins increased the cytotoxicity of toxic PS-ASOs, and treatment of toxic PS-ASOs also altered the localization of these proteins. Together, this study shows for the first time that NAT10 and DDX21 interact with RNase H1 protein and enhance its enzymatic activity, contributing to the potency and safety of PS-ASOs.
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Project description:Antisense oligomers (AOs) are increasingly being used to modulate RNA splicing in live cells, both for research and for the development of therapeutics. While the most common intended effect of these AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping of only part of an exon, through activation of an internal cryptic splice site. In this report, we examined seven AO-induced cryptic splice sites in six genes. Five of these cryptic splice sites were discovered through our own experiments, and two originated from other published reports. We modelled the predicted effects of AO binding on the secondary structure of each of the RNA targets, and how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was disruption of the exon definition signal within the exon's excluded segment.
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