Targeting enhancer RNAs for the treatment of primary brain tumors

Abstract

The invention provides enhancer RNAs (eRNAs) that are expressed specifically in glioma stem cells and whose expression correlates with decreased survival of patients with glioblastomas. The eRNAs are selected, for example, from eTMEM88b, eRTP5, or eNINJ1. The invention also provides an RNA therapy that targets glioma stem cell eRNAs using synthetic oligonucleotides that knock out the eRNA expression. The invention further provides viral vectors that deliver shRNA that inhibit the expression of the eRNA.

Description

FIELD OF THE INVENTION

This invention generally relates to vector systems having a special element relevant for transcription being an enhancer not forming part of the promoter region.

REFERENCE TO RELATED APPLICATIONS

This application is a national phase filing under 35 U.S.C. § 371 of International Patent Application no. PCT/US2021/048671, filed on Sep. 1, 2021, which claims priority to U.S. provisional patent application Ser. No. 63/073,177, filed on Sep. 1, 2020 the entire contents of which are hereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

Glioblastoma is the most prevalent primary brain tumor and one of the most aggressive and lethal cancers. Treatment for glioblastomas includes surgical resection of the tumor mass followed by radiation and temozolomide administration. Stupp et al., N Engl. J. Med., 352, 987-996 (2005). But even with this multi-therapeutic approach, tumor recurrence is inevitable. Stupp & Weber, Onkologie, 28, 315-317 (2005). The aggressiveness attributed to glioblastomas is mainly driven by a population of glioma stem cells (GSCs) within the tumor mass that exhibits high migratory potential, resistance to chemotherapy and radiation, and possess the ability to form secondary tumors. Singh et al., Cancer Res., 63, 5821-5828 (2003); Singh et al., Nature, 432, 396-401 (2004); Lee et al., Cancer Cell 9, 391-403 (2006); and Soni et al., Asian Pac. J. Cancer Prev., 18, 2215-2219 (2017).

Glioma stem cells exhibit remarkable plasticity, can transition between immature and differentiated stages, and can reversibly express various phenotypic markers, depending on the tumor microenvironment. Ben-Porath et al., Nature Genetics, 40, 499-507 (2008); and Jin et al., Nature Medicine, 23, 1352-1361 (2017).

There remains a need in the art for compositions and methods for better treating gliomas.

SUMMARY OF THE INVENTION

The invention targets molecular mechanisms that control glioma stem cell plasticity and affect glioblastoma growth.

In the first embodiment, the invention provides enhancer RNAs (eRNAs) expressed specifically in glioma stem cells and whose expression correlates with decreased survival of patients with glioblastomas. In the second embodiment, the eRNAs are selected from the group consisting of eTMEM88b, eRTP5, and eNINJ1, as shown in the SEQUENCE LISTING below.

In the third embodiment, the invention provides an RNA therapy. The therapy targets glioma stem cell eRNAs using synthetic oligonucleotides that knockout the expression of the eRNA. In the fourth embodiment, the synthetic oligonucleotides are resistant to degradation.

In the fifth embodiment, the invention provides a viral vector that delivers an shRNA that targets and inhibits the expression of the eRNA. In the sixth embodiment, the viral vector is a lentivirus.

The invention has several advantages. In the first aspect, regarding the target specificity, the targeted eRNAs are expressed specifically in glioma stem cells. In the second aspect, regarding drug specificity, the synthesized oligonucleotides or viral vectors are specific for the intended target eRNA. The synthesized oligonucleotides or viral vectors do not have a substantial effect on other genes. In the third aspect, regarding resistance to degradation, the oligonucleotide RNA-therapeutic is chemically modified to be resistant to degradation. The oligonucleotide RNA-therapeutic works for in vivo applications. In the fourth aspect, regarding the specific effect on glioma stem cells, the inhibition of the target eRNA results in reduced expression of transcription factors that define the cancer stem cell phenotype like Nanog, Oct6, and Sox2. This result shows that eRNA targeting can affect cancer stemness, tumor propagation, and aggression.

The genome-wide data analysis has shown there are fifteen glioma stem cell-specific eRNAs compared to differentiated cells. Further analysis has shown these eRNAs are differentially expressed across at least seven glioma stem cells, and high expression of three eRNAs correlates significantly with poor patient survival. Preliminary results on inhibition of one of the three clinically-relevant eRNAs show that inhibition may be important for regulating glioma stem cell essential genes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of eRNA in regulating transcription. The enhancer region (blue box) is marked by H3K27Ac and loaded with transcription factors (TF) and RNA Pol II helping to produce eRNAs in a bidirectional manner. The promoter region (purple box) is marked by H3K4me3 and transcription factors. Looping of enhancer to promoter mediated by Cohesin and Mediator complex brings eRNAs to close proximity with the promoter to induce transcription of the target gene.

FIG. 2 shows a characterization of glioma stem cells and differentiated glioma cells. FIG. 2(A) is a bar chart showing that glioma stem cells express the stem cell-specific transcripts CD133, Sox2, and Olig2, which they lose entirely (CD133, Olig2) or downregulate (Sox2) after differentiation for seven days by removal of EGF, bFGF and Heparin and addition of 10% serum. The differentiated glioma cells gain expression of GFAP, which was not expressed in glioma stem cells. The bar chart presents representative RNA-seq data from one glioma stem cell line. The analysis was performed for all glioma stem cells and differentiated glioma cells. FIG. 2(B) is a chart showing the results of a limiting dilution assay to determine the self-renewal ability of glioma stem cells. The assays were repeated six times, and significance was calculated with a Chi-square test (p<0.008).

FIG. 3 shows comparing survival time in patients with high expression versus low expression of the three glioma stem cell-specific eRNAs. The three Kaplan-Meyer survival curves showing overall patient survival in correlation with eTMEM888 (A), eRTP5 (B), and eNINJ1 (C) eRNAs. The survival time was collected from clinical data of the seventy IDH-wild type glioblastoma patients (Rhode Island Hospital, Departments of Neurosurgery and Pathology). High expression of these eRNAs correlates significantly with lower survival of patients with glioblastoma (p<0.05).

FIG. 4 is a pair of bar graphs showing the inhibition of eTMEM888 and TMEM88B gene. The chart on the left shows the inhibition of eTMEM888 with an Antisense Oligo GapmeR results in inhibition of TMEM88B, NANOG, OCT4, and SOX2 expression. The chart on the right shows the inhibition of TMEM88B mRNA using siRNAs results in inhibition of TMEM88B expression but does not affect NANOG, OCT4, and SOX2.

FIG. 5 is a schematic representation of developing eRNA therapeutics in vivo efficacy measurements.

FIG. 6 is a schematic representation of the lentivirus vector pRSGEP-U6-sg-EF1-Puro.

FIG. 7 is a set of pie charts showing the identification of glioma stem cell-specific eRNAs through a H3K27Ac and RNA Pol II ChIP-seq analysis pipeline. H3K27Ac and RNA Pol II peaks are overlapping the NINJ1 eRNA. H3K27Ac ChIP-seq identified 39,247 and 50,885 sites in GSC1 and GSC2 respectively. These sites were filtered for co-presence of RNA Pol II signal, which resulted in 4284 and 5348 putative eRNA sites in GSC1 and GSC2. See FIG. 7(A). These sites were filtered looking specifically at eRNA sites that have 1:1 relationship with a gene and are located within 10 kb upstream from an annotated promoter of a gene. See FIG. 7(B). This analysis resulted in 229 eRNAs for GSC1 and 213 eRNAs for GSC2. These eRNAs were compared with eRNAs detected (through the same analysis pipeline) in differentiated glioblastoma stromal cells and discovered fifteen glioma stem cell-specific eRNAs that are common in the two patients. See FIG. 7(C).

FIG. 8 is a bar graph showing the expression of eTMEM88b, eNINJ1, and eRTP5 in nine patient-derived glioma stem cells and two human neural stem cell samples.

FIG. 9 is a bar graph showing the expression of TMEM88b, NINJ1, and RTP5 genes in nine patient-derived glioma stem cells and two human neural stem cell samples.

FIG. 10 is a pair of bar graphs showing the treatment of patient derived glioma stem cells with eNINJ1 and eTMEM88B targeting GapmeRs results in 50% inhibition of the eRNA expression. The assays were performed three independent times (n=3 biological replicates) in one patient's glioma stem cells.

FIG. 11 is a bar graph showing the treatment of patient-derived glioma stem cells with 5 μM of the eTMEM88B GapmeR results in significant inhibition of cancer stem cell identity genes. The assay was performed two independent times (n=2 biological replicates).

DETAILED DESCRIPTION OF THE INVENTION

Industrial Applicability

Glioblastoma Multiforme remains one of the most challenging diseases to treat. There is currently no glioma stem cell-specific therapy for glioblastoma. Standard treatments include surgery, followed by combinatorial radiotherapy and chemotherapy. Although conventional GBM therapies have been beneficial to some patients, the average tumor recurrence time is seven months.

Information on epigenetic abnormalities such as DNA methylation, histone modifications, and non-coding RNAs are being evaluated as drivers of cancer, including Glioblastoma Multiforme. See Feng et al., Methods Mol. Biol., 1165, 115-43 (2014).

Using cutting-edge genome-wide technology such as ChIP-seq to identify glioma stem cell-specific eRNAs has shown that eRNAs are essential for glioma stem cell maintenance. The invention provides a way for eRNAs to be a stem cell-specific therapy for Glioblastoma Multiforme. The specific target of eRNAs using Locked Nucleic Acid GapmeRs provides a glioma stem cell-specific, RNA-based therapeutic option for glioblastoma patients.

The successful manipulation of the glioma stem cell-specific eRNAs can benefit the procuring a promising glioma stem cells-specific RNA-based therapy for Glioblastoma Multiforme patients. This specification shows how to determine the functional role of glioma stem cell-specific eRNAs in Glioblastoma Multiforme. See EXAMPLE 2 below. This specification also explains how to determine the contribution of eRNAs to the maintenance of chromatin states in glioma stem cells. See EXAMPLE 3 below. This specification further shows how to evaluate the efficacy of eRNAs as glioma stem cell-specific therapeutic targets. See EXAMPLE 4 below.

Definitions

For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are listed below. Unless stated otherwise or implicit from context, these terms and phrases have the meanings below. These definitions are to aid in describing particular embodiments and are not intended to limit the claimed invention. Unless otherwise defined, all technical and scientific terms have the same meaning as commonly understood by a person having ordinary skill in the molecular neurobiological art or biotechnological art or both arts. For any apparent discrepancy between the meaning of a term in the art and a definition provided in this specification, the meaning provided in this specification shall prevail.

“ATAC-seq” or Assay for Transposase-Accessible Chromatin using sequencing is a molecular biology technique to assess genome-wide chromatin accessibility. See Buenrostro et al., Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins, and nucleosome position. Nature Methods, 10(12), 1213-8 (December 2013).

“Brain tumor” has the neurobiological art-recognized meaning of a growth of abnormal cells in or derived from the tissues of the brain. Brain tumors can be benign (not cancer) or malignant (cancer). See the National Cancer Institute Dictionary of Cancer Terms.

