Modulation Of Enhancer Rna Mediated Gene Expression

Abstract

Disclosed herein are methods and compounds for inhibiting gene expression by inhibiting enhancer RNAs (eRNAs). Such methods and compounds are useful for reducing expression of certain genes, many of which are associated with a variety of diseases and disorders.

Description

SEQUENCE LISTING

[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0202USC1SEQ_ST25.txt created Sep. 30, 2016, which is approximately 80 KB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD

[0002] Certain embodiments are directed to methods and compounds for inhibiting gene expression by inhibiting enhancer RNAs (eRNAs). Such methods and compounds are useful for reducing expression of certain genes, many of which are associated with a variety of diseases and disorders.

BACKGROUND

[0003] Recent high-throughput transcriptomic analyses have revealed that eukaryotic genomes transcribe up to 90% of the genomic DNA. (The ENCODE Project Consortium. The ENCODE (ENCyclopedia of DNA Elements) Project. Science 2004; 306:636-640). Only 1-2% of these transcripts encode for proteins, whereas the vast majority are transcribed as non-coding RNAs (ncRNAs).

[0004] The majority of the non-protein-coding transcripts belong to the group of lncRNAs, which are considered as >200 nucleotides in length. Most lncRNAs are characterized by nuclear localization, low expression, low level of sequence conservation and are composed of both poly A+ and poly A- transcripts. (Kapranov P, et al., "RNA maps reveal new RNA classes and a possible function for pervasive transcription." Science 2007; 316:1484-1488) (Wu Q, et al., "Poly A- transcripts expressed in HeLa cells." PLoS One 2008; 3:0803).

[0005] One subgroup of lncRNAs, termed large intergenic non-coding RNAs (lincRNAs), was described based on distinctive chromatin signature that marks actively transcribed genes. (Khalil A M et al., "Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression." Proc Natl Acad Sci USA 2009; 106:11667-11672) (Guttman M et al., "Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals." Nature 2009; 458:223-227). LincRNAs are marked by trimethylation of lysine 4 of histone H3 (H3K4me3) at their promoter and trimethylation of lysine 36 of histone H3 (H3K36me3) along the transcribed region.

[0006] Another subgroup of lncRNAs, termed enhancer RNAs (eRNAs), was recently reported to be transcribed from genomic enhancer regions. (Kim T K et al., "Widespread transcription at neuronal activity-regulated enhancers." Nature 2010; 465:182-187) (De Santa F et al., "A large fraction of extragenic RNA pol II transcription sites overlap enhancers." PLoS Biol 2010; 8:e1000384). Distinct from eRNAs, another subgroup of lncRNAs, termed ncRNA-activators (ncRNA-a) was classified as mono-directional, polyadenylated, and having a H3K4 trimethylation chromatin signature. (Orom U A, Derrien T, Beringer M, Gumireddy K, Gardini A, Bussotti G et al. Long noncoding RNAs with enhancer-like function in human cells. Cell 2010; 143:46-58.)

SUMMARY

[0007] Several embodiments provided herein relate to the discovery that eRNAs are functional molecules that stimulate gene expression and can be targeted for inhibition to modulate gene expression. Prior to the present discovery manifest in several embodiments described herein, it was unknown and uncertain in the field whether eRNAs are functional molecules or merely products of "transcriptional noise" arising from PolII mediated transcription of genomic sequences adjacent to coding genes. (Struhl K, "Transcriptional noise and the fidelity of initiation by RNA polymerase II." Nat Struct Mol Biol 2007 14: 103-105) (Ponting C P et al., "Evolution and functions of long noncoding RNAs." Cell 2009 136: 629-641). eRNAs are widely considered just a byproduct of a transcriptional "ripple effect," a type of transcriptional noise in which PolII causes a wave of transcription from genes into adjacent sequences including intergenic regions. Ebisuya M et al., "Ripples from neighbouring transcription." Nat Cell Biol 2008 10: 1106-1113.

[0008] The present embodiments demonstrate eRNAs are functional molecules and targeting them for inhibition modulates gene expression. Certain embodiments are directed to methods and compounds for inhibiting gene expression by inhibiting enhancer RNAs (eRNA). In certain several embodiments, a method of inhibiting gene expression in a cell includes contacting the cell with a specific inhibitor of an enhancer RNA (eRNA), thereby inhibiting expression of one or more genes in the cell. In certain aspects, the eRNA is transcribed from a genomic enhancer sequence or region; the genomic enhancer sequence or region comprises any one of the genomic coordinates identified in Mega-Tables 1-9 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012; the eRNA transcription is initiated from a RNA polymerase II (PolII) binding site and is capable of elongating bidirectionally; the eRNA is capable of enhancing transcription of the one or more genes; the genomic enhancer sequence or region has a higher level of monomethylated lysine 4 of histone 3 (H3K4me1) than trimethylated lysine 4 of histone 3 (H3K4me3); the genomic enhancer sequence or region is enriched for bound RNA polymerase II (PolII); the genomic enhancer sequence or region is enriched for bound transcriptional co-activator p300/CBP; the genomic enhancer sequence or region is enriched for bound Rev-Erb.alpha. or Rev-Erb.beta.; the genomic enhancer sequence or region is enriched for bound estrogen receptor, such as estradiol-bound estrogen receptor; eRNA has a relatively short half-life compared to mRNA, such as less than about 10-30 minutes; the transcriptional start site of the one or more genes is located on a chromosome at least about 1 kilobase (kb) from the genomic enhancer sequence or region; the eRNA is not polyadenylated or has a shorter polyA tail than mRNA or ncRNA-a; the cell is a hematopoietic cell, such as a monocyte or macrophage; the cell is a neuron; the cell is a breast cell; the cell is a cancer cell; and/or the cell contacted with a specific inhibitor of an enhancer RNA (eRNA) is in a subject. In several embodiments, the eRNA enhances transcription of matrix metalloproteinase 9 (MMP9), chemokine receptor CX3CR1, CA12, FOXC1, GREB1, P2RY2, SMAD7, PGR, SIAH2, NRIP1, TFF1, or KCNK5.

[0009] In several embodiments, compounds, which can be used in the aforementioned methods of inhibiting gene expression in a cell, comprise a specific inhibitor of an enhancer RNA (eRNA). In certain aspects, the specific inhibitor of an enhancer RNA (eRNA) is an antisense compound. In several aspects, the antisense compound is single-stranded; double-stranded; modified; 8 to 80 nucleosides in length; 12 to 30 nucleosides in length; 16 nucleosides in length; includes at least one modified sugar, such as a bicyclic sugar, a 2'-O-methoxyethyl group, and/or a 4'-CH(CH.sub.3)--O-2' group; includes at least one modified internucleoside linkage, such as a phosphorothioate internucleoside linkage; and/or includes at least one modified nucleobase, such as a 5-methylcytosine.

[0010] In further aspects, the antisense compound includes a gap segment consisting of linked deoxynucleosides; a 5' wing segment consisting of linked nucleosides; and a 3' wing segment consisting of linked nucleosides, wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment and wherein each nucleoside of each wing segment comprises a modified sugar.

[0011] In another aspect, the antisense compound includes a gap segment consisting of ten linked deoxynucleosides; a 5' wing segment consisting of 3 linked nucleosides; and a 3' wing segment consisting of 3 linked nucleosides; wherein the gap segment is positioned between the 5' wing segment and the 3' wing segment, wherein each nucleoside of each wing segment comprises a 2'-O-methoxyethyl sugar or a constrained ethyl sugar; and wherein each internucleoside linkage is a phosphorothioate linkage.

[0012] In certain aspects, the 3 linked nucleosides of the 5' wing segment comprise a 2'-O-methoxyethyl sugar, a constrained ethyl sugar, and a constrained ethyl sugar in the 5' to 3' direction, and the 3 linked nucleosides of the 3' wing segment comprise a constrained ethyl sugar, a constrained ethyl sugar, and a 2'-O-methoxyethyl sugar in the 5' to 3' direction.

[0013] In further aspects, the antisense oligonucleotide comprises a gap segment of ten 2'-deoxynucleotides positioned between wing segments of five 2'-MOE nucleotides.

[0014] In additional aspects, the antisense compound comprises the sequence of any one of SEQ ID NOs: 2, 3, or 124-201 targeted to an eRNA that enhances transcription of MMP9.

[0015] In certain aspects, the antisense compound is targeted to an eRNA that enhances transcription of CX3CR1, CA12, FOXC1, GREB1, P2RY2, SMAD7, PGR, SIAH2, NRIP1, TFF1, or KCNK5.

[0016] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 11-12; SEQ ID NOs: 13-14; or SEQ ID NOs: 15-16, or any one of SEQ ID NOs: 60-63 targeted to an eRNA that enhances transcription of TFF1.

[0017] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 17-18; SEQ ID NOs: 19-20; or SEQ ID NOs: 21-22 targeted to an eRNA that enhances transcription of GREB1.

[0018] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 23-24 or SEQ ID NOs: 25-26 targeted to an eRNA that enhances transcription of PGR.

[0019] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 27-28 or SEQ ID NOs: 29-30 targeted to an eRNA that enhances transcription of SIAH2.

[0020] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 31-32 or SEQ ID NOs: 33-34, or any one of SEQ ID NOs: 68 and 69 targeted to an eRNA that enhances transcription of NRIP1.

[0021] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 35-36 or SEQ ID NOs: 37-38, or any one of SEQ ID NOs: 66 and 77 targeted to an eRNA that enhances transcription of FOXC1.

[0022] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 39-40 or SEQ ID NOs: 41-42 targeted to an eRNA that enhances transcription of P2RY2.

[0023] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 43-44 or SEQ ID NOs: 45-46, or any one of SEQ ID NOs: 64 and 65 targeted to an eRNA that enhances transcription of CA12.

[0024] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs 47-48, SEQ ID NOs: 49-50, or SEQ ID NOs: 51-52 targeted to an eRNA that enhances transcription of SMAD7.

[0025] In several embodiments, antisense compounds comprise any one of the following pairs of sequences: SEQ ID NOs: 53-54; SEQ ID NOs: 55-56, or SEQ ID NOs: 57-5 targeted to an eRNA that enhances transcription of KCNK5.

DETAILED DESCRIPTION

[0026] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "including" as well as other forms, such as "includes" and "included", is not limiting. Also, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0027] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

Definitions

[0028] Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

[0029] Unless otherwise indicated, the following terms have the following meanings: "2'-O-methoxyethyl" (also 2'-MOE and 2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl modification at the 2' position of a furanose ring. A 2'-O-methoxyethyl modified sugar is a modified sugar.

[0030] "2'-MOE nucleoside" (also 2'-O-methoxyethyl nucleoside) means a nucleoside comprising a 2'-MOE modified sugar moiety.

[0031] "2'-substituted nucleoside" means a nucleoside comprising a substituent at the 2'-position of the furanosyl ring other than H or OH. In certain embodiments, 2' substituted nucleosides include nucleosides with bicyclic sugar modifications.

[0032] "3' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 3'-most nucleotide of a particular antisense compound.

[0033] "5' target site" refers to the nucleotide of a target nucleic acid which is complementary to the 5'-most nucleotide of a particular antisense compound.

[0034] "5-methylcytosine" means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.

[0035] "About" means within .+-.7% of a value. For example, if it is stated, "the compounds affected at least about 70% inhibition of eRNA", it is implied that the eRNA levels are inhibited within a range of 63% and 77%.

[0036] "Animal" refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.

[0037] "Antibody" refers to a molecule characterized by reacting specifically with an antigen in some way, where the antibody and the antigen are each defined in terms of the other. Antibody may refer to a complete antibody molecule or any fragment or region thereof, such as the heavy chain, the light chain, F.sub.ab region, and F.sub.c region.

[0038] "Antisense activity" means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

[0039] "Antisense compound" means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, snoRNAs, miRNAs, and satellite repeats.

[0040] "Antisense inhibition" means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.

[0041] "Antisense mechanisms" are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

[0042] "Antisense oligonucleotide" means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

[0043] "Base complementarity" refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

[0044] "Bicyclic sugar" means a furanose ring modified by the bridging of two non-geminal carbon atoms. A bicyclic sugar is a modified sugar.

[0045] "Bicyclic nucleic acid" or "BNA" or "BNA nucleosides" means nucleic acid monomers having a bridge connecting two carbon atoms between the 4' and 2' position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) .alpha.-L-Methyleneoxy (4'-CH.sub.2--O-2') LNA, (B) .beta.-D-Methyleneoxy (4'-CH.sub.2--O-2') LNA, (C) Ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') LNA, (D) Aminooxy (4'-CH.sub.2--O--N(R)-2') LNA and (E) Oxyamino (4'-CH.sub.2--N(R)--O-2') LNA, as depicted below.

##STR00001##

[0046] As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from --[C(R.sub.1)(R.sub.2)].sub.n--, --C(R.sub.1).dbd.C(R.sub.2)--, --C(R.sub.1).dbd.N--, --C(.dbd.O)--, --C(.dbd.S)--, --O--, --Si(R.sub.1).sub.2--, --S(.dbd.O).sub.x-- and --N(R.sub.1)--; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R.sub.1 and R.sub.2 is, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl (S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.

[0047] Examples of 4'-2' bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: --[C(R.sub.1)(R.sub.2)].sub.n--, --[C(R.sub.1)(R.sub.2)].sub.n--O--, --C(R.sub.1R.sub.2)--N(R.sub.1)--O-- or --C(R.sub.1R.sub.2)--O--N(R.sub.1)--. Furthermore, other bridging groups encompassed with the definition of LNA are 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R.sub.1)-2' and 4'-CH.sub.2--N(R.sub.1)--O-2'-bridges, wherein each R.sub.1 and R.sub.2 is, independently, H, a protecting group or C.sub.1-C.sub.12 alkyl.

[0048] Also included within the definition of LNA according to the invention are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4'-CH.sub.2--O-2') bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (--CH.sub.2--) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4'-CH.sub.2--O-2') LNA is used. Furthermore; in the case of the bicyclic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4'-CH.sub.2CH.sub.2--O-2') LNA is used. .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2'), an isomer of methyleneoxy (4'-CH.sub.2--O-2') LNA is also encompassed within the definition of LNA, as used herein.

[0049] "Cap structure" or "terminal cap moiety" means chemical modifications, which have been incorporated at either terminus of an antisense compound.

[0050] "cEt" or "constrained ethyl" means a bicyclic sugar moiety comprising a bridge connecting the 4'-carbon and the 2'-carbon, wherein the bridge has the formula: 4'-CH(CH.sub.3)--O-2'.

[0051] "Constrained ethyl nucleoside" (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4'-CH(CH.sub.3)--O-2' bridge.

[0052] "Chemically distinct region" refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2'-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2'-O-methoxyethyl modifications.

[0053] "Chimeric antisense compounds" means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.

[0054] "Complementarity" means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

[0055] "Comply" means the adherence with a recommended therapy by an individual.

[0056] "Comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0057] "Contiguous nucleobases" means nucleobases immediately adjacent to each other.

[0058] "Deoxyribonucleotide" means a nucleotide having a hydrogen at the 2' position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.

[0059] "Designing" or "Designed to" refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

[0060] "Efficacy" means the ability to produce a desired effect.

[0061] "Enhancer" refers to a DNA sequence or region located at some distance away from a gene and capable of stimulating transcription of the gene. In certain embodiments, an enhancer is capable of binding to transcription factors and stimulating promoters

[0062] "Enhancer RNA (eRNA)" refers to a ribonucleotide transcribed from a genomic enhancer nucleic acid sequence or region.

[0063] "Expression" includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

[0064] "Fully complementary" or "100% complementary" means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

[0065] "Fully modified motif" refers to an antisense compound comprising a contiguous sequence of nucleosides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification.

[0066] "Gapmer" means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the "gap" and the external regions may be referred to as the "wings."

[0067] "Gap-widened" means an antisense compound having a gap segment of 12 or more contiguous 2'-deoxyribonucleotides positioned between 5' and 3' wing segments having from one to six nucleotides having modified sugar moieties.

[0068] "Hybridization" means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.

[0069] "Immediately adjacent" means there are no intervening elements between the immediately adjacent elements.

