Locked Nucleic Acid Gapmers Research Articles

Knockdown of Nuclear-Located Enhancer RNAs and Long ncRNAs Using Locked Nucleic Acid GapmeRs.

The human genome is widely transcribed outside of protein-coding genes, producing thousands of noncoding RNAs from different subfamilies including enhancer RNAs. Functional studies to determine the role of individual genes are challenging with noncoding RNAs appearing to be more difficult to knockdown than mRNAs. One factor that may have hindered progress is that the majority of noncoding RNAs are thought to be located within the nucleus, where the efficiency of traditional RNA interference techniques is debatable. Here we present an alternative RNA interference technique utilizing Locked Nucleic Acids, which is able to efficiently knockdown noncoding RNAs irrespective of intracellular location.

Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

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.

Abstract A48: The lncRNA HOTAIR is overexpressed in Ewing sarcoma and promotes malignant transformation through interactions with histone-modifying complexes

Abstract Introduction: Ewing sarcoma (ES) is an aggressive primitive cancer of the bone and soft tissue in adolescents and young adults, typified by chromosomal translocations resulting in fusions of the EWS gene and an ETS family gene, most commonly FLI1. The resulting EWS-FLI1 fusion protein is the oncogenic driver in this cancer, but its expression alone does not induce malignant transformation in human mesenchymal stem cells (hMSCs), the putative cell of origin. Instead, EWS-FLI1 induces apoptosis in most recipient cell line and animal models. The lncRNA HOTAIR is upregulated in multiple types of cancer and promote tumorigenesis. However, its expression and function have not been studied in Ewing sarcoma. Methods: We used the R2 (http://r2.amc.nl) and Oncogenomics (https://pob.abcc.ncifcrf.gov/cgi-bin/JK) platforms to examine the expression of HOTAIR in Ewing sarcomas as compared to other tumors and normal tissues, as assessed in high-throughput datasets. We directly measured HOTAIR expression by RT-qPCR on total RNA from ES cell lines (n=13), primary marrow-derived hMSCs (n=2), and primary ES tumors (n=22). We repressed HOTAIR expression in cell lines by shRNA and antisense locked nucleic acid GAPmers (Exiqon), and we overexpressed HOTAIR by plasmid transfection into hMSCs. We then evaluated the phenotype of HOTAIR in these models. We performed RNA immunoprecipitation (RIP) on parental cell line extracts to identify interacting proteins with HOTAIR. We transfected mutant forms of HOTAIR into hMSCs and evaluated the effect of loss of interaction with partner proteins on the HOTAIR phenotype. We co-transfected EWS-FLI1 and HOTAIR into hMSCs to evaluate viability and phenotype. Results: HOTAIR was found to be most highly expressed in 3 independent datasets of primary Ewing sarcomas on the R2 platform as compared to all other primary tumors. In one dataset, HOTAIR was overexpressed in Ewing sarcomas (n=68) as compared to normal tissues (n=18, p<0.0001). In the Oncogenomics platform, HOTAIR was overexpressed in all ES cell lines (n=50) and primary tumors (n=72). By RT-qPCR, HOTAIR was overexpressed in 11/13 ES cell lines and 17/22 primary tumors as compared to primary hMSCs. We reduced HOTAIR expression in three ES cell lines by shRNA. We saw no effects on proliferation or chemosensitivity. However, cells with reduced HOTAIR expression had a significant reduction in anchorage-independent tumorsphere formation in soft agar, as compared to nonsilencing control cells (p≤0.01 across all three cell lines). Overexpression of HOTAIR in hMSCs this allowed anchorage independent colony formation in these cells, a phenotype not observed in the parental or vector-control cells. Treatment of the ES cell lines or HOTAIR-overexpressing hMSCs with HOTAIR-specific antisense GAPmers abrogated tumorsphere formation. We confirmed by RIP that, in ES cell lines, HOTAIR interacts with the histone-modifying proteins EZH2 at its 5' domain and LSD1 at its 3' domain, and deletion of each domain disrupts the interaction with each protein specifically. Transfection of hMSCs with HOTAIR mutants lacking either the 5' or 3' domains also failed to allow anchorage-independent tumorsphere formation. Finally, co-transfection of full-length HOTAIR with EWS-FLI1 into hMSCs allowed cell viability and tumorsphere formation, whereas expression of EWS-FLI1 alone or with mutant HOTAIR did not. Conclusions: The lncRNA HOTAIR is significantly overexpressed in Ewing sarcoma and promotes malignant transformation and tumorigenesis through mechanisms dependent on interaction with the histone-modifying proteins EZH2 and LSD1. HOTAIR may promote survival in EWS-FLI1 mediated transformation, and it may be a potential therapeutic target given its high expression in tumors and low expression in most normal tissues. Citation Format: Hasan Siddiqui, Julia Selich-Taylor, Joshua Felgenhauer, Satoru Otsuru, Edwin Horwitz, Nilay Shah. The lncRNA HOTAIR is overexpressed in Ewing sarcoma and promotes malignant transformation through interactions with histone-modifying complexes. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A48.

Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.

Design and Selection of Antisense Oligonucleotides Targeting Transforming Growth Factor Beta (TGF-β) Isoform mRNAs for the Treatment of Solid Tumors.

Transforming growth factor beta isoforms (TGF-β1, -β2, and -β3) are cytokines associated with a wide range of biological processes in oncology including tumor cell invasion and migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Hence, blocking the TGF-β signaling pathways may have a multifold therapeutic benefit for the treatment of solid tumors. Here, we describe the identification and selection processes for the development of highly potent and selective chemically modified antisense oligodeoxynucleotides (fully phosphorothioate locked nucleic acid gapmers) allowing effective and selective suppression of TGF-β isoform expression in cell-based assays and in vivo preclinical models.

DEADSouth protein localizes to germ plasm and is required for the development of primordial germ cells in Xenopus laevis

SummaryDEADSouth mRNA is a component of germ plasm in Xenopus laevis and encodes a DDX25 DEAD-box RNA helicase. To determine the intracellular localization of DEADSouth protein, we injected mRNA encoding DEADSouth tagged with mCherry fluorescent protein into fertilized eggs from transgenic Xenopus expressing EGFP fused with a mitochondrial targeting signal. The DEADSouth-mCherry fusion protein was localized to the germ plasm, a mitochondria-rich region in primordial germ cells (PGCs). DEADSouth overexpression resulted in a reduction of PGC numbers after stage 20. Conversely, DEADSouth knockdown using an antisense locked nucleic acid gapmer inhibited movement of the germ plasm from the cortex to the perinuclear region, resulting in inhibition of PGC division at stage 12 and a decrease in PGC numbers at later stages. The knockdown phenotype was rescued by intact DEADSouth mRNA, but not mutant mRNA encoding inactive DEADSouth helicase. Surprisingly, it was also rescued by mouse vasa homolog and Xenopus vasa-like gene 1 mRNAs that encode DDX4 RNA helicases. The rescue was dependent on the 3′ untranslated region (3′UTR) of DEADSouth mRNA, which was used for PGC-specific expression. The 3′UTR contributed to localization of the injected mRNA to the germ plasm, resulting in effective localization of DEADSouth protein. These results demonstrate that localization of DEADSouth helicase to the germ plasm is required for proper PGC development in Xenopus laevis.

Chemical Modification Study of Antisense Gapmers

A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic--using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.

Natural Antisense Makes Sense for Gene-specific Activation in Brain

The upregulation of specific genes in vivo has been an elusive goal for gene therapy when compared with the wide repertoire of methods available to silence genes or modify mRNA splicing patterns. In the latest issue of Nature Biotechnology, Modarresi and colleagues1 accomplished in vivo upregulation of brain-derived neurotrophic factor (BDNF), a relevant therapeutic target for a number of neurodegenerative diseases. Rather than using small molecules or microRNA inhibitors, which could lead to activation of off-target genes, Modarresi et al.1 upregulated BDNF by inhibiting a natural antisense transcript (NAT) in response to the local delivery of oligonucleotides to the central nervous systems of mice.