“ChIP-seq” or ChIP-sequencing is a method used to analyze protein interactions with DNA. ChIP-seq combines chromatin immunoprecipitation (ChIP) with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins.

“Differentiated glioma cells” (DGCs) have the neurobiological art-recognized meaning of glioma cells differentiated from stem cells. The molecular and genetic classifications of Glioblastoma Multiforme and treatment approaches to Glioblastoma Multiforme have relied predominantly on differentiated glioma cells. In laboratory practice, differentiated glioma cells gain expression of GFAP, which marker was not expressed in glioma stem cells. See, Bramanti et al., Biomarkers of glial cell proliferation and differentiation in culture. Front. Biosci. (Schol. Ed.), 2, 558-70 (2010). See also FIG. 2(A) description, above.

“Enhancer RNA” (eRNA) has the molecular biological art-recognized meaning of a member of a class of relatively short non-coding RNA molecules (50-2000 nucleotides) transcribed from the DNA sequence of enhancer regions. See FIG. 1. The stem-specific eRNAs are named according to the genes in their closest vicinity and denoted with an “e” prefix throughout this specification.

“GapmeR” has the biotechnological art-recognized meaning of short DNA antisense oligonucleotide structures with RNA-like segments on both sides of the sequence. These linear pieces of genetic information are designed to hybridize to a target piece of RNA and silence the gene through the induction of RNase H cleavage. Binding of the gapmer to the target has a higher affinity due to the modified RNA flanking regions, as well as resistance to degradation by nucleases. See Stein et al., Nucleic Acids Res., 38(1), e3 (2010); Crooke et al., Antisense technology: an overview and prospectus. Nature Reviews Drug Discovery, 1-27 (Mar. 24, 2021).

“Glioblastoma Multiforme” brain tumor (GBM) has the neurobiological art-recognized meaning of a fast-growing glioma that develops from star-shaped glial cells (astrocytes and oligodendrocytes) that support the health of the nerve cells within the brain. GBM is often called a grade IV astrocytoma. See National Cancer Institute (NCI) Dictionary of Cancer Terms.

“Glioma stem cell” (GSC)— also known as glioblastoma stem cells for this specification—has the neurobiological art-recognized meaning of self-renewing, tumorigenic cancer stem cells present in glioblastoma and that contribute to tumor initiation and therapeutic resistance. Glioma stem cells exhibit “phenotypic plasticity” which is important because the different cellular states of Glioblastoma Multiforme (stem and differentiated states) are believed by persons having ordinary skill in the molecular neurobiological art to contribute to tumor mechanism and aggression. In laboratory practice, glioma stem cells known markers of stemness, including oligodendrocyte transcription factor (Olig2), Sox2, and prominin-1 (CD133). See Yan et al., Endothelial cells promote the stem-like phenotype of glioma cells through activating the Hedgehog pathway. J. Pathol., 234(1), 11-22 (2014). See also FIG. 2(A) description, above.

“H3K27Ac” has the molecular biological art-recognized meaning of an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the acetylation at the 27th lysine residue of the histone H3 protein. H3K27ac is associated with the higher activation of transcription and therefore defined as an active enhancer mark. H3K27ac is found at both proximal and distal regions of the transcription start site (TSS).

“H3K4me3” has the molecular biological art-recognized meaning of an epigenetic modification to the DNA packaging protein Histone H3. It is a mark that indicates the tri-methylation at the 4th lysine residue of the histone H3 protein and often involved in regulating gene expression. The name denotes the addition of three methyl groups (trimethylation) to the lysine 4 on the histone H3 protein.

“Lentivirus viral vector” has the molecular biological art-recognized meaning of a replication-defective viral vector that comprises a sequence of RNA or DNA nucleotides derived from a lentivirus.

“NINJ1” or Ninjurin 1 is a protein-coding gene. Diseases associated with NINJ1 include Hereditary Sensory Neuropathy and Non-Functioning Pancreatic Endocrine Tumor. An important paralog of this gene is NINJ2. Several identification numbers are GCID: GC09M093121, HGNC: 7824, Entrez Gene: 4814, Ensembl: ENSG00000131669, OMIM: 602062, and UniProtKB: Q92982.

“Non-coding RNAs” have the molecular biological art-recognized meaning of RNA species that are not templates for protein and include: ribosomal RNA (rRNA), microRNA (miRNA), long non-coding RNA (lncRNA) and other forms produced at different regions in the genome. See, Costa, Non-coding RNAs: Meet thy masters. Bioessays, 32(7), 599-608 (2010), and Guttman et al., Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature, 458(7235), 223-7 (2009).

“Primary brain tumor” has the molecular neurobiological art-recognized meaning of a tumor that develops in a brain. Primary brain tumors are classified by type and the World Health Organization (WHO) grade. Doctors often diagnose primary brain tumors by performing a computerized tomography (CT) scan or magnetic resonance imaging (MRI). A full medical exam may also performed and a detailed medical history taken to help doctors plan the best course of management. When human brain tumors are near “eloquent” parts of the brain with important function (i.e., speech and motor areas), a more specialized MRI can be performed by neuroradiologists to better understand the relationship between the tumor and the function in the brain.

“RTP5” or Receptor Transporter Protein 5 (Putative) is a protein-coding gene. Diseases associated with RTP5 include Venous Hemangioma and Amyotrophic Lateral Sclerosis 7. Among its related pathways are Signaling by GPCR and Olfactory transduction. An important paralog of this gene is RTP3. Several identification numbers are GCID: GC02P241869, HGNC: 26585, Entrez Gene: 285093, Ensembl: ENSG00000188011, and UniProtKB: Q14D33

“shRNA” or short hairpin ribonucleic acids have the molecular biological art-meaning of an artificial RNA molecule with a tight hairpin turn that can silence target gene expression via RNA interference (RNAi). Paddison et al., Genes & Development, 16(8), 948-58 (April 2002); Brummelkamp et al., Science, 296(5567), 550-3 (April 2002). The expression of shRNA in cells is typically accomplished by the delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi because it has a relatively low rate of degradation and turnover.

“TMEM88B” or Transmembrane Protein 88B is a protein-coding gene. Diseases associated with TMEM88B include Alexithymia. Several identification numbers are GCID: GC01P001426, HGNC: 37099, Entrez Gene: 643965, Ensembl: ENSG00000205116, and UniProtKB: A6NKF7.

“Viral vector” has the molecular biological art-recognized meaning of a nucleic acid vector construct that includes at least one viral origin element and can be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide or nucleic acid in place of non-essential viral genes. The vector or particle can transfer any nucleic acids into cells either in vitro or in vivo. Many viral vectors are known in the molecular biological art.

Guidance from Materials and Methods

A person having ordinary skill in the molecular neurobiological art can use the following patents, patent applications, and scientific references as guidance to predictable results when making and using the invention:

To ensure rigor and reproducibility, the inventors repeat assays using at least three biological replicates.

eRNA inhibition assay. The effects of eRNA inhibition are compared to glioma stem cells treated with scrambled GapmeRs. Representation of all subtypes is useful for identifying patients who benefit from GapmeR RNA-based therapy.

Cell death assay. Apoptotic cell death is quantified in real-time using the Incucyte live-cell analysis system to measure the cleavage of Caspase-3/7. The assay uses inert, non-fluorescent substrates that freely cross the cell membrane, where they can be cleaved by activated caspase-3/7 to release either a green or red DNA-binding fluorescent label. The appearance of fluorescently-labeled nuclei identifies apoptotic cells.

Proliferation assay. The proliferation rate of cells in culture is quantified in real-time using the Incucyte Cell-by-Cell Analysis Software Module. This label-free, direct cell count allows for identifying individual cells by counting the number of phase objects over time. Cells are classified into subpopulations based on properties such as size and shape.

Self-renewal assay. The ability of glioma stem cells to self-renew is analyzed using the in-vitro extreme limiting dilution analysis (ELDA).

Luciferase reporter assay. Several luciferase assays are commercially available.

Method of Manufacture

In a seventh commercial embodiment, a person having ordinary skill in the biotechnological art can use Qiagen's online algorithm to design the antisense oligonucleotide GapmeR. Qiagen (Hilden, Germany) can then synthesize the custom GapmeR for in vivo applications. Qiagen can synthesize proprietary chemical modifications, so that the antisense oligonucleotides remain stable and resistant to degradation.

Method of Treatment

The method of treatment is based upon the methods of Southwell et al., Trends in Molecular Medicine, 18(11) (November 2012) and several other papers. Oligonucleotide as therapy can be efficiently and safely delivered with intrathecal injections, intra parenchymal, or cerebrospinal fluid (CSG) delivery.

In an eighth particular embodiment, the administration of RNA therapeutics to the brain is intrathecal. In our case, the inventors favor the intrathecal injection in humans or primates. For the rodent models, the inventors use injection in the lateral ventricle, so the GapmeR is delivered through the cerebrospinal fluid.

Pharmaceutical Compositions

One or more of the pharmaceutical and liposomal compositions (e.g., lipid nanoparticles) comprise one or more of the compounds disclosed herein and one or more additional lipids. For example, lipid nanoparticles that comprise or are otherwise enriched with one or more of the compounds disclosed herein may further comprise one or more of DOTAP (1,2-dioleyl-3-trimethylammonium propane), DODAP (1,2-dioleyl-3-dimediylammonium propane), DOTMA (1,2-di-0-octadecenyl-3-Irimethylammonium propane), DLinDMA, DLin-KC2-DMA, CI2-200 and ICE. In a ninth embodiment, the pharmaceutical composition comprises a lipid nanoparticle that comprises HGT4001, DOPE and DMG-PEG2000. In tenth embodiment, the pharmaceutical composition comprises a lipid nanoparticle that comprises HGT4003, DOPE, cholesterol and DMG-PEG2000.

One or more of the pharmaceutical compositions described herein may comprise one or more PEG-modified lipids. For example, lipid nanoparticles that comprise or are otherwise enriched with one or more of the compounds disclosed herein may further comprise one or more of PEG-modified lipids that comprise a poly(ethylene)glycol chain of up to 5 kDa in length covalently attached to a lipid comprising one or more C₆-C₂₀alkyls.

Similarly, the pharmaceutical compositions (e.g., lipid nanoparticles) may comprise or may otherwise be enriched with one or more of the compounds disclosed herein and may further comprise one or more of helper lipids selected from the group consisting of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), DOPE (1,2-dioleoyl-swglycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-s/i-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroylglycero-3-phosphoethanolamine), DPPS (1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine), ceramides, sphingomyelins and cholesterol.

The following EXAMPLES are provided to illustrate the invention and should not be considered to limit its scope.

Example 1

An Oligonucleotide GapmeR that Inhibits the Target eRNA.