[0070] "Individual" means a human or non-human animal selected for treatment or therapy.

[0071] "Induce", "inhibit", "potentiate", "elevate", "increase", "decrease" or the like, generally denote quantitative differences between two states.

[0072] "Inhibiting the expression or activity" refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.

[0073] "Internucleoside linkage" refers to the chemical bond between nucleosides.

[0074] "Lengthened" antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.

[0075] "Linked deoxynucleoside" means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.

[0076] "Linked nucleosides" means adjacent nucleosides linked together by an internucleoside linkage.

[0077] "Mismatch" or "non-complementary nucleobase" refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

[0078] "Modified internucleoside linkage" refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).

[0079] "Modified nucleobase" means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An "unmodified nucleobase" means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0080] "Modified nucleoside" means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.

[0081] "Modified nucleotide" means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.

[0082] "Modified oligonucleotide" means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.

[0083] "Modified sugar" means substitution and/or any change from a natural sugar moiety.

[0084] "Monomer" refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[0085] "Motif" means the pattern of unmodified and modified nucleosides in an antisense compound.

[0086] "Natural sugar moiety" means a sugar moiety found in DNA (2'-H) or RNA (2'-OH).

[0087] "Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage.

[0088] "Non-complementary nucleobase" refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

[0089] "Nucleic acid" refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

[0090] "Nucleobase" means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[0091] "Nucleobase complementarity" refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

[0092] "Nucleobase sequence" means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

[0093] "Nucleoside" means a nucleobase linked to a sugar.

[0094] "Nucleoside mimetic" includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by --N(H)--C(.dbd.O)--O-- or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. "Mimetic" refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.

[0095] "Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0096] "Off-target effect" refers to an unwanted or deleterious biological effect associated with modulation of RNA or protein expression of a gene other than the intended target nucleic acid.

[0097] "Oligomeric compound" means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.

[0098] "Oligonucleoside" means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.

[0099] "Oligonucleotide" means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.

[0100] "Peptide" means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, "peptide" refers to polypeptides and proteins.

[0101] "Phosphorothioate linkage" means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[0102] "Portion" means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound

[0103] "Region" is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

[0104] "Ribonucleotide" means a nucleotide having a hydroxy at the 2' position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

[0105] "Segments" are defined as smaller or sub-portions of regions within a target nucleic acid.

[0106] "Sites," as used herein, are defined as unique nucleobase positions within a target nucleic acid.

[0107] "Specifically hybridizable" refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. "Stringent hybridization conditions" or "stringent conditions" refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.

[0108] "Subject" means a human or non-human animal selected for treatment or therapy.

[0109] "Target" refers to a protein, the modulation of which is desired.

[0110] "Target gene" refers to a gene encoding a target.

[0111] "Targeting" means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

[0112] "Target nucleic acid," "target RNA," "target RNA transcript" and "nucleic acid target" all mean a nucleic acid capable of being targeted by antisense compounds.

[0113] "Target region" means a portion of a target nucleic acid to which one or more antisense compounds is targeted.

[0114] "Target segment" means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. "5' target site" refers to the 5'-most nucleotide of a target segment. "3' target site" refers to the 3'-most nucleotide of a target segment.

[0115] "Unmodified" nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).

[0116] "Unmodified nucleotide" means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. .beta.-D-ribonucleosides) or a DNA nucleotide (i.e. .beta.-D-deoxyribonucleoside).

[0117] "Validated target segment" is defined as at least an 8-nucleobase portion (i.e. 8 consecutive nucleobases) of a target region to which an antisense compound is targeted.

[0118] "Wing segment" means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.

Antisense Compounds

[0119] Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be "antisense" to a target nucleic acid, meaning that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0120] In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5' to 3' direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.

[0121] In certain embodiments, an antisense compound is 10-30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.

[0122] In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5' end (5' truncation), or alternatively from the 3' end (3' truncation). A shortened or truncated antisense compound targeted to an eRNA nucleic acid may have two subunits deleted from the 5' end, or alternatively may have two subunits deleted from the 3' end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5' end and one nucleoside deleted from the 3' end.

[0123] When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5' or 3' end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5' end (5' addition), or alternatively to the 3' end (3' addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5' end and one subunit added to the 3' end.

[0124] It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.

[0125] Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.

[0126] Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.

Certain Antisense Compound Motifs and Mechanisms

[0127] In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.

[0128] Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.

[0129] Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.

[0130] RNase H-Mediated Antisense

[0131] In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.

[0132] Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include .beta.-D-ribonucleosides, .beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such 2'-modified nucleosides may include 2'-MOE and 2'-O--CH.sub.3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2'-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2'-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2'-deoxynucleosides.

[0133] Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as "X--Y--Z", where "X" represents the length of the 5'-wing, "Y" represents the length of the gap, and "Z" represents the length of the 3'-wing. "X" and "Z" may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, "X" and "Y" may include one or more 2'-deoxynucleosides. "Y" may comprise 2'-deoxynucleosides. As used herein, a gapmer described as "X--Y--Z" has a configuration such that the gap is positioned immediately adjacent to each of the 5'-wing and the 3' wing. Thus, no intervening nucleotides exist between the 5'-wing and gap, or the gap and the 3'-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, "X" and "Z" are the same; in other embodiments they are different. In certain embodiments, "Y" is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.

[0134] In certain embodiments, gapmers provided herein include, for example, 11-mers having a motif of 1-9-1.

[0135] In certain embodiments, gapmers provided herein include, for example, 12-mers having a motif of 1-9-2, 2-9-1, or 1-10-1.

[0136] In certain embodiments, gapmers provided herein include, for example, 13-mers having a motif of 1-9-3, 2-9-2, 3-9-1, 1-10-2, or 2-10-1.

[0137] In certain embodiments, gapmers provided herein include, for example, 14-mers having a motif of 1-9-4, 2-9-3, 3-9-2, 4-9-1, 1-10-3, 2-10-2, or 3-10-1.

[0138] In certain embodiments, gapmers provided herein include, for example, 15-mers having a motif of 1-9-5, 2-9-4, 3-9-3, 4-9-2, 5-9-1, 1-10-4, 2-10-3, 3-10-2, or 4-10-1.

[0139] In certain embodiments, gapmers provided herein include, for example, 16-mers having a motif of 4-8-4, 2-9-5, 3-9-4, 4-9-3, 5-9-2, 1-10-5, 2-10-4, 3-10-3, 4-10-2, 3-8-5, or 5-10- 1.

[0140] In certain embodiments, gapmers provided herein include, for example, 17-mers having a motif of 3-9-5, 3-10-4, 4-9-4, 5-9-3, 2-10-5, 3-10-4, 4-10-3, 5-10-2, 2-9-6, 5-8-4, 5-7-5, 6-7-4, or 6-9-2.

[0141] In certain embodiments, gapmers provided herein include, for example, 18-mers having a motif of 4-9-5, 5-9-4, 3-10-5, 4-10-4, or 5-10-3.

[0142] In certain embodiments, gapmers provided herein include, for example, 19-mers having a motif of 5-9-5, 4-10-5, or 5-10-4.

[0143] In certain embodiments, gapmers provided herein include, for example, 20-mers having a motif of 5-10-5, 2-10-8, 8-10-2, 3-10-7, 7-10-3, 4-10-6, or 6-10-4.

[0144] In certain embodiments, the antisense compound has a "wingmer" motif, having a wing-gap or gap-wing configuration, i.e. an X--Y or Y--Z configuration as described above for the gapmer configuration. Thus, wingmer configurations provided herein include, but are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2, 18-1, 10-3, 2-10, 1-10, 8-2, 2-13, 5-13, 5-8, or 6-8.

[0145] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 2-10-2 gapmer motif.

[0146] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 3-10-3 gapmer motif.

[0147] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 4-10-4 gapmer motif.

[0148] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 5-10-5 gapmer motif.

[0149] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 3-10-4 gapmer motif.

[0150] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 2-10-4 gapmer motif.

[0151] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 2-10-8 gapmer motif.

[0152] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 8-10-2 gapmer motif.

[0153] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 3-10-7 gapmer motif.

[0154] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 7-10-3 gapmer motif.

[0155] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 4-10-6 gapmer motif.

[0156] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 6-10-4 gapmer motif.

[0157] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 2-9-6 gapmer motif.

[0158] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 6-9-2 gapmer motif.

[0159] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 4-9-4 gapmer motif.

[0160] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 5-9-3 gapmer motif.

[0161] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 3-9-5 gapmer motif.

[0162] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 5-9-2 gapmer motif.

[0163] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 2-9-5 gapmer motif.

[0164] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 4-9-3 gapmer motif.

[0165] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a 3-9-4 gapmer motif.

[0166] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a gap-widened motif.

[0167] In certain embodiments, the antisense compound targeted to an eRNA nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.

[0168] In certain embodiments, the antisense compounds targeted to an eRNA nucleic acid has any of the following sugar motifs:

[0169] k-d(10)-k

[0170] e-d(10)-k

[0171] k-d(10)-e

[0172] k-k-d(10)-k-k

[0173] k-k-d(10)-e-e

[0174] e-e-d(10)-k-k

[0175] k-k-k-d(10)-k-k-k

[0176] e-e-e-d(10)-k-k-k

[0177] k-k-k-d(10)-e-e-e

[0178] k-k-k-d(10)-k-k-k

[0179] e-k-k-d(10)-k-k-e

[0180] e-e-k-d(10)-k-k-e

[0181] e-d-k-d(10)-k-k-e

[0182] e-k-d(10)-k-e-k-e

[0183] k-d(10)-k-e-k-e-e

[0184] e-e-k-d(10)-k-e-k-e

[0185] e-d-d-k-d(9)-k-k-e

[0186] e-e-e-e-d(9)-k-k-e

[0187] e-e-e-e-e-d(10)-e-e-e-e-e

[0188] k-d-k-d-k-d(9)-e-e

[0189] e-e-k-k-d(9)-e-k-e-e

[0190] k-d-k-d-k-d(10)-e-e-e-e-e

[0191] k-e-k-d(10)-k-e-k

[0192] e-e-e-k-k-d(8)-e-e-e-e

[0193] e-e-e-k-k-d(7)-k-k-e-e-e

[0194] e-e-e-k-d(9)-k-e-e-e

[0195] e-e-e-k-k-d(7)-k-k-e-e-e

[0196] e-e-e-e-k-k-d(7)-e-e-e-e

[0197] e-k-e-k-d(9)-e-e-e-e

[0198] e-k-e-k-d-k-d(7)-e-e-e-e

[0199] e-e-e-k-k-d(7)-k-k-e-e-e

[0200] k-d-k-d-k-d(8)-e-e-e-e-e

wherein, k is a constrained ethyl nucleoside, e is a 2'-MOE substituted nucleoside, and d is a 2'-deoxynucleoside.

[0201] In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows: (J).sub.m-(B).sub.n-(J).sub.p-(B).sub.r-(A).sub.t-(D).sub.g-(A).sub.v-(B)- .sub.w-(B).sub.x-(B).sub.y-(J).sub.z

[0202] wherein:

[0203] each A is independently a 2'-substituted nucleoside;

[0204] each B is independently a bicyclic nucleoside;

[0205] each J is independently either a 2'-substituted nucleoside or a 2'-deoxynucleoside;

[0206] each D is a 2'-deoxynucleoside;

[0207] m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;

[0208] provided that:

[0209] at least one of m, n, and r is other than 0;

[0210] at least one of w and y is other than 0;

[0211] the sum of m, n, p, r, and t is from 2 to 5; and

[0212] the sum of v, w, x, y, and z is from 2 to 5.

[0213] RNAi Compounds

[0214] In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.

[0215] i. ssRNA Compounds

[0216] In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5'-terminal end. In certain such embodiments, the 5'-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5'-phosphate). In certain embodiments, such 5'-terminal nucleosides stabilize the 5'-phosphorous moiety. Certain modified 5'-terminal nucleosides may be found in the art, for example in WO/2011/139702.

[0217] In certain embodiments, the 5'-nucleoside of an ssRNA compound has Formula IIc:

##STR00002##

wherein:

[0218] T.sub.1 is an optionally protected phosphorus moiety;

[0219] T.sub.2 is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;

[0220] A has one of the formulas:

##STR00003##

[0221] Q.sub.1 and Q.sub.2 are each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or N(R.sub.3)(R.sub.4);

[0222] Q.sub.3 is O, S, N(R.sub.5) or C(R.sub.6)(R.sub.7);

[0223] each R.sub.3, R.sub.4 R.sub.5, R.sub.6 and R.sub.7 is, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl or C.sub.1-C.sub.6 alkoxy;

[0224] M.sub.3 is O, S, NR.sub.14, C(R.sub.15)(R.sub.16), C(R.sub.15)(R.sub.16)C(R.sub.17)(R.sub.18), C(R.sub.15).dbd.C(R.sub.17), OC(R.sub.15)(R.sub.16) or OC(R.sub.15)(Bx.sub.2);

[0225] R.sub.14 is H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0226] R.sub.15, R.sub.16, R.sub.17 and R.sub.18 are each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0227] Bx.sub.1 is a heterocyclic base moiety;

[0228] or if Bx.sub.2 is present then Bx.sub.2 is a heterocyclic base moiety and Bx.sub.1 is H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0229] J.sub.4, J.sub.5, J.sub.6 and J.sub.7 are each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0230] or J.sub.4 forms a bridge with one of J.sub.5 or J.sub.7 wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR.sub.19, C(R.sub.20)(R.sub.21), C(R.sub.20).dbd.C(R.sub.21), C[.dbd.C(R.sub.20)(R.sub.21)] and C(.dbd.O) and the other two of J.sub.5, J.sub.6 and J.sub.7 are each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0231] each R.sub.19, R.sub.20 and R.sub.21 is, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0232] G is H, OH, halogen or O--[C(R.sub.8)(R.sub.9)].sub.n--[(C.dbd.O).sub.m--X.sub.1].sub.j--Z;

[0233] each R.sub.8 and R.sub.9 is, independently, H, halogen, C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;

[0234] X.sub.1 is O, S or N(E.sub.1);

[0235] Z is H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or N(E.sub.2)(E.sub.3);

[0236] E.sub.1, E.sub.2 and E.sub.3 are each, independently, H, C.sub.1-C.sub.6 alkyl or substituted C.sub.1-C.sub.6 alkyl;

[0237] n is from 1 to about 6;

[0238] m is 0 or 1;

[0239] j is 0 or 1;

[0240] each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ.sub.1, N(J.sub.1)(J.sub.2), .dbd.NJ.sub.1, SJ.sub.1, N.sub.3, CN, OC(.dbd.X.sub.2)J.sub.1, OC(.dbd.X.sub.2)N(J.sub.1)(J.sub.2) and C(.dbd.X.sub.2)N(J.sub.1)(J.sub.2);

[0241] X.sub.2 is O, S or NJ.sub.3;

[0242] each J.sub.1, J.sub.2 and J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl;

[0243] when j is 1 then Z is other than halogen or N(E.sub.2)(E.sub.3); and

[0244] wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.

[0245] In certain embodiments, M.sub.3 is O, CH.dbd.CH, OCH.sub.2 or OC(H)(Bx.sub.2). In certain embodiments, M.sub.3 is O.

[0246] In certain embodiments, J.sub.4, J.sub.5, J.sub.6 and J.sub.7 are each H. In certain embodiments, J.sub.4 forms a bridge with one of J.sub.5 or J.sub.7.

[0247] In certain embodiments, A has one of the formulas:

##STR00004##

wherein:

[0248] Q.sub.1 and Q.sub.2 are each, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy or substituted C.sub.1-C.sub.6 alkoxy. In certain embodiments, Q.sub.1 and Q.sub.2 are each H. In certain embodiments, Q.sub.1 and Q.sub.2 are each, independently, H or halogen. In certain embodiments, Q.sub.1 and Q.sub.2 is H and the other of Q.sub.1 and Q.sub.2 is F, CH.sub.3 or OCH.sub.3.