Ion Trap Collision-Induced Dissociation of Locked Nucleic Acids

Gas-phase dissociation of model locked nucleic acid (LNA) oligonucleotides and functional LNA-DNA chimeras have been investigated as a function of precursor ion charge state using ion trap collision-induced dissociation (CID). For the model LNA 5 and 8 mer, containing all four LNA monomers in the sequence, cleavage of all backbone bonds, generating a/w-, b/x-, c/y-, and d/z-ions, was observed with no significant preference at lower charge states. Base loss ions, except loss of thymine, from the cleavage of N-glycosidic bonds were also present. In general, complete sequence coverage was achieved in all charge states. For the two LNA-DNA chimeras, however, dramatic differences in the relative contributions of the competing dissociation channels were observed among different precursor ion charge states. At lower charge states, sequence information limited to the a-Base/w-fragment ions from cleavage of the 3'C-O bond of DNA nucleotides, except thymidine (dT), was acquired from CID of both the LNA gapmer and mixmer ions. On the other hand, extensive fragmentation from various dissociation channels was observed from post-ion/ion ion trap CID of the higher charge state ions of both LNA-DNA chimeras. This report demonstrates that tandem mass spectrometry is effective in the sequence characterization of LNA oligonucleotides and LNA-DNA chimeric therapeutics.

Filling the gap in LNA antisense oligo gapmers: the effects of unlocked nucleic acid (UNA) and 4′-C-hydroxymethyl-DNA modifications on RNase H recruitment and efficacy of an LNA gapmer

Stability against nucleases, affinity for the targeted mRNA and the ability to recruit RNase H are prerequisites for antisense oligonucleotide (AON) applications where gene expression knockdown is required. Typically chimeric gapmer AON designs are used with a central continuous stretch of RNase H recruiting nucleotides (e.g. phosphorothioate DNA), flanked by affinity and stability-enhancing modified nucleotides. However, many types of nucleotide modifications in the central DNA gap can disturb RNase H function. Here we present studies into two different types of nucleotide modifications, a flexible acyclic RNA analog named unlocked nucleic acid (UNA) and 4'-C-hydroxymethyl-DNA in the gap of an LNA (locked nucleic acid) flanked gapmer. We compared the efficacy of mRNA degradation by the gap modified LNA antisense gapmers in cell-free assays and cultured cells. This study shows that both UNA and 4'-C-hydroxymethyl-DNA gap insertions are compatible with RNase H activity when used sparingly. However, multiple 4'-C-hydroxymethyl-DNA modifications are better tolerated by RNase H than multiple UNA modifications in the gap. Furthermore, this report shows that LNA gapmer AONs with multiple 4'-C-hydroxymethyl-DNA moieties in the gap can mediate target knockdown in vivo.

Downregulation of p21(WAF1/CIP1) and estrogen receptor alpha in MCF-7 cells by antisense oligonucleotides containing locked nucleic acid (LNA).

Locked nucleic acid (LNA) is a nucleic acid analog with very high affinity to complementary RNA and a promising compound in the field of antisense research. The intracellular localization and quantitative uptake of oligonucleotides containing LNA were found to be equivalent to those of phosphorothioate oligonucleotides (PS AONs). The antisense efficiency of LNA-containing oligonucleotides was systematically compared with standard PS AONs targeting expression of two endogenous proteins in the human breast cancer cell line MCF-7, namely, the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) and the estrogen receptor alpha (ERalpha). For downregulation of both target proteins, the most efficient design was achieved with oligonucleotides containing LNA monomers in the extremities and a central gap of PS-linked DNA monomers, so called LNA gapmers. Such LNA gapmers caused more potent downregulation of the targeted proteins than PS AONs, whereas fully modified LNA AONs or LNA mixmers (LNA nucleotides interspersed) were inactive.