To discover eRNAs expressed specifically in glioma stem cells, the inventors used two patient-derived glioma stem cells that represent the mesenchymal (GSC1) and proneural (GSC2) subtypes of the TOGA-based Verhaak classification scheme. To obtain differentiated glioma cells, the inventors subjected the glioma stem cells to a seven-day differentiation process. They performed transcript expression analysis for glioma stem cells and differentiated cell markers. See FIG. 2(A). All glioma stem cell samples demonstrated self-renewal potential during limiting dilution assays. See FIG. 2(B). After transplantation into immunocompromised mice, the glioma stem cells showed tumor-forming ability.

To identify which eRNAs glioma stem cells specifically express, the inventors performed ChIP-seq using ChIP-validated antibodies against H3K27Ac for active enhancers, and RNAPII for active transcription (Active Motif). Peak calling representing genome-wide antibody binding was achieved for both H3K27Ac and RNAPII in all samples. The subsequent analysis included the removal of overlap with mRNA production; the association of RNAPII and H3K27Ac peak overlaps as putative eRNA production regions; the identification of individual sample-specific eRNAs; the selection of peaks within 10,000 nucleotides (10K) upstream and downstream of the identified putative eRNA sites as these regions best associate with the genes of interest; the isolation of common peaks between the two glioma stem cell and differentiated glioma cell samples; and discovery of unique peaks within the stem and differentiated samples. The unique peaks were defined as RNAPII and H3K27Ac peaks. The inventors observed that the peaks align and overlap exactly within the same enhancer peak boundary. The computational filtration analysis allowed the narrowing of the results down to fifteen eRNAs unique to the two glioma stem cells and twenty-one eRNAs unique to the two differentiated glioma cells.

To validate the expression of the glioma stem cell-specific eRNAs, the inventors performed RT-qPCR using RNA isolated from seven additional patient-derived glioma stem cells (GSC) and two neural stem cell (NSC) lines (H9 & H14 derivatives). The individual eRNAs had varied expression patterns within and across the glioma stem cell samples.

The inventors then probed for the expression of the genes corresponding to all fifteen eRNAs. The inventors observed that the expressions were all amplified in glioma stem cells at varying levels.

Next, the inventors determined the expression of the eRNAs in seventy Glioblastoma Multiforme tissue samples available from the Department of Pathology at Rhode Island Hospital. The inventors probed for fourteen eRNAs instead of fifteen eRNAs because the RT-qPCR results showed that one of the eRNAs [eDAP3] was low or not expressed in various glioma stem cell samples. Using NanoString Technology and their nSolver™ Analysis Software, the inventors generated an eRNA expression heatmap for the seventy Glioblastoma Multiforme tissue samples with high (indicated by red) and low (blue) across all samples. See, e.g., FIG. 5. The inventors correlated the tissue expression of the eRNAs to the survival of patients using the standardized log-rank statistics and maximally selected rank statistics.

Patients with high expression of eTMEM88B (Transmembrane Protein 88B), eRTP5 (Receptor Transporter Protein 5), and eNINJ1 (Nerve Injury-Induced Protein), presented with worst survival at statistically significant levels compared to low expressors. See FIG. 3.

Example 2

Determining the Functional Relevance of Glioma Stem Cell-Specific eRNAs for Glioblastoma Multiforme

The inventors use a pharmacological approach to analyze the effect of eRNA inhibition on apoptosis, proliferation, self-renewal, and differentiation of glioma stem cells. The inventors inhibit three clinically relevant eRNAs and then check the effect of inhibition.

Inhibition of eRNA and functional analysis to check for the effect of inhibition on glioma stem cells. In EXAMPLE 1, using one glioma stem cell sample and two concentrations of Locked Nuclei Acid GapmeRs, targeting eTMEM888 has shown promising knockdown of this eRNA in a concentration-dependent manner. The inventors repeat this inhibition for the other two eRNAs: eRTP5 and eNINJ1.

The following assays can show whether inhibition of eRNAs affects glioma stem cell viability, proliferation, self-renewal, and differentiation:

Additional mechanism of eRNA effects on glioma stem cells. Regarding the functional role of eRNAs in glioma stem cells, the inventors determine how the reported functions are due to the eRNA per se or the cognate gene, TMEM88B. The inventors performed separate single tests where the expression levels of three essential stem-cell genes (NANOG, OCT4, SOX2) upon eTMEM888 inhibition using the GapmeR and inhibition of TMEM88B gene were examined, using a pool of siRNAs. These results showed that inhibition of eTMEM888 results in a significant knockdown of the cognate gene, TMEM88B, especially at the higher concentration of 5 μM. Furthermore, the inventors observed slight inhibition of NANOG, OCT4, and SOX2 despite GapmeR dose. The knockdown of the TMEM88B gene with siRNAs does not affect NANOG, OCT4, and SOX2 expression. See, FIG. 8.

Thus, eTMEM888 expression has a specific role in regulating not only its cognate gene but also in stemness gene expression in glioma stem cells.

The inventors also investigate other parameters of the eRNA effect on glioma stem cells, such as checking for changes in important promoter regions using a luciferase reporter assay.

Alternative strategies. Inhibition using the GapmeR involved a direct cellular uptake method called gymnosis. This method only showed ˜50% knockdown. Therefore, the inventors can use an alternative method such as optimized lipid transfection to increase the GapmeR uptake and achieve a higher knockdown for all eRNA targets. Since each glioma stem cell sample has its own unique cultural needs, the inventors can troubleshoot their transfection protocol to determine the optimal conditions for each sample empirically.

Example 3

Determine the Contribution of eRNAs to Changes in Chromatin States of Glioma Stem Cells

The manipulation of eRNAs can modulate the chromatin state of glioma stem cells. The inventors use a combination of Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq), and Chromatin Immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) genome-wide sequencing tools to test this manipulation. This work is to determine the function of eRNAs as epigenetic regulators of cellular plasticity and cancer stemness.

Research approach. Effects of eRNAs on chromatin accessibility. To determine genome-wide chromatin accessibility patterns, the inventors perform ATAC-seq in glioma stem cells following inhibition of eRNA and compare to ATAC-seq data from control glioma stem cells (treated with scrambled GapmeRs). Data analysis is performed to discover how inhibition of eRNA expression affects global chromatin accessibility over gene regulatory regions. The inventors determine chromatin accessibility over promoter and enhancer regions and functionally cluster the closest genes to the differentially accessible promoters and enhancers.

Effects of eRNAs on transcription regulation. The higher-order eukaryotic genome functional architecture structures and their associated sub-nuclear compartments are recognized by those of ordinary skill in the molecular neurobiological art as the key components contributing to many aspects of nuclear activities, including DNA transcription. Scientific publications have shown that eRNAs augment gene transcription through interaction with various general cofactors, such as CBP, Mediator, BRD4, and Cohesin. To discover the role of the glioma stem cell-specific eRNAs in this transcriptional mechanism, the inventors can perform ChIP-seq for Mediator, cohesin, and CBP and BRD4, respectively, following knockdown of eRNA expression with oligonucleotide GapmeRs as described above.

Alternative strategies. Persons having ordinary skill in the molecular neurobiological art have experience in performing and analyzing ATAC-seq and ChIP-seq. Persons having ordinary skill in the molecular neurobiological art also have experience with an alternative analysis strategy, such as the publicly-available ChromHMM package, to annotate distinct “chromatin states” in the genome of the glioma stem cell samples. The ChromHMM package uses a multivariate hidden Markov model and epigenomic marks given as inputs to identify these chromatin states based on markers absent or present at genomic regions and the spatial relationship of these marks.

Example 4

Evaluate the Efficacy of eRNAs as RNA-Therapeutics.

Glioblastoma Multiforme therapies face many challenges, especially hindrance of drug passage through the blood-brain barrier (BBB). The inventors chemically modified and stable Locked Nucleic Acid GapmeRs that are in-vivo ready to inhibit our target eRNAs. Next, the inventors evaluate the efficacy of inhibition on tumor growth and size using Magnetic Resonance Imaging (MRI) and changes in stem-cell essential gene transcripts using RNA-seq.

To determine the efficacy of eRNA inhibition as a potential therapeutic for glioma stem cells, the inventors perform in vivo studies using orthotopic xenograft models of human glioblastoma. eRNAs may be adequate therapeutic targets for glioma stem cells owing to the identification of a specific group of eRNAs in glioma stem cells and not in their differentiated progeny.

Research Approach. Tumor generation and treatment with eRNA GapmeRs. The inventors can implant our patient-derived glioma stem cells (200,000 cells/mouse) into Nu/J mice under stereotaxic guidance, as described by Zepecki et al., Oncogene (2018). The inventors then allow tumors to grow for a month and then insert a microscopic catheter into the lateral ventricle connected to an osmotic pump (Alzet) to continuously deliver in vivo stable GapmeRs against the targeted eRNAs. See FIG. 5. To compare the efficacy of the treatment, the inventors use twelve mice for the non-treated control group, twelve mice for the scrambled GapmeR negative control group, and twelve mice for the eRNA-targeting GapmeR treated group.

Assays designed (twelve mice per group for each assay). Quantify inhibition for each eRNA and check for expression of stem and differentiation markers. The inventors check for successful inhibition of the target eRNAs using RT-qPCR and examine the changes in stem and differentiated markers: CD44 and GFAP respectively to ensure that in-vivo analyses are due to the inhibition of the eRNAs.

Determine the effect of eRNA inhibition on tumor volume. The inventors can stain serial sections of the brain of mice with HuNu antibody (Abcam) to accurately detect human glioma cells and perform 3D reconstruction and quantification of tumor volume, as shown before by Zepecki et al., Oncogene (2018).

The inventors micro-dissect tumors following a one-month treatment with the eRNA targeting GapmeR and perform RNAseq to determine the effect of eRNA inhibition on the glioblastoma transcriptome. As described in EXAMPLE 2, in vitro inhibition of eRNA results in inhibition of stemness gene expression. The treated tumors should express a less aggressive transcriptomic signature than control tumors.

Relevance to therapies for Glioblastoma Multiforme. Nucleic acid modifications and drug carriers are under development for the delivery of therapeutic molecules to the central nervous system. Alternatively, delivery of therapeutic molecules to the central nervous system can be accomplished by surgical techniques including intraventricular delivery of therapeutics via pumps, convection-enhanced delivery to brain parenchyma for drugs, and direct stereotactic injections of virus or cells remain the technique of choice currently.

Direct targeting of important eRNAs in Glioblastoma Multiforme is attractive for several reasons. First, a low copy number of eRNAs expressed in cells versus mRNAs ensures that fewer inhibitory GapmeRs are necessary to achieve eRNA inhibition. Second, eRNAs impact chromatin accessibility and modify gene expression patterns. Third, eRNAs represent an important node in the alteration of gene expression patterns. Fourth, eRNA expression patterns are linked to the clinical survival of Glioblastoma Multiforme patients in TOGA and other large RNA seq databases.

Alternative strategies. The Alzet pump intraventricular delivery of GapmeRs may not achieve therapeutic levels in the bulk tumor. It may require convection-enhanced delivery, as shown by Souweidane et al., The Lancet Oncology, 19(8), 1040-50 (2018). The inventors can also explore the option of a “cocktail GapmeR” targeting all three eRNAs at once to access the combinatory effect of these eRNAs on glioma stem cell samples. To explore the efficiency of eRNAs as glioma stem cell-specific therapy, the inventors can combine each eRNA with temozolomide (the current chemotherapy for Glioblastoma Multiforme) at various concentrations and then evaluate the effects on glioma stem cells.