[0249] In certain embodiments, T.sub.1 has the formula:

##STR00005##

wherein:

[0250] R.sub.a and R.sub.c are each, independently, protected hydroxyl, protected thiol, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, substituted C.sub.1-C.sub.6 alkoxy, protected amino or substituted amino; and

[0251] R.sub.b is O or S. In certain embodiments, R.sub.b is O and R.sub.a and R.sub.c are each, independently, OCH.sub.3, OCH.sub.2CH.sub.3 or CH(CH.sub.3).sub.2.

[0252] In certain embodiments, G is halogen, OCH.sub.3, OCH.sub.2F, OCHF.sub.2, OCF.sub.3, OCH.sub.2CH.sub.3, O(CH.sub.2).sub.2F, OCH.sub.2CHF.sub.2, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--SCH.sub.3, O(CH.sub.2).sub.2--OCF.sub.3, O(CH.sub.2).sub.3--N(R.sub.10)(R.sub.11), O(CH.sub.2).sub.2--ON(R.sub.10)(R.sub.11), O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11), OCH.sub.2C(.dbd.O)--N(R.sub.10)(R.sub.11), OCH.sub.2C(.dbd.O)--N(R.sub.12)--(CH.sub.2).sub.2--N(R.sub.10)(R.sub.11) or O(CH.sub.2).sub.2--N(R.sub.12)--C(.dbd.NR.sub.13)[N(R.sub.10)(R.sub.11- )] wherein R.sub.10, R.sub.11, R.sub.12 and R.sub.13 are each, independently, H or C.sub.1-C.sub.6 alkyl. In certain embodiments, G is halogen, OCH.sub.3, OCF.sub.3, OCH.sub.2CH.sub.3, OCH.sub.2CF.sub.3, OCH.sub.2--CH.dbd.CH.sub.2, O(CH.sub.2).sub.2--OCH.sub.3, O(CH.sub.2).sub.2--O(CH.sub.2).sub.2--N(CH.sub.3).sub.2, OCH.sub.2C(.dbd.O)--N(H)CH.sub.3, OCH.sub.2C(.dbd.O)--N(H)--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 or OCH.sub.2--N(H)--C(.dbd.NH)NH.sub.2. In certain embodiments, G is F, OCH.sub.3 or O(CH.sub.2).sub.2--OCH.sub.3. In certain embodiments, G is O(CH.sub.2).sub.2--OCH.sub.3.

[0253] In certain embodiments, the 5'-terminal nucleoside has Formula IIe:

##STR00006##

[0254] In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.

[0255] In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2'-F nucleoside. In certain embodiments, each nucleoside of the region is a 2'-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2'-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.

[0256] In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2'-F and 2'-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.

[0257] In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB).sub.xA.sub.y wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:

AABBAA;

ABBABB;

AABAAB;

ABBABAABB;

ABABAA;

AABABAB;

ABABAA;

ABBAABBABABAA;

BABBAABBABABAA; or

ABABBAABBABABAA;

[0258] wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2'-F, 2'-OMe, BNA, and MOE.

[0259] In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5' terminal nucleoside, such as those of formula IIc or IIe.

[0260] In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:

-(A).sub.2-(B).sup.x-(A).sub.2-(C).sub.y-(A).sub.3-

[0261] wherein: A is a first type of modified nucleoside;

[0262] B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;

[0263] x and y are from 1 to 15.

[0264] In certain embodiments, A is a 2'-OMe modified nucleoside. In certain embodiments, B and C are both 2'-F modified nucleosides. In certain embodiments, A is a 2'-OMe modified nucleoside and B and C are both 2'-F modified nucleosides.

[0265] In certain embodiments, oligonucleosides have the following sugar motif:

5'-(Q)-(AB).sub.xA.sub.y-(D).sub.z

wherein:

[0266] Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

[0267] A is a first type of modified nucleoside;

[0268] B is a second type of modified nucleoside;

[0269] D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.

[0270] X is 5-15;

[0271] Y is 0 or 1;

[0272] Z is 0-4.

[0273] In certain embodiments, oligonucleosides have the following sugar motif:

5'-(Q)-(A).sub.x-(D).sub.z

wherein:

[0274] Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;

[0275] A is a first type of modified nucleoside;

[0276] D is a modified nucleoside comprising a modification different from A.

[0277] X is 11-30;

[0278] Z is 0-4.

[0279] In certain embodiments A, B, C, and D in the above motifs are selected from: 2'-OMe, 2'-F, 2'-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.

[0280] In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.

[0281] In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3' end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3' end of the oligonucleotide.

[0282] Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:

TABLE-US-00001 5' most linkage Central region 3'-region PS Alternating PO/PS 6 PS PS Alternating PO/PS 7 PS PS Alternating PO/PS 8 PS

[0283] ii. siRNA Compounds

[0284] In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.

[0285] Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.

[0286] The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is an eRNA. In certain embodiment, the degradation of the targeted eRNA is facilitated by an activated RISC complex that is formed with compositions of the invention.

[0287] Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.

[0288] Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5'-end to enhance its role in one region of the RISC while the 3'-end can be modified differentially to enhance its role in a different region of the RISC.

[0289] The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).

[0290] The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.

[0291] In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.

[0292] As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2'-hydroxy (2'-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2'-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2'-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).

[0293] It is contemplated that compounds and compositions of several embodiments provided herein can target eRNAs by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., "hairpin" or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.

[0294] In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.

[0295] In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5' phosphoryl group of a nucleotide becomes linked to the 2' hydroxyl group of another nucleotide in a loop back fashion.

[0296] In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2' position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2' position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.

[0297] In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.

Occupancy

[0298] In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.

Target eRNAs and Associated Gene Expression

[0299] Several embodiments are directed to methods of modulating gene expression by inhibiting eRNAs.

[0300] In certain embodiments, transcriptional start site of such a gene is located on a chromosome at least about 1 kilobase (kb) from the genomic enhancer nucleic acid sequence. In certain embodiments, eRNAs suitable for inhibition include RNA transcripts characterized by any one or more of the following: (1) transcribed from genomic regions characterized by high levels of monomethylation on lysine 4 of histone 3 (H3K4me1) and low levels of trimethylation on lysine 4 of histone 3 (H3K4me3) (2) transcribed from genomic regions that are enriched for RNA polymerase II (PolII); (3) transcribed from genomic regions that are enriched for transcriptional co-regulators, such as the p300 co-activator; (4) their transcription is initiated from Poll-binding sites and elongated bidirectionally; (5) evolutionarily conserved DNA sequences encoding eRNAs; (6) short half-life; (7) dynamically regulated upon signaling, and/or (7) positively correlated to levels of nearby mRNA expression. In certain aspects, the half-life of the eRNA is less than about 10-30 minutes.

[0301] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs from hematopoietic cells, such as monocytes or macrophages. Examples of such eRNAs include, but are not limited to, those transcribed from enhancers identified by genomic coordinates in Mega-Tables 1 and 2 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Additional information on the eRNAs transcribed from enhancers listed in Mega-Table 2 is reported in De Santa. F, et al., "A large fraction of extragenic RNA pol II transcription sites overlap enhancers," PLoS Biol 2010; 8:e1000384, which is herein incorporated by reference in its entirety.

[0302] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs from neurons. Examples of such eRNAs include, but are not limited to, those transcribed from enhancers identified by genomic coordinates in Mega-Table 3 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Additional information on the eRNAs transcribed from enhancers listed in Mega-Table 3 is reported in Kim T K, et al., "Widespread transcription at neuronal activity-regulated enhancers." Nature 2010; 465:182-187, which is herein incorporated by reference in its entirety.

[0303] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs transcribed from enhancers identified as highly conserved among human, mouse, and rat species. Examples of such eRNAs include, but are not limited to, those transcribed from enhancers identified by genomic coordinates in Mega-Table 4 and Mega-Table 5 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Mega-Tables 4 and 5 are in 4-column format: chr start end log 10(1/pvalue).

[0304] Additional information on the enhancers listed in Mega-Table 4 is reported in Pennacchio et al, Nature 2006; 444:499-502, Prabhakar S, et al. Genome Res. 2006; 16:855-863, and (http://pga.jgi-psf.org/gumby/), each of which is incorporated herein by reference in its entirety. Chromosome coordinates provided in Mega-Table 4 correspond to the hg17 human genome assembly. Additional information on the enhancers listed in Mega-Table 5 is reported in Visel et al., "VISTA Enhancer Browser--a database of tissue-specific human enhancers," Nucleic Acids Research 2007 (35):D88-92 and (http://pga.jgi-psf.org/gumby/), each of which is incorporated herein by reference in its entirety. Chromosome coordinates provided in Mega-Table 5 correspond to the hg18 human genome assembly.

[0305] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs transcribed from human enhancers identified by genomic coordinates in Mega-Table 6 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Chromosome coordinates provided in Mega-Table 6 correspond to the hg19 human genome assembly. Additional information on the enhancers listed in Mega-Table 6 is reported in Visel et al., "VISTA Enhancer Browser--a database of tissue-specific human enhancers," Nucleic Acids Research 2007 (35):D88-92 and the VISTA database website (http://enhancer.lbl.gov/), each of which is incorporated herein by reference in its entirety.

[0306] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs transcribed from mouse enhancers identified by genomic coordinates in Mega-Table 7 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Chromosome coordinates provided in Mega-Table 7 correspond to the mouse mm9 genome assembly. Additional information on the enhancers listed in Mega-Table 7 is reported in Visel et al., "VISTA Enhancer Browser--a database of tissue-specific human enhancers," Nucleic Acids Research 2007 (35):D88-92 and the VISTA database website (http://enhancer.lbl.gov/), each of which is incorporated herein by reference in its entirety.

[0307] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs transcribed from enhancers in human prostate cells. Examples of such eRNAs include, but are not limited to, those identified in LNCaP cells as reported in Wang D et al., "Reprogramming transcription by distinct classes of enhancers functionally defined by eRNA," Nature. 2011 474(7351):390-4 and further described at http://www.nature.com/nature/journal/v474/n7351/full/nature10006.html, each of which is incorporated herein by reference in its entirety. Further description of the eRNAs identified in Wang et al. are available as high-throughput data deposited at Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE27823, which is incorporated herein by reference in its entirety.

[0308] In certain embodiments, eRNAs suitable for inhibition with compounds described herein include eRNAs transcribed from enhancers in human breast cells, such as breast cancer cells. Examples of such eRNAs include, but are not limited to, those transcribed from enhancers identified in MCF-7 human breast cancer cells and listed in Mega-Table 8 and Mega-Table 9 filed in U.S. Provisional Application No. 61/650,426 in electronic format on May 22, 2012. Chromosome coordinates provided in Mega-Table 8 and Mega-Table 9 correspond to the hg18 human genome assembly. Additional examples of such eRNAs include, but are not limited to, those transcribed from enhancers identified in MCF-7 human breast cancer cells as reported in the UCSC Genome Browser located at (http://genome.ucsc.edu/cgi-bin/hgTracks?hgS_doOtherUser=submit&hgS_other- UserName=Bogdantanasa&hgS_otherUserSessionName=EReRNAs), which is incorporated herein by reference in its entirety.

[0309] In certain embodiments, eRNAs suitable for inhibition with compounds described herein exclude ncRNA-activators (ncRNA-a), such as those described in Orom U A et al., "Long noncoding RNAs with enhancer-like function in human cells." Cell 2010; 143:46-58. In certain embodiments, eRNAs suitable for inhibition with compounds described herein exclude transcripts that are characterized by one or more of the following features: (1) produced via unidirectional transcription; (2) polyadenylated; and/or (3), transcribed from a genomic sequence or region having a H3K4 trimethylation chromatin signature.

[0310] In certain embodiments, a method of inhibiting gene expression in a macrophage cell comprises administering to the macrophage cell an antisense compound targeted to an eRNA transcribed from a genomic enhancer nucleic acid sequence or region that is enriched for binding to Rev-Erb.alpha. or Rev-Erb.beta..

[0311] In certain embodiments, a method of inhibiting MMP9 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a MMP9 enhancer. In several embodiments, the MMP9 eRNA sequence is the product of transcription of SEQ ID NO: 1 (GENBANK Accession No. NT_039207.7 truncated from 105809972 to 105810309).

[0312] In certain aspects, the antisense compound is an antisense oligonucleotide that targets the MMP9 eRNA at nucleotides 247 to 262 of the transcribed product of SEQ ID NO:1 In certain aspects, the antisense compound is an antisense oligonucleotide having the nucleic acid sequence of SEQ ID NO:2 In some aspects, the antisense compound is ISIS 566237.

[0313] In certain aspects, the antisense compound is an antisense oligonucleotide that targets the MMP9 eRNA at nucleotides 260 to 275 of the transcribed product of SEQ ID NO:1 In certain aspects, the antisense compound is an antisense oligonucleotide having the nucleic acid sequence of SEQ ID NO:3. In some aspects, the antisense compound is ISIS 566241.

[0314] In certain aspects, the antisense compound is an antisense oligonucleotide that targets the MMP9 eRNA transcribed from SEQ ID NO: 202 (GENBANK Accession number NT_039207.7 truncated from 105809967 to 105810417). In certain aspects, the antisense compound is an antisense oligonucleotide having the nucleic acid sequence of any one of SEQ ID NOs: 2, 3, or 124-201. MMP9 is known to be associated with atherosclerosis, inflammation, apoptosis, and cancer metastasis; methods and compounds provided herein are useful for inhibiting MMP9 gene expression.

[0315] In certain embodiments; a method of inhibiting CX3CR1 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a CX3CR1 enhancer. In several embodiments, the CX3CR1 eRNA sequence is the product of transcription of nucleotides 11132282 to 11132781 of SEQ ID NO: 4 (Genbank Accession No. NT_039482.7), which is incorporated herein by reference. In several embodiments, the CX3CR1 eRNA sequence is the product of transcription of nucleotides 11132134 to 11131599 of SEQ ID NO: 4 (Genbank Accession No. NT_039482.7), which is incorporated herein by reference. CX3CR1 is known to be associated with atherosclerosis; methods and compounds provided herein are useful for inhibiting CX3CR1 gene expression.

[0316] In certain embodiments, a method of inhibiting TFF1 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a TFF1 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 11-12; SEQ ID NOs: 13-14; or SEQ ID NOs: 15-16, In several embodiments, the antisense compound is a single-stranded oligonucleotide having a nucleotide sequence of any one of SEQ ID NOs: 60-63.

[0317] In certain embodiments, a method of inhibiting GREB1 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a GREB1 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 17-18; SEQ ID NOs: 19-20; or SEQ ID NOs: 21-22.

[0318] In certain embodiments, a method of inhibiting PGR gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a PGR enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 23-24 or SEQ ID NOs: 25-26.

[0319] In certain embodiments, a method of inhibiting SIAH2 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a SIAH2 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 27-28 or SEQ ID NOs: 29-30.

[0320] In certain embodiments, a method of inhibiting NRIP1 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a NRIP1 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 31-32 or SEQ ID NOs: 33-34. In several embodiments, the antisense compound is a single-stranded oligonucleotide ha a nucleotide sequence of any one of SEQ ID NOs: 68 or 69.

[0321] In certain embodiments, a method of inhibiting FOXC1 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a FOXC1 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 35-36 or SEQ ID NOs: 37-38. In several embodiments, the antisense compound is a single-stranded oligonucleotide having a nucleotide sequence of any one of SEQ ID NOs: 66 or 67.

[0322] In certain embodiments, a method of inhibiting P2RY2 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a P2RY2 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 39-40 or SEQ ID NOs: 41-42.

[0323] In certain embodiments, a method of inhibiting CA12 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a CA12 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 43-44 or SEQ m NOs: 45-46. In several embodiments, the antisense compound is a single-stranded oligonucleotide having a nucleotide sequence of any one of SEQ ID NOs: 64 or 65.

[0324] In certain embodiments, a method of inhibiting SMAD7 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a SMAD7 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 47-48, SEQ ID NOs: 49-50, or SEQ ID NOs: 51-52.

[0325] In certain embodiments, a method of inhibiting KCNK5 gene expression in a cell comprises administering to the cell an antisense compound targeted to an eRNA transcript of a KCNK5 enhancer. In several embodiments, the antisense compound is a double-stranded siRNA having any one of the following pairs of sequences: SEQ ID NOs: 53-54, SEQ ID NOs: 55-56, or SEQ ID NOs: 57-58.

Hybridization

[0326] In some embodiments, hybridization occurs between an antisense compound disclosed herein and an eRNA. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.

[0327] Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[0328] Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with an eRNA.

Complementarity

[0329] An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as an eRNA nucleic acid).

[0330] Non-complementary nucleobases between an antisense compound and an eRNA nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of an eRNA nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).

[0331] In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to an eRNA nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.

[0332] For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).

[0333] In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to an eRNA nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, "fully complementary" means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be "fully complementary" to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.

[0334] The location of a non-complementary nucleobase may be at the 5' end or 3' end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.

[0335] In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an eRNA nucleic acid, or specified portion thereof.

[0336] In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as an eRNA nucleic acid, or specified portion thereof.

[0337] The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, "portion" refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A "portion" can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.

Identity

[0338] The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

[0339] In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.

[0340] In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

[0341] In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.

Modifications

[0342] A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[0343] Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.

[0344] Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.

Modified Internucleoside Linkages

[0345] The naturally occurring internucleoside linkage of RNA and DNA is a 3' to 5' phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.

[0346] Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.

[0347] In certain embodiments, antisense compounds targeted to an eRNA nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.

Modified Sugar Moieties

[0348] Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5' and 2' substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R.sub.1)(R.sub.2) (R, R.sub.1 and R.sub.2 are each independently H, C.sub.1-C.sub.12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2'-F-5'-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5',2'-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5'-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5'-methyl or a 5'-vinyl group).

[0349] Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3, 2'-OCH.sub.2CH.sub.3, 2'-OCH.sub.2CH.sub.2F and 2'-O(CH.sub.2).sub.2OCH.sub.3 substituent groups. The substituent at the 2' position can also be selected from allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, OCF.sub.3, OCH.sub.2F, O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), and O--CH.sub.2--C(.dbd.O)--N(R.sub.l)--(CH.sub.2).sub.2--N(R.sub.m)(R.sub.n)- , where each R.sub.l, R.sub.m and R.sub.n is, independently, H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl.

[0350] As used herein, "bicyclic nucleosides" refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4' and the 2' ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4' to 2' bridge. Examples of such 4' to 2' bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' (also referred to as constrained ethyl or cEt) and 4'-CH(CH.sub.2OCH.sub.3)-0-2' (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4'-C(CH.sub.3)(CH.sub.3)--O-2' (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4'-CH.sub.2--N(OCH.sub.3)-2' (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4'-CH.sub.2--N(R)--O-2', wherein R is H, C.sub.1-C.sub.12 alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4'-CH.sub.2--C(H)(CH.sub.3)-2' (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH.sub.2--C(.dbd.CH.sub.2)-2' (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).

[0351] Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example .alpha.-L-ribofuranose and .beta.-D-ribofuranose (see PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).

[0352] In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4' and the 2' position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from --[C(R.sub.a)(R.sub.b)].sub.n--, --C(R.sub.a).dbd.C(R.sub.b)--, --C(R.sub.a).dbd.N--, --C(.dbd.O)--, --C(.dbd.NR.sub.a)--, --C(.dbd.S)--, --O--, --Si(R.sub.a).sub.2--, --S(.dbd.O).sub.x--, and --N(R.sub.a)--;

[0353] wherein:

[0354] x is 0, 1, or 2;

[0355] n is 1, 2, 3, or 4;

[0356] each R.sub.a and R.sub.b is, independently, H, a protecting group, hydroxyl, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C.sub.5-C.sub.7 alicyclic radical, substituted C.sub.5-C.sub.7 alicyclic radical, halogen, OJ.sub.1, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, COOJ.sub.1, acyl (C(.dbd.O)--H), substituted acyl, CN, sulfonyl (S(.dbd.O).sub.2-J.sub.1), or sulfoxyl (S(.dbd.O)-J.sub.1); and

[0357] each J.sub.1 and J.sub.2 is, independently, H, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.5-C.sub.20 aryl, substituted C.sub.5-C.sub.20 aryl, acyl (C(.dbd.O)--H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C.sub.1-C.sub.12 aminoalkyl, substituted C.sub.1-C.sub.12 aminoalkyl or a protecting group.

[0358] In certain embodiments, the bridge of a bicyclic sugar moiety is --[C(R.sub.a)(R.sub.b)].sub.n--, --[C(R.sub.a)(R.sub.b)].sub.n--O--, --C(R.sub.aR.sub.b)--N(R)--O-- or --C(R.sub.aR.sub.b)--O--N(R)--. In certain embodiments, the bridge is 4'-CH.sub.2-2', 4'-(CH.sub.2).sub.2-2', 4'-(CH.sub.2).sub.3-2', 4'-CH.sub.2--O-2', 4'-(CH.sub.2).sub.2--O-2', 4'-CH.sub.2--O--N(R)-2' and 4'-CH.sub.2--N(R)--O-2'- wherein each R is, independently, H, a protecting group or C.sub.1-C.sub.12 alkyl.

[0359] In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4'-2' methylene-oxy bridge, may be in the .alpha.-L configuration or in the .beta.-D configuration. Previously, .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).

[0360] In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) .alpha.-L-methyleneoxy (4'-CH.sub.2--O-2') BNA, (B) .beta.-D-methyleneoxy (4'-CH.sub.2--O-2') BNA, (C) ethyleneoxy (4'-(CH.sub.2).sub.2--O-2') BNA, (D) aminooxy (4'-CH.sub.2--O--N(R)-2') BNA, (E) oxyamino (4'-CH.sub.2--N(R)--O-2') BNA, and (F) methyl(methyleneoxy) (4'-CH(CH.sub.3)--O-2') BNA, (G) methylene thio (4'-CH.sub.2--S-2') BNA, (H) methylene-amino (4'-CH.sub.2--N(R)-2') BNA, (I) methyl carbocyclic (4'-CH.sub.2--CH(CH.sub.3)-2') BNA, (J) propylene carbocyclic (4'-(CH.sub.2).sub.3-2') BNA and (K) vinyl BNA as depicted below:

##STR00007## ##STR00008##

[0361] wherein Bx is the base moiety and R is independently H, a protecting group, C.sub.1-C.sub.12 alkyl or C.sub.1-C.sub.12 alkoxy.

[0362] In certain embodiments, bicyclic nucleosides are provided having Formula I:

##STR00009##

wherein:

[0363] Bx is a heterocyclic base moiety;

[0364] -Q.sub.a-Q.sub.b-Q.sub.c- is --CH.sub.2--N(R.sub.c)--CH.sub.2--, --CH.sub.2--O--N(R.sub.c)--, --CH.sub.2--N(R.sub.c)--O-- or --N(R.sub.c)--O--CH.sub.2;

[0365] R.sub.c is C.sub.1-C.sub.12 alkyl or an amino protecting group; and

[0366] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.

[0367] In certain embodiments, bicyclic nucleosides are provided having Formula II:

##STR00010##

wherein:

[0368] Bx is a heterocyclic base moiety;

[0369] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

[0370] Z.sub.a is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.

[0371] In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJ.sub.c, NJ.sub.cJ.sub.d, SJ.sub.c, N.sub.3, OC(.dbd.X)J.sub.c, and NJ.sub.cC(.dbd.X)NJ.sub.cJ.sub.d, wherein each J.sub.c, J.sub.d and J.sub.e is, independently, H, C.sub.1-C.sub.6 alkyl, or substituted C.sub.1-C.sub.6 alkyl and X is O or NJ.sub.c.

[0372] In certain embodiments, bicyclic nucleosides are provided having Formula III:

##STR00011##

wherein:

[0373] Bx is a heterocyclic base moiety;

[0374] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

[0375] Z.sub.b is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.1-C.sub.6 alkyl, substituted C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkynyl or substituted acyl (C(.dbd.O)--).

[0376] In certain embodiments, bicyclic nucleosides are provided having Formula IV:

##STR00012##

wherein:

[0377] Bx is a heterocyclic base moiety;

[0378] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

[0379] R.sub.d is C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl;

[0380] each q.sub.a, q.sub.b, q.sub.c and q.sub.d is, independently, H, halogen, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl, C.sub.1-C.sub.6 alkoxyl, substituted C.sub.1-C.sub.6 alkoxyl, acyl, substituted acyl, C.sub.1-C.sub.6 aminoalkyl or substituted C.sub.1-C.sub.6 aminoalkyl;

[0381] In certain embodiments, bicyclic nucleosides are provided having Formula V:

##STR00013##

wherein:

[0382] Bx is a heterocyclic base moiety;

[0383] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium; [0384] q.sub.a, q.sub.b, q.sub.r and q.sub.f are each, independently, hydrogen, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxy, substituted C.sub.1-C.sub.12 alkoxy, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j, C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.jJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k;

[0385] or q.sub.e and q.sub.f together are .dbd.C(q.sub.g)(q.sub.h);

[0386] q.sub.g and q.sub.h are each, independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.

[0387] The synthesis and preparation of the methyleneoxy (4'-CH.sub.2--O-2') BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.

[0388] Analogs of methyleneoxy (4'-CH.sub.2--O-2') BNA and 2'-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2'-amino-BNA, a novel conformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2'-amino- and 2'-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.

[0389] In certain embodiments, bicyclic nucleosides are provided having Formula VI:

##STR00014##

wherein:

[0390] Bx is a heterocyclic base moiety;

[0391] T.sub.a and T.sub.b are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;

[0392] each q.sub.j, q.sub.j, q.sub.k and q.sub.l is, independently, H, halogen, C.sub.1-C.sub.12 alkyl, substituted C.sub.1-C.sub.12 alkyl, C.sub.2-C.sub.12 alkenyl, substituted C.sub.2-C.sub.12 alkenyl, C.sub.2-C.sub.12 alkynyl, substituted C.sub.2-C.sub.12 alkynyl, C.sub.1-C.sub.12 alkoxyl, substituted C.sub.1-C.sub.12 alkoxyl, OJ.sub.j, SJ.sub.j, SOJ.sub.j, SO.sub.2J.sub.j, NJ.sub.jJ.sub.k, N.sub.3, CN, C(.dbd.O)OJ.sub.j, C(.dbd.O)NJ.sub.jJ.sub.k, C(.dbd.O)J.sub.j, O--C(.dbd.O)NJ.sub.jJ.sub.k, N(H)C(.dbd.NH)NJ.sub.JJ.sub.k, N(H)C(.dbd.O)NJ.sub.jJ.sub.k or N(H)C(.dbd.S)NJ.sub.jJ.sub.k; and

[0393] q.sub.i and q.sub.j or q.sub.l and q.sub.k together are .dbd.C(q.sub.g)(q.sub.h), wherein q.sub.g and q.sub.h are each, independently, H, halogen, C.sub.1-C.sub.12 alkyl or substituted C.sub.1-C.sub.12 alkyl.

[0394] One carbocyclic bicyclic nucleoside having a 4'-(CH.sub.2).sub.3-2' bridge and the alkenyl analog bridge 4'-CH.dbd.CH--CH.sub.2-2' have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).

[0395] As used herein, "4'-2' bicyclic nucleoside" or "4' to 2' bicyclic nucleoside" refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2' carbon atom and the 4' carbon atom of the sugar ring.

[0396] As used herein, "monocyclic nucleosides" refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.

[0397] As used herein, "2'-modified sugar" means a furanosyl sugar modified at the 2' position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2' modifications are selected from substituents including, but not limited to: O[(CH.sub.2).sub.nO].sub.mCH.sub.3, O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nF, O(CH.sub.2).sub.nONH.sub.2, OCH.sub.2C(.dbd.O)N(H)CH.sub.3, and O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3].sub.2, where n and m are from 1 to about 10. Other 2'-substituent groups can also be selected from: C.sub.1-C.sub.12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, F, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2'-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2'-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2'-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2'-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).

[0398] As used herein, a "modified tetrahydropyran nucleoside" or "modified THP nucleoside" means a nucleoside having a six-membered tetrahydropyran "sugar" substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:

##STR00015##

[0399] In certain embodiments, sugar surrogates are selected having Formula VII:

##STR00016##

wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

[0400] Bx is a heterocyclic base moiety;

[0401] T.sub.a and T.sub.b are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T.sub.a and T.sub.b is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T.sub.a and T.sub.b is H, a hydroxyl protecting group, a linked conjugate group or a 5' or 3'-terminal group;

[0402] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl or substituted C.sub.2-C.sub.6 alkynyl; and each of R.sub.1 and R.sub.2 is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ.sub.1J.sub.2, SJ.sub.1, N.sub.3, OC(.dbd.X)J.sub.1, OC(.dbd.X)NJ.sub.1J.sub.2, NJ.sub.3C(.dbd.X)NJ.sub.1J.sub.2 and CN, wherein X is O, S or NJ.sub.1 and each J.sub.1, J.sub.2 and J.sub.3 is, independently, H or C.sub.1-C.sub.6 alkyl.

[0403] In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 are each H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is other than H. In certain embodiments, at least one of q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6 and q.sub.7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R.sub.1 and R.sub.2 is fluoro. In certain embodiments, R.sub.1 is fluoro and R.sub.2 is H; R.sub.1 is methoxy and R.sub.2 is H, and R.sub.1 is methoxyethoxy and R.sub.2 is H.

[0404] In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term "morpholino" means a sugar surrogate having the following formula:

##STR00017##

In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as "modified morpholinos."

[0405] Combinations of modifications are also provided without limitation, such as 2'-F-5'-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5', 2'-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2'-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5'-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4'-CH.sub.2--O-2' bicyclic nucleoside is further substituted at the 5' position with a 5'-methyl or a 5'-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).

[0406] In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horvath et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.

##STR00018##

[0407] wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:

[0408] Bx is a heterocyclic base moiety;

[0409] T.sub.3 and T.sub.4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T.sub.3 and T.sub.4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T.sub.3 and T.sub.4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5'- or 3'-terminal group; and

[0410] q.sub.1, q.sub.2, q.sub.3, q.sub.4, q.sub.5, q.sub.6, q.sub.7, q.sub.8 and q.sub.9 are each, independently, H, C.sub.1-C.sub.6 alkyl, substituted C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl, substituted C.sub.2-C.sub.6 alkenyl, C.sub.2-C.sub.6 alkynyl, substituted C.sub.2-C.sub.6 alkynyl or other sugar substituent group.

[0411] As used herein, "2'-modified" or "2'-substituted" refers to a nucleoside comprising a sugar comprising a substituent at the 2' position other than H or OH. 2'-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2' carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2' substituents, such as allyl, amino, azido, thio, O-allyl, O--C.sub.1-C.sub.10 alkyl, --OCF.sub.3, O--(CH.sub.2).sub.2--O--CH.sub.3, 2'-O(CH.sub.2).sub.2SCH.sub.3, O--(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), or O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and R.sub.n is, independently, H or substituted or unsubstituted C.sub.1-C.sub.10 alkyl. 2'-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.

[0412] As used herein, "2'-F" refers to a nucleoside comprising a sugar comprising a fluoro group at the 2' position of the sugar ring.

[0413] As used herein, "2'-OMe" or "2'-OCH.sub.3" or "2'-O-methyl" each refers to a nucleoside comprising a sugar comprising an --OCH.sub.3 group at the 2' position of the sugar ring.

[0414] As used herein, "MOE" or "2'-MOE" or "2'-OCH.sub.2CH.sub.2OCH.sub.3" or "2'-O-methoxyethyl" each refers to a nucleoside comprising a sugar comprising a --OCH.sub.2CH.sub.2OCH.sub.3 group at the 2' position of the sugar ring.

[0415] As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).

[0416] Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.

[0417] Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.

[0418] In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.

[0419] In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2'-MOE. In certain embodiments, the 2'-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4'-CH(CH.sub.3)--O-2') bridging group. In certain embodiments, the (4'-CH(CH.sub.3)--O-2') modified nucleosides are arranged throughout the wings of a gapmer motif.

Modified Nucleobases

[0420] Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

[0421] Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (--C.ident.C--CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0422] Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.

[0423] In certain embodiments, antisense compounds comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

Conjugated Antisense Compounds

[0424] Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.

[0425] Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3' terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5'-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.

[0426] In certain embodiments, antisense compounds, including, but not limited to those particularly suited for use as ssRNA, are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0427] For additional conjugates including those useful for ssRNA and their placement within antisense compounds, see e.g., US Application No. 61/583,963.