Example 5

Identification of Glioma Stem Cell Specific eRNAs

To identify glioma stem cell-specific eRNAs, the inventors performed H3K27Ac ChIP-seq and RNA PolII ChiP-seq on glioma stem cells and differentiated glioma cells from two patients. H3K27Ac is a histone mark that identifies active enhancers and RNA PolII denotes active transcription. Detection of overlapping peaks on the genome where H3K27Ac and RNA PolII are co-present is an indication of the presence of an eRNA. H3K27Ac ChIP-seq identified 39,247 and 50,885 sites in GSC1 and GSC2 respectively. These sites were filtered for co-presence of RNA Pol II signal, which resulted in 4284 and 5348 putative eRNA sites in GSC1 and GSC2. See FIG. 7(A). The inventors filtered these sites looking specifically at eRNA sites that have 1:1 relationship with a gene and are located within 10 kb upstream from an annotated promoter of a gene. See FIG. 7(B). This analysis resulted in 229 eRNAs for GSC1 and 213 eRNAs for GSC2. The inventors then compared these eRNAs with eRNAs detected (through the same analysis pipeline) in differentiated glioblastoma stromal cells and discovered fifteen glioma stem cell-specific eRNAs that are common in the two patients. See FIG. 7(C). The inventors visualized the presence of H3K27Ac and RNA Pol II signal on the enhancer region of NINJ1 gene, denoting the presence of NINJ1 eRNA in GSC1 and GSC2.

Example 6

Expression of eRNAs in Patients with Glioblastoma

Expression of eTMEM88b, eNINJ1 and eRTP5 (three of the fifteen GSC-specific eRNAs that most significantly correlate with patient survival) was examined in GSCs isolated from nine patients with glioblastoma and in two human neural stem cell lines (H9 and H4 derivatives from NIH) as controls. See FIG. 5. All patients express the three eRNAs in variable levels. eTMEM88b shows high expression in GSCs and control cells, eNINJ1 is expressed in moderate levels in GSCs and not detected in control cells, while eRTP5 is barely detected in GSCs or neural stem cells. See FIG. 8.

The inventors determined the expression of the cognate genes of the eRNAs in GSCs and control human neural stem cells, since eRNAs usually regulate the expression of their cognate genes. Ninj1 gene is expressed in variable levels in all nine GSC samples and in the two neural stem cell samples. TMEM88b shows little expression in two of the nine GSCs, while RTP5 is not expressed in GSCs. See FIG. 9. To determine the expression of the three eRNAs in glioblastoma tumors, the inventors performed Nanostring expression analysis in glioblastoma tumor samples from seventy patients that were operated in Rhode Island Hospital. This result showed that eNINJ1 and eTMEM88b have variable but high expression in all seventy glioblastoma tumor samples, while eRTP5 shows low expression. Clustering of eRNAs was determined using Euclidean distance.

The inventors correlated the tissue expression of the three eRNAs to survival of the patients using the standardized log-rank statistics and maximally selected rank statistic. Patients with high expression of eNINJ1 show the most significant correlation with decreased survival (p<0.002), while high expression of eTMEM88b and eRTP5 also correlated with decreased survival but the correlation was less significant than eNINJ1. See FIG. 5.

Example 7

Design and Synthesis of Oligonucleotide GapmeRs Targeting eNINJ1 and eTMEM88b

The inventors designed LNA-modified oligonucleotide GapmeRs targeting various areas of the eNINJ1 and eTMEM88b sequence and tested them in patient-derived GSCs to verify inhibition of the eRNA expression. The initial candidate GapmeRs show significant inhibition of eNINJ1 and eTMEM88b expression, but only up to 50% of the total eRNA expression. See FIG. 10.

To increase GapmeR induced inhibition of the Ninj1 eRNA, the inventors designed ninety-six candidates GapmeRs spanning the entire sequence of the Ninj1 eRNA. See TABLE 1 for some of the sequences of the GapmeRs to be used in a high-throughput in vitro assay for discovering the best inhibitory GapmeR for further in vitro and in vivo studies as an eRNA therapeutic against eNinj1.

TABLE 1
Name of	Antisense	SEQ
enhancer RNA	LNA GapmeR sequence	ID NO:
eTMEM88B	5′-CGGGAGTAGAAGTTGG-3′	1
eNINJ1	5′-GCGTCTAGGCTGGCAG-3′	2

Example 8

Inhibition of eTMEM888 Results in Inhibition of Glioma Stem Cell Identity Genes.

The significance of these genes in glioblastoma has been shown previously. Notch1 regulates glioblastoma growth, resistance to apoptosis, and maintenance of GSC properties. Notch1 regulates expression of PTCH1, Sox2, and Nanog. Klf4 regulates growth of GSCs and is upregulated following treatment of glioblastoma with chemotherapy. Fold-change of these genes is calculated in comparison to GSCs treated with non-targeting GapmeRs and normalized to housekeeping gene expression. See FIG. 11.

SEQUENCE LISTING
eTMEM88B: Chromosome 1 (GSC1-start: 1430292-GSC2-end: 1430960).
Length: 668nucleotides.
ATCTAGTACATCATATTGCCAGCAGGGCTCAGCCTGTGACCAGCAAGGTCTGGGCTCCGTCTGGG
GCCAGGGTCAAGGCTCATTCCGTGGCCTTGAGCACAGCCTGGTGTGTGGCTGGGTCGAGTCCAGC
AGAAGTCTCGGGGTTTTCCAGCCTCCATCCGAGTCCGGCTTTGATGCCTTTCCTGGTCAGACAGG
AGCCGGGCCAGTGGCCAAGCTGCCAGGATGGCTTCCCCGGGGCCGCGGCCGCCTTCCTTCCCCTC
CTGCCCGGGCTGGCTCTGGTCGCCACTAGGGGTTGAAGATGAGGGCTCTCCTGGGAAGCTCTTGG
CTGAGGATCCGCCTGGTCCCGGAGCCGTGAAAACAACAGCTGGGCCTGGTGGGGGTGGGGAGGCT
GAGCGGAGAGGCCAGCTCCCTTGGCTGCTGGGCAGGGTCTTCTGTCCACAGCTGCCTCAGGCGGC
TGTTTCCAAAGGTGTTTCCAGCTTCCCAGGCCCACCCTGAGGCCCCGCACCGCCAGGGAGGTGGA
AGGCACGGAGCAGCGAAGCCCGGCCCCGGCCCCGGCCGCCCGACCAGCTCACAGAGGAACACCTG
TGGGGGGGCCTGTGGGCGGTTCACAGAGGGATGTAGGAACGTGCCTGTGGGAGGCCGTAGCCCCG
GAGAGCAGAGGCCTGGCC [SEQ ID NO: 3].
eRTP5: Chromosome 2 (GSC1-start: 242088593-GSC2-end: 242088884).
Length: 291 nucleotides.
CGGCAGCAACACTGCCACGCGAATCCGCGCCGGCCAATCAGCATGGCCAGGGGCGGGGCTTCCCT
GAGGCGCGCCGAGAGGCGGTGGCCCACTTCCGGCAATAATCGCCTGGTCGCCGTCAGGTGCCGGC
CCAGGTGGCAGGCGCGCCCGTTGGGCACTGGGGGACGCGGGCGCGTCAGGTGAAGACTGGGGGCT
GCAGGCGCGCTAGGTAGGTACGGGGTGCCGCGGGCGCGTCAGGTGAAGACTGGGCGCCGCAGGCG
CCTTAGGTGAAGATTGGGGATCGCGGGCGCG [SEQ ID NO: 4].
eNINJ1: Chromosome 9 (GSC1-start: 93143058-GSC2-end: 93144787).
Length: 1,832 nucleotides.
agaattgcctgaaccaggggttggaggttgcagcgggtggaaattttgccactgcactccagcct
gggtgacagagtgagaccctgtctcaaagaaaaaaaaaaaaagaTACATCATCTGGGACAATAAC
CTTGAAAAGCAGGGGTCCCAGACGACCTTATTTGCAGAGAATGCGACTGCAGACGGCAAGCAGGG
GGGCATGCCCTTCGCTCTCTGTCCTCTGCTCTTTGCCCCGGCCACTGTCGGCCTCATCTGAAGGC
CACCTGTGCCTCACGGTCTGAAAACGCTGGTTTCCACAGCTGCTTCTCCTTCCAAATTTCCCTGG
AGTCTTGCTTTGGTGGCGAACCTCTGGTTCTATTCCTTCCTTTTAACTGAAGCCTATAGGAAAAA
TTTGGAAGTTGAAATATGCCAGTCCAAGGAAGGTGGACGTGGAGTTCTGAGGGTGGGGGTGCTGC
CAGGGAAGTGGCACTGTGCAGGGGACCGCCCCTGGGACCCCCTGGTTCCTGTCAAGAGCAGGTAG
Gggctgggcgcggtggctcacgcctataatcccagcactttgggaggtcaaggagagtggatcac
ctaaggtcaggagttcaagaccagctgaccaacatggtgaaaccccgtctctactaaaaatacaa
aaattagctgggcgtggtggcaggcgcctataatcccaggtattcaggaggctgaggcaggagaa
tagcttgaacccaggaggcagaggttgcagtgagctgagatcgcaccactgcactccagcctggg
cgacagagcgagactctgtctacacacacacacacacacacacagacacacacacacacacaGAT
CAGGTAGGATGTGAGGTGTGTCCTCATGGCCGGACATGGGGTGGGTGGGGCCAAACAACCACAGG
GACTCGTCCTGTGGCCACTGCTGCTCAGGAAGTGGATCCCAAGGAGCAGAGTCGCCAGACCCCTC
AGTTCCCAGCTCCACATTTAAGGCAGGTCTGGCCATGAGCCAGGCCTCTGCATGTGACCTGGGGC
CTCACTGTGGCATGGCTGCCTGTCCCACCTGTGGATGTTGCCTGTGCTGTGTAGAAGCCACATAG
CCTCCGGGGCGGCTCCCCAGAATGCCACATTTCCTGTCTCTGGCTCTGATGGCGTCTAGGCTGGC
AGGGGTCCCGGCCCCAGCAGTACTGTTGCCGGGCAGAGCTCAGGGCCACGTGCAGTTGGGTCTGG
CTGAGAGCATCTCATGGGTTTATGAGAACCCTTCCAGCACAAAGGGGCATTTATCAGGCAGGGAT
GGCATGTCTTGGTCTGAACACAGGAAACACAGAAATAGCCTTTCACAGAGTGCCAGCAGGGCTGG
GCTCGCCTGCTGTGGAGGGTGTCGGCTTTCCAACTCCTTCTCCAAGCTGTGCGACCCGTCCATGT
TCCCCTGTGAGTTGTTCTGTCCCAGACAGGGCATTCCCTGAGAACGCTCCTGCTGCAACTGgagg
gagagaggcagggaggggaaaggggaagacttgcagggagaaggaagaagggagacagatggaga
cagacagaaggagggatggaagaaatgagagagagagagggagggagaTGGAAACATAGATTGTC
TCCACTGTGACACCCTGCCTCCATGGTTCTCCATGATGGGAATGGAATTCATGTCTCACCTGGCA
GCAAGGGTCTCCACAATATGACTTCACCTTCTTTCTCTTAGTAAGGCGAGAGCAGACAGGCAGAC
ACTCCAGGAAGTAACTGATGTGTCCCTGAAAATGCCTGTCTTTCCCAGCTTGTACAACTTTGCTT
GTGTTATTTCTT [SEQ ID NO: 5].