In Vitro Testing of Antisense Oligonucleotides

[0428] Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.

[0429] Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.

[0430] One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

[0431] Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.

[0432] Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.

[0433] Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.

[0434] The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

[0435] RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.

EXAMPLES

Non-Limiting Disclosure and Incorporation by Reference

[0436] While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.

Example 1: Determination of Genome-Wide Binding Profiles in Engineered RAW264.7 Macrophages

[0437] To understand the mechanisms underlying Rev-Erb regulation of macrophage gene expression, genome-wide binding profiles in RAW264.7 macrophages engineered to contain biotin-tagged Rev-Erb.alpha. and Rev-Erb.beta. were determined. Rev-Erb.alpha. and Rev-Erb.beta. were tagged with biotin-ligase recognition peptide for in vivo biotinylation in RAW264.7 macrophages expressing biotin ligase from E. coli (Heinz, S. et al., Mol. Cell. 2010. 38: 576-589). Biotin-based or antibody-based Chromatin immunoprecipitation linked to deep sequencing (ChIP-Seq) was performed. ChIP-Seq indicated enrichment for both Rev-Erb.alpha. and Rev-Erb.beta. at the promoter of the circadian target gene BmalI. Previous studies (Preitner, N. et al., Cell. 2002. 110: 251-260; Yin, L. and Lazar, M. A. Mol. Endocrinol. 2005. 19: 1452-1459) have also demonstrated a high degree of overlap in the Rev-Erb.alpha. and Rev-Erb.beta. cistromes in several independent cell lines.

[0438] To focus on transcriptional programs common to Rev-Erb.alpha. and Rev-Erb.beta., a core set of 2,079 of the highest confidence peaks occupied by both proteins was selected for detailed analysis. As seen in Table 1, the majority (.about.88%) of these peaks were in intra- and intergenic regions at least 1 kilobase away from annotated transcription start sites.

TABLE-US-00002 TABLE 1 Distribution of Rev-Erb localization relative to the nearest protein-coding genes. % Promoter (1 kb-500 bp) 10 Intergenic 49 Exon 2 intron 39

[0439] This was exemplified by intergenic Rev-Erb binding sites vicinal to the Mmp9 and Cxcr1 genes. In addition, 70% of Rev-Erb bound sites were in regions demarcated by high enrichment for H3K4me1 and low enrichment for H3K4me3, a histone signature for enhancer elements (Feng, D. et al., Science. 2011. 331: 1315-1319). De novo motif discovery of Rev-Erb-bound loci returned significant enrichment for binding sites for Rev-Erb, PU.1, AP1 and C/EBP, as presented in Table 2, with significant p-values and percentage of expected vs. observed instances of motifs.

TABLE-US-00003 TABLE 2 Top-enriched transcription factor motifs identified % % Transcription Factor p-value observed expected AP1 1e-449 31 4 PU.1 1e-354 40 9 Rev-Erb DR2 1e-174 7 1 C/EBP 1e-49 6 1

[0440] PU.1, AP-1 and C/EBP transcription factors are required for macrophage differentiation (Solt, L. A. et al., Nature. 2012. doi:10.1038/nature11030) and have recently been shown to establish the majority of the enhancer-like elements in macrophages (Fontaine, C. et al., Mol. Endocrinol. 2008. 22: 1797-1811). Co-localization of Rev-Erbs with PU.1 and C/EBP in macrophages was confirmed by comparison with direct binding data for these factors. Consistent with these findings, the Rev-Erb bound sites defined above localized to enhancer-like elements specific for macrophages, as defined by H3K4me1. The data is presented in Table 3, which shows the results of distribution of averaged ChIP-Seq signal of H3K4me1 at Rev-Erb bound H3K4me1.sup.hi H3K4me3.sup.lo (n=1,388) region in macrophages (Heinz, S. et al., Mol. Cell 2010. 38: 576-589), B-cell (Lin, Y. C. et al., Nat. Immunol. 2010. 11: 635), liver (Creyghton, M. P. et al., Proc. Natl. Acad. Sci. USA 2010. 107: 21931-21936), mouse embryonic stell cells (mES), and neural progenitor (NP) (Meissner, A. et al., Nature. 2008. 454: 766-770).

TABLE-US-00004 TABLE 3 Normalized Tag density in different cell types vs. distance from center of Rev-Erb bound enhancers Distance from Center (bp) Macrophages Liver mES NP B Cell -1800 0.17 0.06 0.07 0.05 0.10 -1600 0.19 0.06 0.07 0.06 0.11 -1400 0.21 0.06 0.07 0.06 0.12 -1200 0.24 0.06 0.07 0.07 0.14 -1000 0.28 0.07 0.07 0.07 0.15 -800 0.35 0.08 0.07 0.08 0.17 -600 0.41 0.09 0.08 0.09 0.20 -400 0.48 0.09 0.08 0.10 0.22 -200 0.40 0.11 0.08 0.11 0.22 0 0.18 0.11 0.08 0.10 0.18 200 0.40 0.10 0.08 0.10 0.22 400 0.48 0.10 0.08 0.10 0.23 600 0.42 0.08 0.08 0.08 0.20 800 0.34 0.08 0.07 0.08 0.18 1000 0.29 0.07 0.07 0.06 0.16 1200 0.24 0.07 0.07 0.06 0.14 1400 0.21 0.06 0.06 0.06 0.13 1600 0.19 0.06 0.06 0.06 0.12 1800 0.18 0.06 0.06 0.06 0.12

Example 2: Gene Expression Analysis in Wild-Type Macrophages and in Macrophages Deficient for Both Rev-Erb.alpha. and Rev-Erb.beta.

[0441] Gene expression analysis was performed in wild-type (WT) macrophages and macrophages deficient for both Rev-Erb.alpha. and Rev-Erb.beta..

[0442] Bone marrow derived macrophages (BMDMs) and thioglycollate-elicited (ThioMac) macrophages were generated from 4-6 week old Rev-Erb.alpha..sup.flox/flox; Rev-Erb.beta..sup.flox/flox with or without Tie2-Cre as previously described (Huang. W. et al., Nature. 2011. 470: 414-418). Rev-Erb double knockout (DKO) macrophages were generated from bone marrow differentiation of Tie2-Cre; Rev-Erb.alpha..sup.flox/flox; Rev-Erb.beta..sup.flox/flox animals (RevErb DKO) and were compared to control macrophages derived from Cre-negative littermates (WT). Tie2-Cre expression in hematopoietic stem cells (Schlaeger, T. M. et al., Blood. 2005. 105: 3871-3874) resulted in excision efficiencies in DKO macrophages of 85% of Rev-Erb.alpha. and 92% for Rev-Erb.beta., as determined by Q-PCR. Knockout of Rev-Erb.beta. dramatically increased Rev-Erb.alpha. expression. Excision of Rev-Erb.alpha. was estimated by comparing the derepression between the floxed region versus a non-excised region.

[0443] Global Run-On sequencing (GRO-Seq) was performed to investigate the relationship between Rev-Erb binding sites and target genes. Preparation for GRO was performed as described previously (Wang, D. et al., Nature. 2011. doi:10.1038/nature10006). GRO-Seq analysis indicated that 142 genes were significantly up-regulated in DKO macrophages (p-value <0.005), while 71 genes were down-regulated (p-value <0.005). The distance between transcription start sites of annotated genes to the nearest Rev-Erb peaks in all, up-, or down-regulated genes is presented in Table 4. The results indicate that genes that were up-regulated in DKO macrophages were significantly closer to Rev-Erb binding sites than down-regulated genes, consistent with the primary roles of Rev-Erbs as dedicated transcriptional repressors. However, only 3 of the 142 up-regulated genes had Rev-Erb peaks within 2 kb of annotated transcription start sites, suggesting that Rev-Erbs primarily act to repress gene expression at enhancer-like elements.

TABLE-US-00005 TABLE 4 Distance of nearest Rev-Erb peaks to TSS (bp in log2 scale) compared to gene expression in Rev-Erb DKO macrophages Gene expression in Rev-Erb DKO Up-regulated Down-regulated All genes genes average distance (bp) 3181221 755558 3457211 1st quartile 112798 45767 122946 3rd quartile 998603 627405 1603662 gene # 24741 142 71

[0444] For expression analysis, RNA was harvested from macrophages using RNEasy kit (Qiagen), treated with Turbo-DNases (Ambion) and reverse-transcribed, using SuperScript III Reverse-Trasncriptase (Invitrogen) with random hexamers. Values determined for mRNA, using Quantitative reverse transcriptase-dependent PCR (Q-PCR), were normalized to 36B4 mRNA content from at least three independent experiments in triplicates. Quantitative reverse transcriptase-dependent PCR was used to validate GRO-Seq data in WT and DKO macrophages for the Mmp9 and Cx3cr1 genes at the level of mature mRNA. The results are presented in Table 5 and present the analysis in Rev-Erb DKO (WT=8 and DKO=7 in number) and Rev-Erb.alpha. (control=17 and alpha phenotype=18 independent lines), or Rev-Erb.beta. (control=13 and beta phenotype=18 independent lines) over-expressing macrophages. Statistical significance was determined by two tail Student's t-test. P-value is represented as ** for P<0.01 and by .sctn. for P<0.05 versus the control. Mature mRNA for both genes was significantly increased in the Rev-Erb DKO macrophages. Conversely, constitutive expression of either Rev-Erb.alpha. or Rev-Erb.beta. in RAW264.7 macrophages resulted in repression of Mmp9 and Cx3cr1 expression.

TABLE-US-00006 TABLE 5 Q-PCR analysis of relative normalized expression of Mmp9 and Cx3cr1 mRNA in Rev-Erb DKO and Rev-Erb.alpha. or Rev- Erb.beta. overexpressing macrophages Mmp9 Cx3cr1 WT 0.43 0.19 DKO 1.65.sctn. 1.93.sctn. Control 1.72 1.18 Rev-Erb.alpha. 0.32** 0.83.sctn. Control 1.69 1.26 Rev-Erb.beta. 0.50** 0.81**

[0445] By evaluating multiple independent clones, the extent of Mmp9 and Cx3cr1 repression was observed to be inversely correlated with Rev-Erb expression levels. The data was generated by Q-PCR analysis of Mmp9 and Cx3cr1 mRNA expression in independent RAW264.7 macrophage cell lines expressing Rev-Erb.alpha. (n=17), Rev-Erb.beta. (n=18), or empty expression vector. The data was found to be statistically significant, as determined by Spearman rank correlation test.

Example 3: Testing of Genomic Regions Containing Rev-Erb Binding Sites for Enhancer Activity

[0446] A 983 bp region surrounding the Rev-Erb-bound site at -5 kb from the Mmp9 transcription start site (TSS) was cloned downstream of a luciferase reporter driven by a TATA-like promoter. The region was cloned into a pGL4-based reporter and tested in RAW264.7, as described previously (Heinz, S. et al., Mol. Cell. 2010. 38: 576-589). The presence of this Rev-Erb-containing region increased reporter gene activity in RAW264.7 macrophages, while a control genomic region devoid of Rev-Erb binding sites or other enhancer-like features did not. This enhancer activity was also sensitive to Rev-Erb repression, as determined by luciferase reporter activity.

[0447] Sequencing libraries were prepared by ligating separate modified single-stranded adapters to 3' and 5'-RNA ends using mutant (K227Q) truncated RNA ligase 2 (NEB) and RNA ligase 1, respectively, reverse-transcribed with SuperScript III reverse transcriptase (Invitrogen), and PCR-amplified with primers bearing primer landing sites compatible with Illumina sequencing. RAR-related orphan nuclear receptors (RORs) also bind to Rev-Erb response elements and constitutively activate gene expression (Giguere, V et al., Genes Dev. 1994. 8: 538-553). Consistent with this, constitutive expression of ROR.alpha. increased activity of the Mmp9 enhancer element. Co-expression of wild type Rev-Erb.beta., but not Rev-Erb.beta. with a mutation disrupting sequence-specific DNA binding, antagonized ROR.alpha. activation. For this assay, luciferase activity of reporters containing Rev-Erb bound enhancer from the genomic regions was measured after co-transfection of ROR.alpha. and Rev-Erb.alpha. or Rev-Erb.beta. with mutation in the DNA binding domain. BmalI promoter was used as the positive control. A 1 kb genomic region without enhancer-like element was used as a negative control. Data generated was the mean of at least 3 independent experiments and was found to be statistically significant, as determined by one-way ANOVA followed by Tukey HSD test.

[0448] Similar experiments were performed for six other Rev-Erb-bound distal regions, all of which were sensitive to ROR.alpha. activation. Four of the six could be antagonized by Rev-Erb co-transfection. Collectively, these findings suggest that Rev-Erbs confer a macrophage-specific program of repression by regulating the activities of enhancers that are established by collaborative interactions between macrophage lineage-determining transcription factors, such as PU.1 and C/EBPs, and signal-dependent factors, such as RORs.

[0449] Among the 2,079 common Rev-Erb binding sites, 1,388 loci were located at least .+-.1 kb from annotated transcription start sites and were associated with the enhancer histone signature H3K4me1.sup.hi/H3K4me3.sup.lo. ChIP-sequencing experiments indicated that many of these sites were co-enriched for the engaged, serine-5 phosphorylated RNA Polymerase II (RNAPII). The data was derived from a cluster plot of ChIP-Seq signal for H3K4me1, H3K4me3, and serine-5 phosphorylated RNA polymerase II (RNAPII) at 1,388 Rev-Erb bound, H3K4me1.sup.hiH3K4me31.sup.o regions.

[0450] In addition, examination of GRO-Seq data at these sites indicated the presence of bi-directional RNA transcription, consistent with recent studies indicating that RNAs are transcribed from distal enhancer elements on a genome-wide scale (Kim, T.-K. et al., Nature. 2010. 465: 182; Wang, D. et al., Nature. 2011. doi:10.1038/nature10006; Hah, N. et al., Cell. 2011. 145: 622-634). The data is presented in Table 6 as the distribution of averaged macrophage GRO-Seq eRNA signal flanking Rev-Erb intergenic sites defined in macrophages (n=722) and liver (n=521). No significant RNA signal was detected from the locations of intergenic Rev-Erb.alpha. peaks defined in liver (Feng, D. et al., Science. 2011. 331: 1315-1319), indicating that RNA expression at distal regulatory elements is cell-type specific.

TABLE-US-00007 TABLE 6 Distribution of macrophage GRO-Seq eRNA signal flanking Rev-Erb intergenic sites in macrophages and liver macrophages Liver distance from Rev-Erb Rev-Erb.alpha. Rev-Erb.alpha. Rev-Erb.alpha. Rev-Erb.alpha. intergenic sites (bp) Plus Minus Plus Minus -1820 0.0028 0.0025 0.0010 0.0007 -1700 0.0031 0.0028 0.0011 0.0007 -1500 0.0032 0.0030 0.0010 0.0008 -1300 0.0032 0.0033 0.0009 0.0007 -1100 0.0031 0.0041 0.0010 0.0008 -900 0.0030 0.0052 0.0010 0.0009 -700 0.0030 0.0066 0.0009 0.0010 -500 0.0031 0.0076 0.0009 0.0013 -300 0.0036 0.0087 0.0009 0.0014 -100 0.0042 0.0071 0.0011 0.0014 -20 0.0054 0.0059 0.0012 0.0013 20 0.0060 0.0053 0.0012 0.0013 100 0.0070 0.0039 0.0013 0.0012 300 0.0087 0.0033 0.0012 0.0009 500 0.0079 0.0032 0.0010 0.0009 700 0.0071 0.0030 0.0011 0.0009 900 0.0055 0.0030 0.0010 0.0009 1100 0.0046 0.0030 0.0008 0.0009 1300 0.0039 0.0027 0.0010 0.0009 1500 0.0032 0.0026 0.0011 0.0009 1700 0.0028 0.0026 0.0009 0.0009 1820 0.0026 0.0026 0.0007 0.0008

Example 4: Determination of the Role of Rev-Erbs in Regulating Enhancer-RNA (eRNA) Expression

[0451] To determine whether Rev-Erbs regulate eRNA expression, transcription of nascent RNA at Rev-Erb bound enhancers was examined in both loss of function and gain of function models.