The following sequences were generated using bioinformatics methods whereby the target eRNA sequence (eNINJ1) a 1,832 nt non-coding RNA was screened to find ideal/target regions that were 20 nucleotides long. The ideal/target regions were selected using criteria such as % GC content (˜30-60%), number of miRNA hits (score of was selected); Gene hits (scores of 1 or less were selected) and a rank system (this showed whether a target sequence was good or not; bioinformatics stuff really). The top (50) target regions were selected to synthesize and generate GapmeRs (LNA and 2′MOE) that target the selected regions of eNINJ1. (lN)# is a LNA nucleotide. (eN)# is a 2′MOE nucleotide. (dN)# is a deoxyribonucleotide (DNA).

LNCRNA_22_LNA. Target 20 nucleotides (nt) (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGUACAAGCUGGGAAAGACA [SEQ ID NO: 6].

LNCRNA_22_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGUCUUUCCCAGCUUGUACA [SEQ ID NO: 7].

LNCRNA_22_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lT)#(lC)#(lT)#(dT)#(dT)#(dC)#(dC)#(dC)#(dA) #(dG)#(dC)#(dT)#(dT)#(lG)#(lT)#(lA)#(lC)#(lA) [SEQ ID NO: 8].

LNCRNA_22_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eT)#(eC)#(eT)#(dT)#(dT)#(dC) #(dC)#(dC)#(dA)#(dG)#(dC)#(dT)#(dT)#(eG)#(eT)#(eA)#(eC)#(eA) [SEQ ID NO: 9].

LNCRNA_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CUGGGAAAGACAGGCAUUUU [SEQ ID NO: 10].

LNCRNA_30_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAAAUGCCUGUCUUUCCCAG [SEQ ID NO: 11].

LNCRNA_30_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lA)#(lA)#(lT)#(dG)#(dC)#(dC)#(dT)#(dG)#(dT) #(dC)#(dT)#(dT)#(dT)#(lC)#(lC)#(lC)#(lA)#(lG) [SEQ ID NO: 12].

LNCRNA_30_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eA)#(eA)#(eT)#(dG)#(dC) #(dC)#(dT)#(dG)#(dT)#(dC)#(dT)#(dT)#(dT)#(eC)#(eC)#(eC)#(eA)#(eG) [SEQ ID NO: 13].

LNCRNA_40_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CAGGCAUUUUCAGGGACACA [SEQ ID NO: 14].

LNCRNA_40_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGUGUCCCUGAAAAUGCCUG [SEQ ID NO: 15].

LNCRNA_40_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lT)#(lG)#(lT)#(dC)#(dC)#(dC)#(dT)#(dG)#(dA) #(dA)#(dA)#(dA)#(dT)#(lG)#(lC)#(lC)#(lT)#(lG) [SEQ ID NO: 16].

LNCRNA_40_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eT)#(eG)#(eT)#(dC)#(dC) #(dC)#(dT)#(dG)#(dA)#(dA)#(dA)#(dA)#(dT)#(eG)#(eC)#(eC)#(eT)#(eG) [SEQ ID NO: 18].

LNCRNA_49_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCAGGGACACAUCAGUUACU [SEQ ID NO: 19].

LNCRNA_49_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGUAACUGAUGUGUCCCUGA [SEQ ID NO: 20].

LNCRNA_49_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lT)#(lA)#(lA)#(dC)#(dT)#(dG)#(dA)#(dT)#(dG) #(dT)#(dG)#(dT)#(dC)#(lC)#(lC)#(lT)#(lG)#(lA) [SEQ ID NO: 21].

LNCRNA_49_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eT)#(eA)#(eA)#(dC)#(dT) #(dG)#(dA)#(dT)#(dG)#(dT)#(dG)#(dT)#(dC)#(eC)#(eC)#(eT)#(eG)#(eA) [SEQ ID NO: 22].

LNCRNA_51_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AGGGACACAUCAGUUACUUC [SEQ ID NO: 23].

LNCRNA_51_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GAAGUAACUGAUGUGUCCCU [SEQ ID NO: 24].

LNCRNA_51_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lA)#(lA)#(lG)#(lT)#(dA)#(dA)#(dC)#(dT)#(dG)#(dA) #(dT)#(dG)#(dT)#(dG)#(lT)#(lC)#(lC)#(lC)#(lT) [SEQ ID NO: 25].

LNCRNA_51_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eA)#(eA)#(eG)#(eT)#(dA)#(dA) #(dC)#(dT)#(dG)#(dA)#(dT)#(dG)#(dT)#(dG)#(eT)#(eC)#(eC)#(eC)#(eT) [SEQ ID NO: 26].

LNCRNA_109_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AAAGAAGGUGAAGUCAUAUU [SEQ ID NO: 27].

LNCRNA_109_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAUAUGACUUCACCUUCUUU [SEQ ID NO: 28].

LNCRNA_109_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lT)#(lA)#(lT)#(dG)#(dA)#(dC)#(dT)#(dT)#(dC) #(dA)#(dC)#(dC)#(dT)#(lT)#(lC)#(lT)#(lT)#(lT) [SEQ ID NO: 29].

LNCRNA_109_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eT)#(eA)#(eT)#(dG)#(dA)#(dC) #(dT)#(dT)#(dC)#(dA)#(dC)#(dC)#(dT)#(eT)#(eC)#(eT)#(eT)#(eT) [SEQ ID NO: 30].

LNCRNA_118_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). GAAGUCAUAUUGUGGAGACC [SEQ ID NO: 31].

LNCRNA_118_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GGUCUCCACAAUAUGACUUC [SEQ ID NO: 32].

LNCRNA_118_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lT)#(lC)#(lT)(dC)#(dC)#(dA)#(dC)#(dA)#(dA) #(dT)#(dA)#(dT)#(dG)#(lA)#(lC)#(lT)#(lT)#(lC) [SEQ ID NO: 33].

LNCRNA_118_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eT)#(eC)#(eT)(dC)#(dC)#(dA) #(dC)#(dA)#(dA)#(dT)#(dA)#(dT)#(dG)#(eA)#(eC)#(eT)#(eT)#(eC) [SEQ ID NO: 34].

LNCRNA_141_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). GCUGCCAGGUGAGACAUGAA [SEQ ID NO: 35].

LNCRNA_141_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UUCAUGUCUCACCUGGCAGC [SEQ ID NO: 36].

LNCRNA_141_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lT)#(lC)#(lA)(lT)#(dG)#(dT)#(dC)#(dT) #(dC)#(dA)#(dC)#(dC)#(dT)#(dG)#(lG)#(lC)#(lA)#(lG)#(lC) [SEQ ID NO: 37].

LNCRNA_141_ LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eT)#(eC)#(eA)(eT)#(dG)#(dT)#(dC) #(dT)#(dC)#(dA)#(dC)#(dC)#(dT)#(dG)#(eG)#(eC)#(eA)#(eG)#(eC) [SEQ ID NO: 38].

LNCRNA_150_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGAGACAUGAAUUCCAUUCC [SEQ ID NO: 39].

LNCRNA_150_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GGAAUGGAAUUCAUGUCUCA [SEQ ID NO: 40].

LNCRNA_150_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lA)#(lA)(lT)#(dG)#(dG)#(dA)#(dA)#(dT)#(dT) #(dC)#(dA)#(dT)#(dG)#(lT)#(lC)#(lT)#(lC)#(lA) [SEQ ID NO: 41].

LNCRNA_150_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eA)#(eA)(eT)#(dG)#(dG) #(dA)#(dA)#(dT)#(dT)#(dC)#(dA)#(dT)#(dG)#(eT)#(eC)#(eT)#(eC)#(eA) [SEQ ID NO: 42].

LNCRNA_196_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGUCACAGUGGAGACAAUCU [SEQ ID NO: 43].

LNCRNA_196_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGAUUGUCUCCACUGUGACA [SEQ ID NO: 44].

LNCRNA_196_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lA)#(lT)#(lT)#(dG)#(dT)#(dC)#(dT)#(dC)#(dC) #(dA)#(dC)#(dT)#(dG)#(lT)#(lG)#(lA)#(lC)#(lA) [SEQ ID NO: 45].

LNCRNA_196_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eA)#(eT)#(eT)#(dG)#(dT) #(dC)#(dT)#(dC)#(dC)#(dA)#(dC)#(dT)#(dG)#(eT)#(eG)#(eA)#(eC)#(eA) [SEQ ID NO: 46].

LNCRNA_250_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCUUCCAUCCCUCCUUCUGU [SEQ ID NO: 47].

LNCRNA_250_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. ACAGAAGGAGGGAUGGAAGA [SEQ ID NO: 48].

LNCRNA_250_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lC)#(lA)#(lG)#(lA)#(dA)#(dG)#(dG)#(dA)#(dG)#(dG) #(dG)#(dA)#(dT)#(dG)#(lG)#(lA)#(lA)#(lG)#(lA) [SEQ ID NO: 49].

LNCRNA_250_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eC)#(eA)#(eG)#(eA)#(dA)#(dG) #(dG)#(dA)#(dG)#(dG)#(dG)#(dA)#(dT)#(dG)#(eG)#(eA)#(eA)#(eG)#(eA) [SEQ ID NO: 50].

LNCRNA_377_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCUGGGACAGAACAACUCAC [SEQ ID NO: 51].

LNCRNA_377_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GUGAGUUGUUCUGUCCCAGA [SEQ ID NO: 52].

LNCRNA_377_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lT)#(lG)#(lA)#(lG)(dT)#(dT)#(dG)#(dT)#(dT)#(dC) #(dT)#(dG)#(dT)#(dC)#(lC)#(lC)#(lA)#(lG)#(lA) [SEQ ID NO: 53].

LNCRNA_377_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eT)#(eG)#(eA)#(eG)(dT)#(dT)#(dG) #(dT)#(dT)#(dC)#(dT)#(dG)#(dT)#(dC)#(eC)#(eC)#(eA)#(eG)#(eA) [SEQ ID NO: 54].

LNCRNA_485_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CUGUGAAAGGCUAUUUCUGU [SEQ ID NO: 55].

LNCRNA_485_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. ACAGAAAUAGCCUUUCACAG [SEQ ID NO: 56].