[0452] Using GRO-Seq, analysis of averaged RNA signal distribution from the 100 highest enriched Rev-Erb bound intergenic enhancers indicated an overall increase of RNA signal in Rev-Erb DKO macrophages compared to WT control. Conversely, overexpression of Rev-Erb.alpha. resulted in decreased eRNA expression at the most confident Rev-Erb binding sites. To precisely define the eRNA start sites, the GRO-Seq protocol was modified to detect nascent RNA with a 5' 7-methylguanylated cap (5'GROseq), thus focusing on detecting transcriptional events at initiation sites. 5'GRO-Seq was performed with the following modifications from the GRO-Seq protocol (Wang, D. et al., Nature. 2011. doi:10.1038/nature10006). Briefly, GRO-RNA was 3' and 5'-dephosphorylated with polynucleotide kinase (Enzymatics) and calf intestinal phosphatase (NEB), respectively, and then capped fragments were de-capped with tobacco acid pyrophosphatase (Epicentre) to leave 5' phosphates. Analysis of averaged RNA signal distribution at the top 100 Rev-Erb intergenic enhancers showed a striking decrease of eRNA initiation in macrophages overexpressing Rev-Erb.alpha. compared to control macrophages. The data is presented in Tables 7 and 8. Table 7 presents the distribution of averaged GRO-Seq eRNA signal from Rev-Erb DKO and control BMDM at the top 100 Rev-Erb bound intergenic sites. Table 8 shows the distribution of average 5' capped RNA (5' GRO-Seq) signal from Rev-Erb.alpha. overexpressing and control RAW264.7 macrophages flanking the top 100 Rev-Erb-bound intergenic sites.

TABLE-US-00008 TABLE 7 Distribution of averaged GRO-Seq eRNA signal from Rev-Erb DKO and control BMDM distance from center of Wild type DKO Rev-Erb sites (bp) plus minus plus minus -1820 0.002 0.003 0.003 0.003 -1700 0.003 0.003 0.003 0.003 -1500 0.003 0.003 0.003 0.003 -1300 0.003 0.004 0.003 0.004 -1100 0.002 0.005 0.003 0.006 -900 0.002 0.006 0.003 0.008 -700 0.003 0.008 0.004 0.009 -500 0.004 0.008 0.004 0.009 -300 0.004 0.009 0.004 0.011 -100 0.004 0.009 0.005 0.010 -20 0.005 0.008 0.007 0.008 20 0.006 0.007 0.007 0.008 100 0.007 0.005 0.008 0.006 300 0.009 0.004 0.010 0.005 500 0.008 0.004 0.009 0.005 700 0.008 0.004 0.009 0.004 900 0.006 0.003 0.008 0.003 1100 0.005 0.003 0.007 0.003 1300 0.004 0.002 0.005 0.003 1500 0.004 0.003 0.004 0.004 1700 0.003 0.004 0.004 0.004 1820 0.003 0.003 0.003 0.004

TABLE-US-00009 TABLE 8 Distribution of 5'GRO-Seq eRNA signal from Rev-Erb.alpha. macrophages and control macrophages flanking Rev-Erb-bound intergenic sites distance from center of Control Rev-Erb Rev-Erb sites (bp) plus minus plus minus -480 0.034 0.027 0.013 0.016 -400 0.032 0.044 0.021 0.018 -300 0.032 0.050 0.026 0.031 -200 0.015 0.078 0.008 0.044 -100 0.020 0.309 0.012 0.172 -10 0.175 0.169 0.108 0.120 0 0.198 0.148 0.117 0.109 10 0.245 0.161 0.153 0.107 100 0.242 0.032 0.162 0.027 200 0.114 0.017 0.055 0.013 300 0.058 0.047 0.030 0.025 400 0.045 0.015 0.026 0.006 480 0.055 0.011 0.033 0.005

[0453] In either loss or gain of function experiment, the eRNA signal at the global set of PU.1-bound enhancers showed no significant changes, as presented in Table 9, indicating that the changes in eRNA are specific to Rev-Erb-bound enhancer elements. Table 9 presents the distribution of average 5' capped RNA (5'GRO-Seq) signal from Rev-Erb.alpha. overexpressing and control RAW264.7 macrophages flanking the top 100 PU.1-bound intergenic sites.

TABLE-US-00010 TABLE 9 Distribution of 5'GRO-Seq eRNA signal from Rev-Erb.alpha. macrophages and control macrophages flanking PU.1-bound intergenic sites distance from center of Control RevErb.alpha. PU.1 sites (bp) plus minus plus minus -480 0.027 0.023 0.020 0.018 -400 0.015 0.029 0.010 0.026 -300 0.034 0.035 0.036 0.033 -200 0.012 0.049 0.009 0.074 -100 0.013 0.199 0.014 0.188 -10 0.105 0.163 0.097 0.161 0 0.152 0.127 0.161 0.129 10 0.180 0.085 0.189 0.079 100 0.225 0.013 0.233 0.019 200 0.059 0.013 0.050 0.020 300 0.046 0.018 0.039 0.017 400 0.022 0.015 0.022 0.014 480 0.013 0.010 0.012 0.013

[0454] Moreover, the de-repression level of eRNA in Rev-Erb DKO is inversely correlated to eRNA repression level upon constitutive expression of Rev-Erb.alpha. (.rho.=-0.39, p-value=0.008), indicating a strong agreement between the experimental sets. For this assay, 53 Rev-Erb bound enhancers with de-repressed eRNA expression in Rev-Erb DKO macrophages was plotted against changes of eRNA level upon overexpression of Rev-Erb.alpha. in RAW264.7 macrophages. Changes in eRNA was determined by the log.sub.2 difference of expression between DKO and WT macrophages, or between Rev-Erb.alpha. over-expressing and control macrophages. The data was considered statistically significant, as determined by Spearman rank correlation test.

[0455] GRO-Seq results indicating Rev-Erb mediated negative regulation of eRNA expression were confirmed using RT-PCR for eRNA of Mmp9 -5 kb and Cx3cr1 28 kb enhancers. Q-PCR analysis of the -5 kb Mmp9 and 28 kb Cx3cr1 enhancer RNA in Rev-Erb DKO (wild type=6) and DKO-5 in number) and Rev-Erb.alpha. overexpressing RAW264.7 macrophages (control=13 and alpha=14 independent cell lines) are presented in Table 10. The statistical significance was determined by two tail Student's t-test where * represents P<0.01 and .sctn.represents P<0.05, versus the control.

TABLE-US-00011 TABLE 10 Relative expression of Mmp9 and Cx3cr1 enhancer RNA Mmp9 Cx3cr1 eRNA eRNA WT 0.67 0.33 DKO 1.38* 1.75.sctn. Control 1.37 1.37 Rev-Erb 0.65 0.66.sctn.

[0456] To investigate mechanisms by which Rev-Erbs regulate eRNA expression, the effects of gain and loss of Rev-Erb function on enhancer assembly and histone modification were evaluated. ChIP-Seq experiments demonstrated increased H3K9 acetylation (H3K9ac) at Rev-Erb-occupied enhancers in DKO macrophages and reduced H3K9ac enrichment in macrophages with constitutively expressed Rev-Erb.alpha.. H3K9ac enrichment was not changed at the global set of PU.1-enhancers, consistent with Rev-Erb-mediated recruitment of NCoR/HDAC3 complexes mediating local deacetylation of H3K9 (Yin, L. and Lazar, M. A. Mol. Endocrinol. 2005. 19: 1452-1459). The assay measured the average distribution of H3K9ac ChIP-Seq signal flanking 53 Rev-Erb bound enhancers or the top 500 enriched PU.1 bound enhancers for WT and Rev-Erb DKO macrophages. The assay also measured the same parameters for 266 Rev-Erb bound enhancer which have repressed eRNA expression or the top 500 enriched PU.1 bound enhancer for control and Rev-Erb.alpha. overexpressing RAW264.7 macrophages.

[0457] In contrast, constitutive expression of Rev-Erb.alpha. had no significant effect on H3K4me1 or PU.1 enrichment at Rev-Erb bound enhancer elements, despite the profound changes in eRNA initiation. The assay measured the average distribution of PI.1 and H3K4me1 and ChIP-Seq signal flanking the 266 Rev-Erb bound enhancers with repressed eRNA level upon overexpression of Rev-Erb.alpha..

[0458] Collectively, these results raise the possibility that Rev-Erbs repressed gene expression at a distance by regulating enhancer-directed transcription. Consistent with this possibility, changes in eRNA expression at Rev-Erb-bound sites due to gain or loss of Rev-Erb function were correlated with changes in expression of the nearest mRNA.

Example 5: Determination of the Role of eRNA Activity In Vitro

[0459] Even though levels of eRNA are low at steady state (Kim, T.-K. et al., Nature. 2010. 465: 182), transcriptional initiation from enhancers, as measured by 5' GRO-Seq, was often comparable to that at promoters of protein-coding genes, as exemplified by Cx3cr1 and Mmp9, suggesting robust production of short-lived transcripts. Three experimental approaches were used to investigate whether the synthesis of enhancer-directed RNA transcripts contributed to enhancer activity.

[0460] First, RNA interference (RNAi) was utilized to target eRNA transcripts of Mmp9 and Cx3cr1. Non-targeting siRNA oligos or siRNA directed against Mmp9 and Cxcr1 eRNA (Dharmacon) were transfected using DeliverX (Affymetrix) or LipofectAMINE2000.RTM. (Invitrogen) into ThioMac or BMDM, respectively. siRNAs were identified that specifically reduced expression of eRNAs associated with the Mmp9 or Cx3cr1 enhancers in primary WT macrophages. Q-PCR analysis of Mmp9 eRNA, Mmp9 mRNA and NCoA5 (negative control) for WT and Rev-Erb DKO ThioMac transfected with control or Mmp9 eRNA siRNA (WT=4 and DKO=4 in number) was conducted. The results are presented in Table 11. Q-PCR analysis was also conducted of Cx3cr1 eRNA, Cxc3cr1 mRNA and Csrnp1 (negative control) for WT and Rev-Erb DKO BMDM transfected with siRNA targeting Cx3cr1 eRNA (WT=6 and DKO=5 in number). The results are presented in Table 12. The results demonstrate the reduction in eRNA expression was associated with a corresponding reduction of Mmp9 and Cx3cr1 mRNAs, but not mRNAs from the nearest expressing genes such as NCoA5 and Csrnp1, respectively. Furthermore, these siRNAs reversed the de-repression phenotype associated with increased eRNA expression in Rev-Erb DKO macrophages.

TABLE-US-00012 TABLE 11 Relative expression of Mmp9 eRNA and mRNA after treatment with siRNA Mmp9 Mmp9 eRNA mRNA NCoA5 Control siRNA WT 0.4 1.1 0.8 siRNA to Mmp9 eRNA WT 0.3 0.4 0.8 Control siRNA DKO 1.3 2.0 0.8 siRNA to Mmp9 eRNA DKO 0.5 0.4 1.0

TABLE-US-00013 TABLE 12 Relative expression of Cx3cr1 eRNA and mRNA after treatment with siRNA Cx3cr1 Cx3cr1 eRNA mRNA CsrnpI Control siRNA WT 0.8 0.3 0.9 siRNA to Cx3cr1 eRNA WT 0.2 0.1 0.9 Control siRNA DKO 2.1 2.5 1.0 siRNA to Cx3cr1 eRNA DKO 0.9 1.4 1.2

[0461] As a second approach, chemically modified antisense oligonucleotides (ASO) were utilized to knock down the plus strand Mmp9 -5 kb eRNA. Seventy seven overlapping ASOs targeting the proximal 450 bp of the plus-strand Mmp9 -5 kb eRNA were systematically screened and the most effective ASOs were selected for detailed analysis.

[0462] ISIS 566237 is an oligonucleotide with deoxy, MOE and (S)-cEthyl units with a sequence 5'-ATTGTGTGACCCCAGC-3' (SEQ ID NO:3) and a chemistry notation 5'-Aes Tes Tks Gds Tds Gds Tds Gds Ads mCds mCds mCds mCds Aks Gks mCe-3' (e=2'-O-methoxyethyl ribose; s=thioate ester; k=(S)-cEt; d=2'-deoxyribose). ISIS 566237 is targeted to an Mmp9 eRNA sequence, SEQ ID NO: 1 (GENBANK Accession No. NT_039207.7 truncated from 105809972 to 105810309). ISIS 566237 targets SEQ ID NO: 1 at nucleotides 247 to 262.

[0463] ISIS 566241 is an oligonucleotide with deoxy, MOE and (S)-cEthyl units with a sequence 5'-CAAGCTTCAGCTCATT-3' (SEQ ID NO:4) and a chemistry notation 5'-mCes Aes Aks Gds mCds Tds Tds mCds Ads Gds mCds Tds mCds Aks Tks Te-3' (e=2'-O-methoxyethyl ribose; s=thioate ester; k=(S)-cEt; d=2'-deoxyribose). ISIS 566241 is targeted to an Mmp9 eRNA sequence, SEQ ID NO: 1 at nucleotides 260 to 275.

[0464] ISIS 129700 is a 5-10-5 MOE gapmer and is 20 nucleosides in length, wherein the central gap segment comprises of ten 2'-deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and each nucleoside in the 3' wing segment has a 2'-MOE modification. The internucleoside linkages throughout the gapmer are phosphorothioate (P.dbd.S) linkages. All cytosine residues throughout the gapmer are 5-methylcytosines. ISIS 129700 has the sequence 5'-TAGTGCGGACCTACCCACGA-3' (SEQ ID NO: 5) and does not target any known murine target. Hence, it was used as a negative control.

[0465] ISIS 535522 is a 5-10-5 MOE gapmer with a sequence 5'-GCACCTTTCCCTCGGATGGG-3' (SEQ ID NO: 6) and is targeted to the murine Mmp9 mRNA sequence (GENBANK Accession No. NM_013599.2) (SEQ ID NO:203) at nucleotides 2275 to 2294. ISIS 535522 also served as a control in the assay.

[0466] The ASOs were transfected into ThioMac using Cytofectin (Gene Therapy System). The results of the Q-PCR analysis of Mmp9 eRNA expression in thioglycollate-elicited primary macrophages transfected with ASOs are presented in Table 13. Both ISIS 566237 and ISIS 566241 reduced eRNA expression. Statistical significance was determined by one-way ANOVA with Tukey HSD test. In case of the P value, * represents P<0.05 and .sctn.represents P<0.005, versus the control.

TABLE-US-00014 TABLE 13 Relative expression of Mmp9 eRNA and mRNA after treatment with ASOs % inhibition ISIS 129700 0 ISIS 566237 15* ISIS 566241 42.sctn.

[0467] Furthermore, titrating the concentration of ASO resulted in dose dependent reduction of the corresponding Mmp9 mRNA. Table 14 presents the Q-PCR analysis of Mmp9 mRNA expression in ThioMac transfected with titrated concentration of ASO (n=3 per condition).

TABLE-US-00015 TABLE 14 Dose-dependent inhibition of Mmp9 mRNA after treatment with ASOs Dose % ISIS No (nM) inhibition ASO targeting 566237 12.5 0 Mmp9 eRNA 566237 25 0 566237 50 10 566237 100 45 566237 200 54 ASO targeting 566241 12.5 0 Mmp9 eRNA 566241 25 0 566241 50 9 566241 100 55 566241 200 49 ASO targeting 535522 100 88 Mmp9 mRNA

[0468] As a third approach, the functional significance of the Mmp9 -5 kb eRNAs was examined using the enhancer reporter assay guided by the definition of eRNA start sites provided by 5'GRO-Seq. The 983 bp sequence upstream of Mmp9 that conferred Rev-Erb-regulated enhancer activity in RAW264.7 cells encompassed a 388 bp central region of open chromatin containing the binding sites for PU.1, C/EBPs, AP-1 and Rev-Erbs, as well as start sites of the plus and minus-strand eRNAs. The 983 bp of the Mmp9 enhancer was cloned downstream of the luciferase reporter gene driven by the Mmp9 promoter. The luciferase activity of the enhancer reporter driven by Mmp9 promoter containing the indicated DNA fragments and transfected in RAW264.7 macrophages (n=8) was measured and is presented in Table 15. The 388 bp core was significantly less active than the 983 bp sequence, which encoded the eRNAs. Statistical significance was determined by one-way ANOVA followed by Tukey HSD test. In case of the P value, .sctn. represents P<0.005 versus all other indicated conditions.