LNCRNA_485_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lC)#(lA)#(lG)#(lA)#(dA)#(dA)#(dT)#(dA)#(dG)#(dC) #(dC)#(dT)#(dT)#(dT)#(lC)#(lA)#(lC)#(lA)#(lG) [SEQ ID NO: 57].

LNCRNA_485_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eC)#(eA)#(eG)#(eA)#(dA)#(dA) #(dT)#(dA)#(dG)#(dC)#(dC)#(dT)#(dT)#(dT)#(eC)#(eA)#(eC)#(eA)#(eG) [SEQ ID NO: 58].

LNCRNA_491_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AAGGCUAUUUCUGUGUUUCC [SEQ ID NO: 59].

LNCRNA_491_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GGAAACACAGAAAUAGCCUU [SEQ ID NO: 60].

LNCRNA_491_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lA)#(lA)#(lA)#(dC)#(dA)#(dC)#(dA)#(dG)#(dA) #(dA)#(dA)#(dT)#(dA)#(lG)#(lC)#(lC)#(lT)#(lT) [SEQ ID NO: 61].

LNCRNA_491_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eA)#(eA)#(eA)#(dC)#(dA) #(dC)#(dA)#(dG)#(dA)#(dA)#(dA)#(dT)#(dA)#(eG)#(eC)#(eC)#(eT)#(eT) [SEQ ID NO: 62].

LNCRNA_499_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUCUGUGUUUCCUGUGUUCA [SEQ ID NO: 63].

LNCRNA_499_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGAACACAGGAAACACAGAA [SEQ ID NO: 64].

LNCRNA_499_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lA)#(lA)#(lC)#(dA)#(dC)#(dA)#(dG)#(dG)#(dA) #(dA)#(dA)#(dC)#(dA)#(lC)#(lA)#(lG)#(lA)#(lA) [SEQ ID NO: 65].

LNCRNA_499_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eA)#(eA)#(eC)#(dA)#(dC) #(dA)#(dG)#(dG)#(dA)#(dA)#(dA)#(dC)#(dA)#(eC)#(eA)#(eG)#(eA)#(eA) [SEQ ID NO: 66].

LNCRNA_500_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCUGUGUUUCCUGUGUUCAG [SEQ ID NO: 67].

LNCRNA_500_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. CUGAACACAGGAAACACAGA [SEQ ID NO: 68].

LNCRNA_500_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lC)#(lT)#(lG)#(lA)#(lA)#(dC)#(dA)#(dC)#(dA)#(dG)#(dG) #(dA)#(dA)#(dA)#(dC)#(lA)#(lC)#(lA)#(lG)#(lA) [SEQ ID NO: 69].

LNCRNA_500_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eC)#(eT)#(eG)#(eA)#(eA)#(dC)#(dA) #(dC)#(dA)#(dG)#(dG)#(dA)#(dA)#(dA)#(dC)#(eA)#(eC)#(eA)#(eG)#(eA) [SEQ ID NO: 70].

LNCRNA_517_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CAGACCAAGACAUGCCAUCC [SEQ ID NO: 71].

LNCRNA_517_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GGAUGGCAUGUCUUGGUCUG [SEQ ID NO: 72].

LNCRNA_517_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lA)#(lT)#(lG)#(dG)#(dC)#(dA)#(dT)#(dG)#(dT) #(dC)#(dT)#(dT)#(dG)#(lG)#(lT)#(lC)#(lT)#(lG) [SEQ ID NO: 73].

LNCRNA_517_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eA)#(eT)#(eG)#(dG)#(dC) #(dA)#(dT)#(dG)#(dT)#(dC)#(dT)#(dT)#(dG)#(eG)#(eT)#(eC)#(eT)#(eG) [SEQ ID NO: 74].

LNCRNA_527_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CAUGCCAUCCCUGCCUGAUA [SEQ ID NO: 75].

LNCRNA_527_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UAUCAGGCAGGGAUGGCAUG [SEQ ID NO: 76].

LNCRNA_527_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lA)#(lT)#(lC)#(lA)#(dG)#(dG)#(dC)#(dA)#(dG)#(dG) #(dG)#(dA)#(dT)#(dG)#(lG)#(lC)#(lA)#(lT)#(lG) [SEQ ID NO: 77].

LNCRNA_527_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eA)#(eT)#(eC)#(eA)#(dG)#(dG) #(dC)#(dA)#(dG)#(dG)#(dG)#(dA)#(dT)#(dG)#(eG)#(eC)#(eA)#(eT)#(eG) [SEQ ID NO: 78].

LNCRNA_536_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CCUGCCUGAUAAAUGCCCCU [SEQ ID NO: 79].

LNCRNA_536_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGGGGCAUUUAUCAGGCAGG [SEQ ID NO: 80].

LNCRNA_536_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lG)#(lG)#(lG)#(dC)#(dA)#(dT)#(dT)#(dT)#(dA) #(dT)#(dC)#(dA)#(dG)#(lG)#(lC)#(lA)#(lG)#(lG) [SEQ ID NO: 81].

LNCRNA_536_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eG)#(eG)#(eG)#(dC)#(dA) #(dT)#(dT)#(dT)#(dA)#(dT)#(dC)#(dA)#(dG)#(eG)#(eC)#(eA)#(eG)#(eG) [SEQ ID NO: 82].

LNCRNA_555_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUUGUGCUGGAAGGGUUCUC [SEQ ID NO: 83].

LNCRNA_555_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GAGAACCCUUCCAGCACAAA [SEQ ID NO: 84].

LNCRNA_555_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lA)#(lG)#(lA)#(lA)#(dC)#(dC)#(dC)#(dT)#(dT)#(dC) #(dC)#(dA)#(dG)#(dC)#(lA)#(lC)#(lA)#(lA)#(lA) [SEQ ID NO: 85].

LNCRNA_555_LNA. 5-10-5 MOE Gapmer contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eA)#(eG)#(eA)#(eA)#(dC)#(dC) #(dC)#(dT)#(dT)#(dC)#(dC)#(dA)#(dG)#(dC)#(eA)#(eC)#(eA)#(eA)#(eA) [SEQ ID NO: 86].

LNCRNA_556_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUGUGCUGGAAGGGUUCUCA [SEQ ID NO: 87].

LNCRNA_556_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGAGAACCCUUCCAGCACAA [SEQ ID NO: 88].

LNCRNA_556_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lA)#(lG)#(lA)#(dA)#(dC)#(dC) #(dC)#(dT)#(dT)#(dC)#(dC)#(dA)#(dG)#(lC)#(lA)#(lC)#(lA)#(lA) [SEQ ID NO: 89].

LNCRNA_556_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eA)#(eG)#(eA)#(dA)#(dC) #(dC)#(dC)#(dT)#(dT)#(dC)#(dC)#(dA)#(dG)#(eC)#(eA)#(eC)#(eA)#(eA) [SEQ ID NO: 90].

LNCRNA_569_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). GUUCUCAUAAACCCAUGAGA [SEQ ID NO: 91].

LNCRNA_569_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UCUCAUGGGUUUAUGAGAAC [SEQ ID NO: 92].

LNCRNA_569_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lC)#(lT)#(lC)#(lA)#(dT)#(dG)#(dG)#(dG)#(dT)#(dT) #(dT)#(dA)#(dT)#(dG)#(lA)#(lG)#(lA)#(lA)#(lC) [SEQ ID NO: 93].

LNCRNA_569_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eC)#(eT)#(eC)#(eA)#(dT)#(dG) #(dG)#(dG)#(dT)#(dT)#(dT)#(dA)#(dT)#(dG)#(eA)#(eG)#(eA)#(eA)#(eC) [SEQ ID NO: 94].

LNCRNA_571_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCUCAUAAACCCAUGAGAUG [SEQ ID NO: 95].

LNCRNA_571_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. CAUCUCAUGGGUUUAUGAGA [SEQ ID NO: 96].

LNCRNA_571_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lC)#(lA)#(lT)#(lC)#(lT)#(dC)#(dA)#(dT)#(dG)#(dG)#(dG) #(dT)#(dT)#(dT)#(dA)#(lT)#(lG)#(lA)#(lG)#(lA) [SEQ ID NO: 97].

LNCRNA_571_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eC)#(eA)#(eT)#(eC)#(eT)#(dC)#(dA) #(dT)#(dG)#(dG)#(dG)#(dT)#(dT)#(dT)#(dA)#(eT)#(eG)#(eA)#(eG)#(eA) [SEQ ID NO: 98].

LNCRNA_972_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CCUCACAUCCUACCUGAUCU [SEQ ID NO: 99].

LNCRNA_972_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGAUCAGGUAGGAUGUGAGG [SEQ ID NO: 100].

LNCRNA_972_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lA)#(lT)#(lC)#(dA)#(dG)#(dG)#(dT)#(dA)#(dG) #(dG)#(dA)#(dT)#(dG)#(lT)#(lG)#(lA)#(lG)#(lG) [SEQ ID NO: 101].

LNCRNA_972_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eA)#(eT)#(eC)#(dA)#(dG) #(dG)#(dT)#(dA)#(dG)#(dG)#(dA)#(dT)#(dG)#(eT)#(eG)#(eA)#(eG)#(eG) [SEQ ID NO: 102].

LNCRNA_1027_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGUGUAGACAGAGUCUCGCU [SEQ ID NO: 103].

LNCRNA_1027_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGCGAGACUCUGUCUACACA [SEQ ID NO: 104].

LNCRNA_1027_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lC)#(lG)#(lA)#(dG)#(dA)#(dC)#(dT)#(dC)#(dT) #(dG)#(dT)#(dC)#(dT)#(lA)#(lC)#(lA)#(lC)#(lA) [SEQ ID NO: 105].

LNCRNA_1027_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eC)#(eG)#(eA)#(dG)#(dA) #(dC)#(dT)#(dC)#(dT)#(dG)#(dT)#(dC)#(dT)#(eA)#(eC)#(eA)#(eC)#(eA) [SEQ ID NO: 106].

LNCRNA_1207_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUCACCAUGUUGGUCAGCUG [SEQ ID NO: 107].

LNCRNA_1207_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. CAGCUGACCAACAUGGUGAA [SEQ ID NO: 108].

LNCRNA_1207_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lC)#(lA)#(lG)#(lC)#(lT)#(dG)#(dA)#(dC)#(dC)#(dA)#(dA) #(dC)#(dA)#(dT)#(dG)#(lG)#(lT)#(lG)#(lA)#(lA) [SEQ ID NO: 109].

LNCRNA_1207_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eC)#(eA)#(eG)#(eC)#(eT)#(dG)#(dA) #(dC)#(dC)#(dA)#(dA)#(dC)#(dA)#(dT)#(dG)#(eG)#(eT)#(eG)#(eA)#(eA) [SEQ ID NO: 110].

LNCRNA_1216_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUGGUCAGCUGGUCUUGAAC [SEQ ID NO: 111].

LNCRNA_1216_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GUUCAAGACCAGCUGACCAA [SEQ ID NO: 112].

LNCRNA_1216_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lT)#(lT)#(lC)#(lA)#(dA)#(dG)#(dA)#(dC)#(dC)#(dA) #(dG)#(dC)#(dT)#(dG)#(lA)#(lC)#(lC)#(lA)#(lA) [SEQ ID NO: 113].

LNCRNA_1216_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eT)#(eT)#(eC)#(eA)#(dA)#(dG) #(dA)#(dC)#(dC)#(dA)#(dG)#(dC)#(dT)#(dG)#(eA)#(eC)#(eC)#(eA)#(eA) [SEQ ID NO: 114].

LNCRNA_1415_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CUUGGACUGGCAUAUUUCAA [SEQ ID NO: 115].

LNCRNA_1415_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UUGAAAUAUGCCAGUCCAAG [SEQ ID NO: 116].

LNCRNA_1415_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lT)#(lG)#(lA)#(lA)#(dA)#(dT)#(dA)#(dT)#(dG)#(dC) #(dC)#(dA)#(dG)#(dT)#(lC)#(lC)#(lA)#(lA)#(lG) [SEQ ID NO: 117].

LNCRNA_1415_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eT)#(eG)#(eA)#(eA)#(dA)#(dT)#(dA) #(dT)#(dG)#(dC)#(dC)#(dA)#(dG)#(dT)#(eC)#(eC)#(eA)#(eA)#(eG) [SEQ ID NO: 118].

LNCRNA_1421_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CUGGCAUAUUUCAACUUCCA [SEQ ID NO: 119].

LNCRNA_1421_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGGAAGUUGAAAUAUGCCAG [SEQ ID NO: 120].

LNCRNA_1421_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lG)#(lA)#(lA)#(dG)#(dT)#(dT)#(dG)#(dA)#(dA) #(dA)#(dT)#(dA)#(dT)#(lG)#(lC)#(lC)#(lA)#(lG) [SEQ ID NO: 121].

LNCRNA_1421_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eG)#(eA)#(eA)#(dG)#(dT) #(dT)#(dG)#(dA)#(dA)#(dA)#(dT)#(dA)#(dT)#(eG)#(eC)#(eC)#(eA)#(eG) [SEQ ID NO: 122].

LNCRNA_1422_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGGCAUAUUUCAACUUCCAA [SEQ ID NO: 123].

LNCRNA_1422_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UUGGAAGUUGAAAUAUGCCA [SEQ ID NO: 124].

LNCRNA_1422_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lT)#(lG)#(lG)#(lA)#(dA)#(dG)#(dT)#(dT)#(dG)#(dA) #(dA)#(dA)#(dT)#(dA)#(lT)#(lG)#(lC)#(lC)#(lA) [SEQ ID NO: 125].

LNCRNA_1422_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eT)#(eG)#(eG)#(eA)#(dA)#(dG) #(dT)#(dT)#(dG)#(dA)#(dA)#(dA)#(dT)#(dA)#(eT)#(eG)#(eC)#(eC)#(eA) [SEQ ID NO: 126].

LNCRNA_1427_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UAUUUCAACUUCCAAAUUUU [SEQ ID NO: 127].

LNCRNA_1427_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAAAUUUGGAAGUUGAAAUA [SEQ ID NO: 128].

LNCRNA_1427_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lA)#(lA)#(lT)#(dT)#(dT)#(dG)#(dG)#(dA)#(dA) #(dG)#(dT)#(dT)#(dG)#(lA)#(lA)#(lA)#(lT)#(lA) [SEQ ID NO: 129].

LNCRNA_1427_LNA. 5-10-5 MOE GapmeR contains 2′ MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eA)#(eA)#(eT)#(dT)#(dT)#(dG) #(dG)#(dA)#(dA)#(dG)#(dT)#(dT)#(dG)#(eA)#(eA)#(eA)#(eT)#(eA) [SEQ ID NO: 130].

LNCRNA_1445_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUUCCUAUAGGCUUCAGUUA [SEQ ID NO: 131].

LNCRNA_1445_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UAACUGAAGCCUAUAGGAAA [SEQ ID NO: 132].

LNCRNA_1445_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lA)#(lA)#(lC)#(lT)#(dG)#(dA)#(dA)#(dG)#(dC)#(dC) #(dT)#(dA)#(dT)#(dA)#(lG)#(lG)#(lA)#(lA)#(lA) [SEQ ID NO: 133].

LNCRNA_1445_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eA)#(eA)#(eC)#(eT)#(dG)#(dA)#(dA) #(dG)#(dC)#(dC)#(dT)#(dA)#(dT)#(dA)#(eG)#(eG)#(eA)#(eA)#(eA) [SEQ ID NO: 134].

LNCRNA_1447_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCCUAUAGGCUUCAGUUAAA [SEQ ID NO: 135].

LNCRNA_1447_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UUUAACUGAAGCCUAUAGGA [SEQ ID NO: 136].

LNCRNA_1447_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lT)#(lT)#(lA)#(lA)#(dC)#(dT)#(dG)#(dA)#(dA)#(dG) #(dC)#(dC)#(dT)#(dA)#(lT)#(lA)#(lG)#(lG)#(lA) [SEQ ID NO: 137].

LNCRNA_1447_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eT)#(eT)#(eA)#(eA)#(dC)#(dT)#(dG) #(dA)#(dA)#(dG)#(dC)#(dC)#(dT)#(dA)#(eT)#(eA)#(eG)#(eG)#(eA) [SEQ ID NO: 138].

LNCRNA_1460_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AGUUAAAAGGAAGGAAUAGA [SEQ ID NO: 139].

LNCRNA_1460_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UCUAUUCCUUCCUUUUAACU [SEQ ID NO: 140].

LNCRNA_1460_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lC)#(lT)#(lA)#(lT)#(dT)#(dC)#(dC)#(dT)#(dT)#(dC) #(dC)#(dT)#(dT)#(dT)#(lT)#(lA)#(lA)#(lC)#(lT) [SEQ ID NO: 141].

LNCRNA_1460_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eC)#(eT)#(eA)#(eT)#(dT)#(dC)#(dC) #(dT)#(dT)#(dC)#(dC)#(dT)#(dT)#(dT)#(eT)#(eA)#(eA)#(eC)#(eT) [SEQ ID NO: 142].

LNCRNA_1488_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUCGCCACCAAAGCAAGACU [SEQ ID NO: 143].

LNCRNA_1488_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGUCUUGCUUUGGUGGCGAA [SEQ ID NO: 144].

LNCRNA_1488_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lT)#(lC)#(lT)#(dT)#(dG)#(dC)#(dT)#(dT)#(dT) #(dG)#(dG)#(dT)#(dG)#(lG)#(lC)#(lG)#(lA)#(lA) [SEQ ID NO: 145].

LNCRNA_1488_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eT)#(eC)#(eT)#(dT)#(dG)#(dC) #(dT)#(dT)#(dT)#(dG)#(dG)#(dT)#(dG)#(eG)#(eC)#(eG)#(eA)#(eA) [SEQ ID NO: 146].

LNCRNA_1532_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AGCUGUGGAAACCAGCGUUU [SEQ ID NO: 147].

LNCRNA_1532_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAACGCUGGUUUCCACAGCU [SEQ ID NO: 148].

LNCRNA_1532_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lA)#(lC)#(lG)#(dC)#(dT)#(dG)#(dG)#(dT)#(dT) #(dT)#(dC)#(dC)#(dA)#(lC)#(lA)#(lG)#(lC)#(lT) [SEQ ID NO: 149].

LNCRNA_1532_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eA)#(eC)#(eG)#(dC)#(dT) #(dG)#(dG)#(dT)#(dT)#(dT)#(dC)#(dC)#(dA)#(eC)#(eA)#(eG)#(eC)#(eT) [SEQ ID NO: 150].

LNCRNA_1664_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). CUCUGCAAAUAAGGUCGUCU [SEQ ID NO: 151].

LNCRNA_1664_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGACGACCUUAUUUGCAGAG [SEQ ID NO: 152].

LNCRNA_1664_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lA)#(lC)#(lG)#(dA)#(dC)#(dC)#(dT)#(dT)#(dA) #(dT)#(dT)#(dT)#(dG)#(lC)#(lA)#(lG)#(lA)#(lG) [SEQ ID NO: 153].

LNCRNA_1664_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eA)#(eC)#(eG)#(dA)#(dC) #(dC)#(dT)#(dT)#(dA)#(dT)#(dT)#(dT)#(dG)#(eC)#(eA)#(eG)#(eA)#(eG) [SEQ ID NO: 154].

LNCRNA_1683_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGGGACCCCUGCUUUUCAAG [SEQ ID NO: 155].

LNCRNA_1683_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. CUUGAAAAGCAGGGGUCCCA [SEQ ID NO: 156].

LNCRNA_1683_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lC)#(lT)#(lT)#(lG)#(lA)#(dA)#(dA)#(dA)#(dG)#(dC)#(dA) #(dG)#(dG)#(dG)#(dG)#(lT)#(lC)#(lC)#(lC)#(lA) [SEQ ID NO: 157].

LNCRNA_1683_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eC)#(eT)#(eT)#(eG)#(eA)#(dA)#(dA)#(dA) #(dG)#(dC)#(dA)#(dG)#(dG)#(dG)#(dG)#(eT)#(eC)#(eC)#(eC)#(eA) [SEQ ID NO: 158].

LNCRNA_1695_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUUUCAAGGUUAUUGUCCCA [SEQ ID NO: 159].

LNCRNA_1695_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. UGGGACAAUAACCUUGAAAA [SEQ ID NO: 160].

LNCRNA_1695_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lG)#(lG)#(lG)#(lA)#(dC)#(dA)#(dA)#(dT)#(dA)#(dA) #(dC)#(dC)#(dT)#(dT)#(lG)#(lA)#(lA)#(lA)#(lA) [SEQ ID NO: 161].

LNCRNA_1695_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eG)#(eG)#(eG)#(eA)#(dC)#(dA) #(dA)#(dT)#(dA)#(dA)#(dC)#(dC)#(dT)#(dT)#(eG)#(eA)#(eA)#(eA)#(eA) [SEQ ID NO: 162].

LNCRNA_1706_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AUUGUCCCAGAUGAUGUAUC [SEQ ID NO: 163].

LNCRNA_1706_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GAUACAUCAUCUGGGACAAU [SEQ ID NO: 164].

LNCRNA_1706_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lA)#(lT)#(lA)#(lC)#(dA)#(dT)#(dC)#(dA)#(dT)#(dC) #(dT)#(dG)#(dG)#(dG)#(lA)#(lC)#(lA)#(lA)#(lT) [SEQ ID NO: 165].

LNCRNA_1706_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eA)#(eT)#(eA)#(eC)#(dA)#(dT) #(dC)#(dA)#(dT)#(dC)#(dT)#(dG)#(dG)#(dG)#(eA)#(eC)#(eA)#(eA)#(eT) [SEQ ID NO: 166].

LNCRNA_1707_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UUGUCCCAGAUGAUGUAUCU [SEQ ID NO: 167].