TABLE-US-00016 TABLE 15 Relative promoter activity of the Mmp9 enhancer activity Random 1 kb 0.14 Mmp9 enhancer 983 bp 1.96.sctn. Mmp9 enhancer 388 bp 0.90

[0469] Expression of the plus-strand eRNA from the 983 bp enhancer was confirmed by RT-PCR of Mmp9 plus eRNA normalized to the copy number of the transfected plasmid DNA using reporter-specific primer for first strand cDNA synthesis Enhancer reporter assays were performed as described above with different DNA fragments cloned in the luciferase reporter and the data is presented in Table 16. Addition of DNA encoding the plus-strand eRNA to the core enhancer, but not the DNA encoding the minus strand eRNA, restored transcriptional activity. Statistical significance was determined by one-way ANOVA followed by Tukey HSD test. In case of the P value, .sctn. represents P<0.005 versus all other indicated conditions.

[0470] In another experiment, 983 bp of the Mmp9 enhancer was cloned downstream of the luciferase reporter gene driven by the Mmp9 promoter in the opposite orientation from that used in the study above. Enhancer reporter constructs driven by Mmp9 promoter containing the DNA fragments were transfected into RAW264.7 macrophages. A similar activity of the plus strand eRNA was observed when the 1 kb or core enhancer elements were inserted in the reverse orientation.

TABLE-US-00017 TABLE 16 Relative promoter activity of the Mmp9 enhancer Activity Mmp9 enhancer 388 bp 0.77 Plus eRNA WT 1.91.sctn. Flipped 0.70 Minus eRNA WT 0.84 Flipped 0.79

[0471] These observations were consistent with the finding that siRNAs and ASOs directed against the plus-strand eRNA resulted in reduction of Mmp9 mRNA expression. These findings also suggested that either the eRNA sequence is an important determinant of enhancer function, or the sequences encoding the plus strand eRNA contain binding sites for additional transcription factors that contribute to enhancer activity.

[0472] To address this question, the orientation of the sequences encoding the plus strand eRNA enhancer was inverted relative to the 388 bp-core. This strategy would retain any putative transcription factor binding sites but completely change the sequence of any potential eRNA product. In the "flipped" construct, enhancer activity was reduced to a level comparable to the 388 bp-enhancer construct despite production of eRNA from an alternative start site. Collectively, these data suggest that the DNA element encoding the plus eRNA contributes to Mmp9 enhancer activity.

Example 6: Determination of the Role of eRNA Activity In Vivo

[0473] The findings of the studies described above raised the question of whether enhancers might be considered as targets for cell-specific manipulation of gene expression in vivo. To explore this possibility, sterile peritonitis was induced in mice and the ability of siRNAs directed against the Mmp9 -5 kb plus strand eRNA to alter Mmp9 mRNA expression was investigated. Thioglycollate-elicited sterile peritonitis and in vivo RNAi assays were performed as described in previous studies (Huang, W. et al., Nature. 2011. 470: 414-418). Briefly, 2 ml thioglycollate medium was delivered to each animal by intra-peritoneal injection on day 1. On day 2, 100 .mu.g scrambled control siRNAs or Mmp9 eRNA-specific siRNA was complexed in LipofectAMINE2000.RTM. and serum-depleted medium (in 1 ml final volume) and delivered to animals by intraperitoneal injection. Elicited macrophages in the peritoneal cavities were collected with 10 ml of PBS on day 4 for RNA analysis (n=8 per condition). Values were normalized to the average of 36B4 and Cyclophilin A mRNA. Statistical significance was determined by two tail Student's t-test. In case of the P value, * represents P<0.05 versus the control siRNA. The results are presented in Table 17 and indicate that the eRNA-specific siRNA, but not a control siRNA, reduced expression of the -5 kb plus strand eRNA and the Mmp9 primary transcript, but not the NCoA5 mRNA, as was observed in primary macrophages in vitro. Cx3cr1 mRNA was also measured as a negative control.

TABLE-US-00018 TABLE 17 Relative RNA expression in mice control siRNA to siRNA Mmp9 eRNA Mmp9 eRNA 1.17 0.83 Mmp9 pre-mRNA 1.21 0.79 NcoA5 1.07 0.93 Cx3cr1 1.05 0.95

Example 7: Role of eRNAs in Enhancer-Dependent Activation of Coding Genes Regulated by Sex Steroid Receptors

[0474] The role of eRNAs in the enhancer-dependent activation of coding genes regulated by sex steroid receptors was studied by performing experiments designed to determine whether liganded estrogen receptors induce eRNA transcription on ER.alpha.-bound enhancers.

[0475] MCF-7 cells were initially obtained from ATCC and were incubated in .alpha.-MEM media supplemented with 10% FBS in a 7% CO.sub.2 humidified incubator. Once the cells reached 60% confluency, they were hormone-stripped for 3 days by culturing in phenol-free media plus charcoal-depleted FBS. The cells were treated or not with 100 nM Estradiol (E.sub.2) for 1 hr to induce estrogen signaling (Prasanth, K. V. and Spector, D. L. Genes Dev. 2007. 21: 11-42).

[0476] A ChIP-seq analysis (>100.times.10.sup.6 uniquely mapped reads) of ER.alpha. binding sites was performed, as previously described (Wang, D. et al., Nature. 2011. 474: 390-394). Briefly, approximately 10.sup.7 treated cells were cross-linked with 1% formaldehyde at room temperature for 10 min. After sonication, the soluble chromatin was incubated with 1-5 .mu.g of antibody (HC-20 and H-184, Santa Cruz Biotechnology) at 40.degree. C. overnight. Immunoprecipitated complexes were collected using Dynabeads A/G (Invitrogen). Subsequently, immunocomplexes were washed, DNA extracted and purified by QIAquick Spin columns (Qiagen). The extracted DNA was ligated to specific adaptors, followed by deep sequencing with the Illumina HiSeq 2000 system, according to the manufacturer's instructions. The first 48 bp for each sequence tag returned by the Illumina pipeline was aligned to the hg18 assembly (National Center for Biotechnology Information, build 36.1), using BFast, allowing up to two mismatches. Only uniquely mapped tags were selected for further analysis.

[0477] The data was visualized by preparing custom tracks on the University of California, Santa Cruz genome browser, using HOMER (http://biowhat.ucsd.edu/homer) (Heinz, S. et al., Mol. Cell. 2010. 38: 576-589). Given that the peak distribution of transcription factors and histone marks were markedly different, parameters were optimized for the narrow tag distribution characteristic of transcription factors by searching for high read density regions with a 200 bp sliding window. Regions of maximal density exceeding a given threshold were called `peaks`, and it was required for adjacent peaks to be at least 500 bp away to avoid redundant detection. The common artifacts derived from clonal amplification were circumvented by considering only one tag from each unique genomic position, as determined from the mapping data. The threshold for the number of tags that determined a valid peak was selected at a false discovery rate of 0.001, determined by peak finding using randomized tag positions in a genome with an effective size of 2.times.10.sup.9 bp. It was also required for peaks to have at least four-fold more tags (normalized to total count) than input control samples. In addition, it was required to obtain four-fold more tags relative to the local background region (10 kb) to avoid identifying regions with genomic duplications or non-localized binding.

[0478] In the case of histone marks, the parameters were modified to search for enrichment in wide genomic segments as, unlike transcription factors, they can occupy large segments in the magnitude of several kb. Seed regions were initially found using a peak size of 500 bp at a false discovery rate of 0.001 to identify enriched loci. Enriched regions separated by 1 kb were merged and considered as blocks of variable lengths. All called peaks meeting the criteria established for transcription factors and histone marks were then associated with genes by cross-referencing the RefSeq TSS database as available in the UCSC genome browser. Peaks from individual experiments were considered equivalent if their peak centers were located within 200 bp.

[0479] The analysis (>100.times.10.sup.6 uniquely mapped reads) of ER.alpha. binding sites using vehicle or E.sub.2-treated MCF-7 breast cancer cells increased the number of known genomic ER.alpha. binding sites three-fold, to 23,255, genome-wide. In this robust analysis, ER.alpha. was found to preferentially bind distal intergenic (53%) and intronic sites (39%), with only 3% being bound to promoters, suggesting that ER.alpha. is able to exert transcriptional effects based on binding to genomic elements other than the regulated coding gene promoters. A corresponding `deep` ChIP-seq analysis was done for H3K4me (Newman J. J. and Young, R. A. Cold Spring Harb. Symp. Quant. Biol. 2011. 75: 227-235), a histone modification considered to mark enhancers (Heintzman, et al., Nat. Genet. 2007. 39: 311-318). Analysis was also done for H3K27ac, a modification considered to mark potentially active enhancers (Creyghton, M. P. et al., Proc. Natl. Acad. Sci. USA. 2010. 107: 21931-6). Analysis of these two markers was used to identify 7,797 potential estrogen-responsive enhancers.

Example 8: GRO-Seq Analysis of Genome-Wide Transcription Units Regulated by ER.alpha.

[0480] The transcriptional consequences of a one hour E.sub.2 treatment at 100 nM, compared to the vehicle, was determined by GRO-Seq (Core, L. J. et al., Science. 2008. 322: 1845-8) to provide a genome-wide catalogue of transcription units regulated by ER.alpha..

[0481] GRO-sequencing of nascent RNAQ was achieved using MCF-7 cells hormone-stripped for 3 days and treated or not with 100 nM Estradiol (E.sub.2) for 1 hr to induce estrogen signaling. The cells were then washed three times with cold PBS buffer and then swelled in swelling buffer (10 mM Tris-HCl, pH 7.5, 2 mM MgCl.sub.2, 3 mM CaCl.sub.2) for 5 min on ice, and harvested. Cells were re-suspended and lysed in lysis buffer (swelling buffer with 0.5% IGEPAL and 10% glycerol). Nuclei were washed once with 10 mM lysis buffer and re-suspended in 100 .mu.L freezing buffer (50 mM Tris-HCl, pH 8.3, 40% glycerol, 5 mM MgCl.sub.2, 0.1 mM EDTA). For the run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl.sub.2, 1 mM DTT, 300 mM KCl, 20 units SUPERase-In.TM., 1% sarkosyl, 500 .mu.M ATP, GTP, Br-UTP, 2 .mu.M CTP), and incubated for 5 min at 30.degree. C. The nuclear run-on RNA (NRO-RNA) was then extracted with TRIzol LS reagent (Invitrogen), following the manufacturer's instructions. NRO-RNA was then subjected to base hydrolysis on ice for 40 min, followed by treatment with DNase I and Antarctic phosphatase. To purify the Br-UTP labeled RNA, the NRO-RNA was immunoprecipitated with anti-BrdU agarose beads (Santa Cruz Biotech) in binding buffer (0.5.times.SSPE, 1 mM EDTA, 0.05% Tween-20) for 1 hour at 40.degree. C. with rotation. To repair the end, the immunoprecipitated BrU-RNA was re-suspended in 50 .mu.L reaction (45 .mu.L DEPC water, 5.2 .mu.L T4 PNK buffer, 1 .mu.L SUPERase-In and 1 .mu.L T4 PNK [NEB]) and incubated at 37.degree. C. for 1 hr. The RNA was extracted and precipitated using acidic phenol-chloroform.

[0482] cDNA synthesis was performed, as described previously (Tsai, M. C. et al., Science. 2010. 329: 689-93), with some modifications. The RNA fragments were subjected to poly-A tailing reaction by poly-A polymerase (NEB) for 30 min at 37.degree. C. Subsequently, reverse transcription was performed using oNTI223 primer, the sequence of which is presented in Table 18. Tailed RNA (8.0 .mu.L) was subjected to reverse transcription using Superscript III (Invitrogen). The cDNA products were separated on a 10% polyacrylamide TBE-urea gel. The extended first-strand product (100-500 bp) was excised and recovered by gel extraction. The first-strand cDNA was then circularized by CircLigase.TM. (Epicentre) and re-linearized by Ape1 (NEB). Re-linearized single strand cDNA (sscDNA) was separated in a 10% polyacrylamide TBE gel and the product of needed size was excised (.about.120-320 bp) for gel extraction. Finally, sscDNA template was amplified by PCR using the Phusion High-Fidelity enzyme (NEB), according to the manufacturer's instructions. The oligonucleotide primers oNTI200 and oNTI201 were used to generate DNA for deep sequencing (Table 18). `;` indicated an abasic dSpacer furan; `N` indicates degenerate nucleotides.

TABLE-US-00019 TABLE 18 Oligos used in the Gro-Seq protocol SEQ ID Name Sequence 5' to 3' NO oNTI223 GATCGTCGGACTGTAGAACTCT;CAAGCAGAAGAC 7 GGCATACGATTTTTTTTTTTTTTTTTTTTN oNTI200 CAAGCAGAAGACGGCATA 8 oNTI201 AATGATACGGCGACCACCGACAGGTTCAGAGTTCT 9 ACAGTCCGACG Illumina CGACAGGTTCAGAGTTCTACAGTCCGACGATC 10 small RNA-seq

[0483] Transcript identification and assignment to genomic regions, including annotated genes was accomplished using HOMER. GRO-Seq read densities were analyzed in a similar manner to ChIP-Seq, except that in this case, all the GRO-Seq libraries corresponding to the same experiment were merged in order to maximize read density for transcript identification. Provided GRO-Seq generated strand-specific data, separate tracks were uploaded onto the UCSC genome browser, once tag-enriched sites were identified using a sliding window of 250 bp. The portion of GRO-Seq tags that mapped to repeat regions was excluded and instead, the read density for these regions was approximated with values from flanking regions to avoid having to end transcripts prematurely. Transcript initiation sites were identified as regions where the GRO-Seq read density increased three-fold relative to the preceding 1 kb region. Transcript termination sites were defined by either a reduction in reads below 10% of the start of the transcript or when another transcript's start site was identified on the same strand. Individual high-density peaks spanning a region less than 250 bp were considered as artifacts, and thus removed from the analysis. Transcripts were defined as putative eRNAs if their TSS was located distal to RefSeq TSS (.gtoreq.3 kb) and were associated with ER.alpha. and H3K4me1 regions. To identify differentially regulated transcripts, strand-specific read counts from each GRO-Seq experiment were determined for each transcript using HOMER. EdgeR (http://www.bioconductor.org/) was then used to calculate differential genomic and non-genic expression (.gtoreq.1.5-fold, .ltoreq.0.01 false discovery rate).

[0484] The sequencing resulted in the finding of 1,033 up-regulated genes that exhibited E.sub.2/ER.alpha.-binding in one or in multiple adjacent enhancers, while only 112 of these ER.alpha. up-regulated coding genes exhibited ER.alpha. binding to their promoters, consistent with initial suggestions (Jin, V. X. et al., Nucleic Acids Res. 2004. 32: 6627-35; Carroll, J. S. et al., Nat. Genet. 2006. 38: 1289-97; Kwon, Y. S. et al., Proc. Natl. Acad. Sci. USA 2007. 104: 4852-7; Lin, C. Y. et al., PLoS Genet. 2007. 3: e87; Welboren, W. J. et al., EMBO J. 2009. 28: 1418-28) that ER.alpha. occupancy on enhancers is likely to be the key strategy underlying estrogen-induced gene expression.