LNCRNA_1707_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AGAUACAUCAUCUGGGACAA [SEQ ID NO: 168].

LNCRNA_1707_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lG)#(lA)#(lT)#(lA)#(dC)#(dA)#(dT)#(dC)#(dA)#(dT) #(dC)#(dT)#(dG)#(dG)#(lG)#(lA)#(lC)#(lA)#(lA) [SEQ ID NO: 169].

LNCRNA_1707_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eG)#(eA)#(eT)#(eA)#(dC)#(dA)#(dT) #(dC)#(dA)#(dT)#(dC)#(dT)#(dG)#(dG)#(eG)#(eA)#(eC)#(eA)#(eA) [SEQ ID NO: 170].

LNCRNA_1708_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UGUCCCAGAUGAUGUAUCUU [SEQ ID NO: 171].

LNCRNA_1708_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAGAUACAUCAUCUGGGACA [SEQ ID NO: 172].

LNCRNA_1708_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lG)#(lA)#(lT)#(dA)#(dC)#(dA)#(dT)#(dC)#(dA) #(dT)#(dC)#(dT)#(dG)#(lG)#(lG)#(lA)#(lC)#(lA) [SEQ ID NO: 173].

LNCRNA_1708_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eG)#(eA)#(eT)#(dA)#(dC) #(dA)#(dT)#(dC)#(dA)#(dT)#(dC)#(dT)#(dG)#(eG)#(eG)#(eA)#(eC)#(eA) [SEQ ID NO: 174].

LNCRNA_1709_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). GUCCCAGAUGAUGUAUCUUU [SEQ ID NO: 175].

LNCRNA_1709_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAAGAUACAUCAUCUGGGAC [SEQ ID NO: 176].

LNCRNA_1709_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lA)#(lG)#(lA)#(dT)#(dA)#(dC)#(dA)#(dT) #(dC)#(dA)#(dT)#(dC)#(dT)#(lG)#(lG)#(lG)#(lA)#(lC) [SEQ ID NO: 177].

LNCRNA_1709_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eA)#(eG)#(eA)#(dT)#(dA) #(dC)#(dA)#(dT)#(dC)#(dA)#(dT)#(dC)#(dT)#(eG)#(eG)#(eG)#(eA)#(eC) [SEQ ID NO: 178].

LNCRNA_1710_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). UCCCAGAUGAUGUAUCUUUU [SEQ ID NO: 179].

LNCRNA_1710_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. AAAAGAUACAUCAUCUGGGA [SEQ ID NO: 180].

LNCRNA_1710_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lA)#(lA)#(lA)#(lA)#(lG)#(dA)#(dT)#(dA)#(dC)#(dA)#(dT) #(dC)#(dA)#(dT)#(dC)#(lT)#(lG)#(lG)#(lG)#(lA) [SEQ ID NO: 181].

LNCRNA_1710_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eA)#(eA)#(eA)#(eA)#(eG)#(dA)#(dT)#(dA) #(dC)#(dA)#(dT)#(dC)#(dA)#(dT)#(dC)#(eT)#(eG)#(eG)#(eG)#(eA) [SEQ ID NO: 182].

LNCRNA_1775_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). AGUGCAGUGGCAAAAUUUCC [SEQ ID NO: 183].

LNCRNA_1775_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. GGAAAUUUUGCCACUGCACU [SEQ ID NO: 184].

LNCRNA_1775_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lA)#(lA)#(lA)#(dT)#(dT)#(dT)#(dT)#(dG)#(dC) #(dC)#(dA)#(dC)#(dT)#(lG)#(lC)#(lA)#(lC)#(lT) [SEQ ID NO: 185].

LNCRNA_1775_LNA. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eA)#(eA)#(eA)#(dT)#(dT)#(dT) #(dT)#(dG)#(dC)#(dC)#(dA)#(dC)#(dT)#(eG)#(eC)#(eA)#(eC)#(eT) [SEQ ID NO: 186].

LNCRNA_1783_LNA. Target 20 nt (5′-3′) contains the original eRNA sequence that was selected for GapmeR design (20 nucleotides each). GGCAAAAUUUCCACCCGCUG [SEQ ID NO: 187].

LNCRNA_1783_LNA. AS sequence (5′-3′) contains the antisense sequence that targets the eRNA region above. The antisense sequences are provided as RNA nucleotides. CAGCGGGUGGAAAUUUUGCC [SEQ ID NO: 188].

LNCRNA_1783_LNA. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lC)#(lA)#(lG)#(lC)#(lG)#(dG)#(dG)#(dT)#(dG)#(dG) #(dA)#(dA)#(dA)#(dT)#(dT)#(lT)#(lT)#(lG)#(lC)#(lC) [SEQ ID NO: 189].

LNCRNA_1783_LNA. 5-10-5 MOE Gapmer contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eC)#(eA)#(eG)#(eC)#(eG)#(dG)#(dG) #(dT)#(dG)#(dG)#(dA)#(dA)#(dA)#(dT)#(dT)#(eT)#(eT)#(eG)#(eC)#(eC) [SEQ ID NO: 190].

MALAT1. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lG)#(lG)#(lG)#(lT)#(lC)#(dA)#(dG)#(dC)#(dT)#(dG)#(dC)#(dC)#(dA)#(dA) #(dT)#(lG)#(lC)#(lT)#(lA)#(lG) [SEQ ID NO: 191].

MALAT1. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eG)#(eG)#(eG)#(eT)#(eC)#(dA)#(dG)#(dC)#(dT)#(dG)#(dC) #(dC)#(dA)#(dA)#(dT)#(eG)#(eC)#(eT)#(eA)#(eG) [SEQ ID NO: 192].

NTC_LNAGapmer. 5-10-5 LNA GapmeR contains LNA GapmeR sequences in this structural order: (5) LNA sequences followed by (10) DNA sequences and finally (5) LNA sequences. (lT)#(lA)#(lA)#(lT)#(lC)#(dG)#(dT)#(dA)#(dT)#(dT)#(dT) #(dG)#(dT)#(dC)#(dA)#(lA)#(lT)#(lC)#(lA)#(lT) [SEQ ID NO: 193].

NTC_LNAGapmer. 5-10-5 MOE GapmeR contains 2′MOE GapmeR sequences in this structural order: (5) 2′MOE sequences followed by (10) DNA sequences and finally (5) 2′MOE sequences. (eT)#(eA)#(eA)#(eT)#(eC)#(dG)#(dT)#(dA) #(dT)#(dT)#(dT)#(dG)#(dT)#(dC)#(dA)#(eA)#(eT)#(eC)#(eA)#(eT) [SEQ ID NO: 194].

List of Embodiments

Specific compositions and methods of targeting enhancer RNAs for the treatment of primary brain tumors have been described. The detailed description in this specification is illustrative and not restrictive or exhaustive. The detailed description is not intended to limit the disclosure to the precise form disclosed. Other equivalents and modifications besides those already described are possible without departing from the inventive concepts described in this specification, as persons having skilled in the molecular neurobiological art will recognize. When the specification or claims recite method steps or functions in an order, alternative embodiments may perform the functions in a different order or substantially concurrently. The inventive subject matter, therefore, is not to be restricted except in the spirit of the disclosure.

When interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by persons having ordinary skill in the molecular neurobiological art. This invention is not limited to the particular methodology, protocols, reagents, and the like described in this specification and, as such, can vary in practice. The terminology used in this specification is not intended to limit the scope of the invention, which is defined solely by the claims.

The terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, used, or combined with other elements, components, or steps. The singular terms “a,” “an,” and “the” include plural referents unless context indicates otherwise. Similarly, the word “or” should cover “and” unless the context indicates otherwise. The abbreviation “e.g.” is used to indicate a non-limiting example and is synonymous with the term “for example.”

When a range of values is provided, each intervening value, to the tenth of the unit of the lower limit, unless the context dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that range of values.

Some embodiments of the technology described can be defined according to the following numbered paragraphs:

• • 1. The GapmeRs disclosed in this patent specification for use in the treatment of brain tumors.
  • 2. The GapmeRs disclosed in this patent specification for use in the treatment of primary brain tumors.
  • 3. A method of targeting enhancer RNAs (eRNA), using synthetic oligonucleotides or a lentivirus carrying a specific shRNA for the treatment of primary brain tumors.
  • 4. The method of embodiment 3, wherein the synthetic oligonucleotides are synthetic RNA oligonucleotides.
  • 5. A synthetic oligonucleotide that hybridizes with enhancer RNAs (eRNAs) that are expressed specifically in glioma stem cells and which expression correlates with decreased survival of patients with glioblastomas.
  • 6. The synthetic oligonucleotide of embodiment 5, wherein the eRNAs are selected from the group consisting of eTMEM88b, eRTP5, and eNINJ1.
  • 7. A method of treating glioma, using synthetic oligonucleotides to knock out the expression of glioma stem cell eRNAs.
  • 8. The method of embodiment 5, wherein the synthetic oligonucleotides are resistant to degradation.
  • 9. The method of embodiment 5, wherein the administration is systemic or intrathecal.
  • 10. A viral vector that delivers an shRNA that targets and inhibits the expression of the eRNA.
  • 11. The viral vector of embodiment 8, wherein the viral vector is a lentivirus.

CITATION LIST

A person having ordinary skill in the molecular neurobiological art can use these patents, patent applications, and scientific references as guidance to predictable results when making and using the invention.

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All patents and publications cited throughout this specification are expressly incorporated by reference to disclose and describe the materials and methods that might be used with the technologies described in this specification. The publications discussed are provided solely for their disclosure before the filing date. They should not be construed as an admission that the inventors may not antedate such disclosure under prior invention or for any other reason. If there is an apparent discrepancy between a previous patent or publication and the description provided in this specification, the present specification (including any definitions) and claims shall control. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicants and constitute no admission as to the correctness of the dates or contents of these documents. The dates of publication provided in this specification may differ from the actual publication dates. If there is an apparent discrepancy between a publication date provided in this specification and the actual publication date supplied by the publisher, the actual publication date shall control.

Claims

1. A method of targeting enhancer RNAs (eRNA) in a subject in need thereof, the method comprising administering a synthetic modified oligonucleotide for the treatment of primary brain tumors to the subject, wherein the synthetic modified oligonucleotide is a GapmeR comprising SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, or SEQ ID NO: 22.

2. A synthetic modified oligonucleotide that hybridizes with enhancer RNAs (eRNAs) that are expressed specifically in glioma stem cells and which expression correlates with decreased survival of patients with glioblastomas, wherein the synthetic modified oligonucleotide is a GapmeR comprising SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21, or SEQ ID NO: 22.

3. The method of claim 1, wherein the synthetic modified oligonucleotide further comprises a synthetic modified oligonucleotide that is a GapmeR selected from the group consisting of the following GapmeRs: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: , SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, and SEQ ID NO: 194.

4. The synthetic modified oligonucleotide of claim 2, further comprising a synthetic modified oligonucleotide that is a GapmeRs selected from the group consisting of the following GapmeRs: SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: , SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, SEQ ID NO: 193, and SEQ ID NO: 194.