[0485] The E.sub.2-regulated enhancers generally displayed a basal expression of bidirectional eRNAs and those in proximity to up-regulated coding genes displayed a characteristic bidirectional activation of eRNAs, exemplified by the FOXC1 locus, in general agreement with recent findings (Hah, N. et al., Cell. 2011. 145: 622-34). In contrast to the more 1:1 enhancer: promoter ratio for AR-regulated genes (Prasanth, K. V. and Spector, D. L. Genes Dev. 2007. 21: 11-42), there was often more than one ER.alpha.-bound enhancer adjacent to up-regulated coding genes. This raised the possibility that for many estrogen-regulated coding genes, more than one enhancer might be involved in up-regulation events and might even cross-regulate. The eRNA transcripts varied in apparent length but were generally .about.1.5 kb, although .about.10% exhibited an apparent predominance of unidirectional eRNA transcripts. Analysis of the GRO-Seq data confirmed the overall up-regulation of eRNAs in response to ligand, generally with bidirectional transcription, robust at 1 h after E.sub.2, and subsequently diminishing, and being highly diminished by 24 hr. Overall, >83% of ER.alpha.-bound enhancers adjacent to up-regulated coding genes exhibited E.sub.2 induced eRNA transcription by GRO-Seq. The median distance between enhancers exhibiting E.sub.2-dependent up-regulation of their eRNAs and their closest up-regulated coding gene was .about.52 kb, with most <215 kb from the coding gene cap site, compared with a median distance of >270 kb for enhancers exhibiting ligand-insensitive enhancer eRNAs with corresponding non-responsive coding genes. Examining the strength of ER.alpha. binding, based on normalized ChIP-seq data on these cohorts of enhancers with upregulation of the eRNAs, exhibited significantly stronger binding than on enhancers not exhibiting eRNA upregulation.

[0486] Based on these GRO-seq data analyses, ten robustly up-regulated transcription units were selected for further experimentation, each associated with enhancers exhibiting clearly increased eRNAs--CA12, FOXC1, GREB1, P2RY2, SMAD7, PGR, SIAH2, NRIP1, TFF1 and KCNK5. For these transcription units, there was a .about.2-5-fold increase in coding gene expression, with a corresponding .about.2.5-5-fold increase in eRNA expression on associated enhancers, assessed 1 hr following addition of E.sub.2 to MCF-7 cell cultures.

Example 9: Investigation of the Potential Role of Ligand-Induced eRNAs on Gene Activation Events Using Transcription Inhibition Assays

[0487] To investigate the potential roles, if any, of ligand-induced eRNAs on gene activation events, two different technologies were employed to down-regulate eRNAs: siRNAs and locked nucleic acid antisense oligonucleotides (LNAs).

[0488] Specific siRNAs directed at several regions of each transcript were used to assess possible effects on gene expression (Table 19). Similarly, LNAs were utilized by placing the LNA-modified bases at key positions to ensure target specificity as well as stability of the oligonucleotides (Table 20). The LNAs were designed for this purpose with complete phosphorothioate backbones to trigger RNAse H cleavage of the targeted sequences (Vester, B. et al., Bioorg. Med. Chem. Lett. 2008. 18: 2296-300) (Table 20). In both cases, the siRNAs or LNAs were designed based on the location of the eRNA CAP sites .about.200 bp 5' of ER.alpha. binding sites, determined using the modified GRO-seq protocol described above. For both LNAs and siRNAs, scrambled sequences were used as controls. For all transcription units examined, experiments were performed with two different LNAs or siRNAs, with similar knock-down efficacy, to exclude any off-target effects.

TABLE-US-00020 TABLE 19 siRNA sequences SEQ SEQ Name ID Name ID (Sense) Sequence (Sense) 5' to 3' NO: (Antisense) Sequence (Antisense) 5' to 3' NO: Overhangs IFF1e_1 CAGAGUCAGAGAGUCAGAGAGAGAU 11 TFF1e_1 AUCUCUCUCUGACUCUCUGACUCUG 12 None TFF1e_2 GAGUUUGGACCUGUGACCUUCCUAA 13 TFF1e_2 UUAGGAAGGUCACAGGUCCAAACUC 14 None TFF1e_3 AAUCUCCUGGGAGGAUGAAGCUGUU 15 TFF1e_3 AACAGCUUCAUCCUCCCAGGAGAUU 16 None GREB1e1_1 ACCACUGUUUCUGACUGCUUUCUCA 17 GREB1e1_1 UGAGAAAGCAGUCAGAAACAGUGGU 18 None GREB1e1_2 GGAUUGAGAGUGACCAGGACAUUUA 19 GREB1e1_2 UAAAUGUCCUGGUCACUCUCAAUCC 20 None GREB1e1_3 CGCCCAGCCUAAUUGUAGUACUUUA 21 GREB1e1_3 UAAAGUACUACAAUUAGGCUGGGCG 22 None PGRe_1 GCAAAUUCUUUCAUGACAA 23 PGRe_1 UUGUCAUGAAAGAAUUUGC 24 UU PGRe_2 GCAAAGAUGGAUAGAGAUA 25 PGRe_2 UAUCUCUAUCCAUCUUUGC 26 UU SIAH2e1_1 GCACAUACCUCAUUAGAGA 27 SIAH2e1_1 UCUCUAAUGAGGUAUGUGC 28 UU SIAH2e2_2 GGUAUUAAUAGCUCUGAAA 29 SIAH2e2_2 UUUCAGAGCUAUUAAUACC 30 UU NRIP1e1_1 GGGAGAGGGUCUACAAUUA 31 NRIP1e1_1 UAAUUGUCGACCCUCUCCC 32 UU NRIP1e3_2 GGCCAGAUCUCCUGUGAUA 33 NRIP1e3_2 UAUCACAGGAGAUCUGGCC 34 UU FOXC1e_1 GCUCCAUUCUGCUGCUCAA 35 FOXC1e_1 UUGAGCAGCAGAAUGGAGC 36 UU FOXC1e_2 CUAACGUGACAGUGACAUA 37 FOXC1e_2 UAUGUCACUGUCACGUUAG 38 UU P2RY2e_1 GCAAAAGGUAGGAGGGUUU 39 P2RY2e_1 AAACCCUCCUACCUUUUGC 40 UU P2RY2e_2 GGAGAUGAAUUGAUAGAGA 41 P2RY2e_2 UCUCUAUCAAUUCAUCUCC 42 UU CA12e_1 CAGAAGAGCUAUUUGGUAU 43 CA12e_1 AUACCAAAUAGCUCUUCUG 44 UU CA12e_2 GAGUGGACUUCACAAGAAA 45 CA12e_2 UUUCUUGUGAAGUCCACUC 46 UU SMAD7e1_1 AGAGAAGAAUGAAGGUGAA 47 SMAD7e1_1 UUCACCUUCAUUCUUCUCU 48 UU SMAD7e2_2 CCACAGGUGAGCAGAAAUU 49 SMAD7e2_2 AAUUUCUGCUCACCUGUGG 50 UU SMAD7e3_3 CCUAAUUCCCAGAAGCAGA 51 SMAD7e3_3 UCUGCUUCUGGGAAUUAGG 52 UU KCNK5e1_1 CGAAAUGGCCUAAAGAUGA 53 KCNK5e1_1 UCAUCUUUAGGCCAUUUCG 54 UU KCNK5e2_2 ACACAAAGGUGGAAGGAAA 55 KCNK5e2_2 UUUCCUUCCACCUUUGUGU 56 UU KCNK5e3_3 GGAAGAACCUGCAGAGAUG 57 KCNK5e3_3 CAUCUCUGCAGGUUCUUCC 58 UU

TABLE-US-00021 TABLE 20 LNA sequences LNA name Sequence SEQ ID NO: LNA_Ctrl CACGTCTATACACCAC 59 TFF1E_1 GAATTAACGCCTGAGG 60 TFF1E_2 GAACTGACAAAGGTGG 61 TFF1E_3 ATCTCCCCACTCAAGG 62 TFF1E_4 CATTTTTCTGCTGACC 63 CA12E_1 ACAAGACAGAGGCAGA 64 CA12E_2 TCAGTTGGAGGACAGT 65 FOXC1E_1 GAAGGAGCAGGTGAAA 66 FOXC1E_2 GGTATTTCCGCTTCAC 67 NRIP1E3_1 AGGATACCAGGACACA 68 NRIP1E3_2 TGATAAAAGCAGGGTC 69

[0489] Twenty four hours after cell seeding, MCF-7 cells were hormone-stripped for one day, followed by siRNA transfection (40 nM) using LipofectAMINE2000.RTM.. Cells were washed twice with PBS and maintained in hormone-deprived phenol-free supplemented stripped media for 2 days, and then treated with EtOH or E2 for 1 hr. LNA transfections (40 nM) were performed 2 days after starvation in stripped media and the LNA treatment lasted 6 or 24 hrs, after which cells were treated as described above.

[0490] For RT-QPCR, RNA was isolated using TRIzol reagent (Invitrogen), and total RNA was reverse-transcribed using SuperScript.RTM. III Reverse Transcriptase (Invitrogen), as per the manufacturer's instructions. Quantitative PCRs were performed in MX3000P (Stratagene), using Q-PCR master mix (Agilent Inc.). For normalization, .DELTA.Ct values were calculated using the formula: .DELTA.Ct=(Ct Target-Ct input) where input corresponds to the level of ACTB transcript. Fold differences in normalized gene expression were calculated by dividing the level of expression of the treated sample with the untreated sample or between siRNA/LNA and Control siRNA/control-LNA transfected cells. A list of primers used for Q-PCR is provided in Tables 21 and 22. `F` and `R` indicate the forward and reverse primers, respectively.

TABLE-US-00022 TABLE 21 List of primers used to measure eRNA Name Sequence 5' to 3' SEQ ID NO: TFF1e_F TCAGTTCCCAGCATTCTCATC 70 TFF1e_R TTGAGCCTTGGAGACAGAAAG 71 GREB1e1_F TCCAAAGCATCCCATTCCTG 72 GREB1e1_R TGAGCAAAACAAGACAAACCG 73 PGRe_F TTATGTTGCTCTTGATAGACTCCC 74 PGRe_R GCTAGGTGCTGTCTGAGATTC 75 SIAH2e1_F TTCAAGCAAAGATTATAGCCATGTG 76 SIAH2e1_R ATCCAGTGCAGAGTAACATCAG 77 SIAH2e2_F AGATGCCTCTGCATACTGGTT 78 SIAH2e2_R CAGACCATATTGGGCCACAG 79 NRIP1e1_F CCACAGCAGAAAACCACTGA 80 NRIP1e1_R TTCCCTCTGCACTGACTCCT 81 NRIP1e3_F CGTCTTTTCCCACTGACACA 82 NRIP1e3_R CCCCTCCCCAGAAGAAAATA 83 FOXC1e_F CTGAGGAACACAAGACTAGCC 84 FOXC1e_R ACTGGACTCATTTTGGGACATC 85 P2RY2e_F ATTGTGCATGGCTCTTACCC 86 P2RY2e_R CTTGGTGCATGTGAGCTTGT 87 P2RY2e_F AGCTTCTGGTTCCAAGGTCA 88 P2RY2e_R CATGTGCTGTTGTTGCTGTG 89 CA12e_F TGAAAGGGAAGACGCAGATG 90 CA12e_R TTGTATCCTTTGACTGGGCAG 91 SMAD7e1_F AAAGAAGGCAGGGGAACAAT 92 SMAD7e1_R CACTTGGGCAATCCAGAAAT 93 SMAD7e2_F TCACCTGTGGAAAGAGACAAC 94 SMAD7e2_R AGAACCTTTTGCTCCCTAGTG 95 SMAD7e3_F TTAAACGAGCCTGGAGTTGG 96 SMAD7e3_R AAATTCCTCAGAGCCCAGTG 97 KCNK5e1_F GGCTCAGAGAGGCCAAAA 98 KCNK5e1_R TGGACCCTATCATCTCCTTTAACT 99 KCNK5e2_F GGAAAGGAATTGCTGGATCA 100 KCNK5e2_S_R GTGCAACCACTTGGGAAACT 101 KCNK5e3_S_F CAGAGATGAGGAAAGGTTTGC 102 KCNK5e3_S_R ATCTGCTTCACGGTCTCATG 103

TABLE-US-00023 TABLE 22 List of primers used to measure mRNA Name Sequence 5' to 3' SEQ ID NO: FOXC1_F AGTAGCTGTCAAATGGCCTTC 104 FOXC1_R TTAGTTCGGCTTTGAGGGTG 105 P2RY2_F GGGGACCTGTTTTTCCTGTT 106 P2RY2_R GACTTGGATCTGGACCTGGA 107 CA12_F TCTGTCTGCCAACAAGCAGT 108 CA12_R GCACTGTAGCGAGACTGGAG 109 SMAD7_F TCCTGCTGTGCAAAGTGTTC 110 SMAD7_R AAATCCATCGGGTATCTGGA 111 KCNK5_F TGCCAAGAGACTAGGGCAGT 112 KCNK5_R GAATACGAAGGGTGGGATCA 113 PGR_F CATCACAGGGAACCAGACCT 114 PGR_R CACCCCGAAGAGACCATAGA 115 SIAH2_F TCAGGAACCTGGCTATGGAG 116 SIAH2_R GGCAGGAGTAGGGACGGTAT 117 NRIP1_F GCCAGAAGATGCACACTTGA 118 NRIP1_R CAAGCTCTGAGCCTCTGCTT 119 TFF1_F CACCATGGAGAACAAGGTGA 120 TFF1_R TGACACCAGGAAAACCACAA 121 GREB1_F GGCAGGACCAGCTTCTGA 122 GREB1_R CTGTTCCCACCACCTTGG 123

[0491] Both siRNA knockdown and LNA treatment of the TFF1, FOXC1, CA12 and NRIP1 enhancers revealed that, for each transcription unit, the induction of both the eRNA and of the adjacent coding gene transcript, as assessed by Q-PCR and GRO-seq, respectively, was significantly inhibited or fully abolished. In contrast, these LNAs or siRNAs caused no inhibitory effects on housekeeping genes tested or on E.sub.2-regulated or non-E2-regulated adjacent transcription units located distal to the regulated coding genes, as exemplified by measuring levels of RSPH1 (TFF1e), APH1b (CA12e), and USP25 (NRIP1e). Ligand-induced increase of ER.alpha. binding occurred even after eRNA knockdown. Similar eRNA requirements for coding genes were observed based on knockdown of the eRNAs for PGR, SIAH2, KCNK5, P2RY2 and SMAD7, using either of two LNAs or siRNAs designed for each targeted enhancer. GRO-Seq analysis of effects of siRNAs or LNAs on E.sub.2-regulated coding transcription units gave similar results as those quantitated by QPCR.

Example 10: Investigation of the Potential Role eRNA in Enhancer: Promoter Looping Events

[0492] To investigate whether eRNAs might be required for enhancer: promoter looping events, generally considered to be part of the E.sub.2-activation process, an open-ended (3D-DSL) approach was employed for studying the spatial organization of genomes (Harismendy, O. et al., Nature. 2011. 470: 264-8), conceptually analogous to 5C methodology (Fullwood, M. J. et al., Nature. 2009. 462: 58-64).

[0493] In the 3D-DSL method, oligonucleotides corresponding to genomic sites that are to be analyzed for participation in a network contain a 5'-phosphate (referred to as acceptors), while oligonucleotides corresponding to genomic sites of potential interaction have a 5'-OH (referred to as donors). This method permits interrogation of both short (<10 kb), as well as long-distance, genomic interactions with specific genomic regions including promoters and enhancers. Therefore, "donor" pools of oligonucleotides spanning .about.200 kb flanking the promoter of four up-regulated ER.alpha. target genes were designed based on the HindIII restriction site. The "acceptor" pool constituted all ER.alpha. binding sites and promoters in the interval. The housekeeping gene GAPDH was used as a control (data not shown).

[0494] 3D-DSL was performed as described previously (Fullwood, M. J. et al., Nature 2009. 462: 58-64). Briefly, equal amount of 3C chromatin was biotinylated using the Photoprobe Kit (Vector Lab). Donor and acceptor probe pools (2.5 fmol per probe) were annealed to the biotinylated 3C samples at 45.degree. C. for 2 hrs, followed by 10 min at 95.degree. C. The biotinylated DNA was immunoprecipitated with magnetic beads conjugated to streptavidin. During this process, unbound oligonucleotides were removed by stringent washes. The 5'-phosphate of acceptor probes and 3'-OH of donor probes were ligated using Taq DNA ligase at 45.degree. C. for 1 hr. These ligated products were washed and eluted from beads and then amplified by PCR using primers A and B-AD (or Primer B-BC1 and -BC2 if bar coding was used) for deep sequencing on the Illumina HiSeq 2000, using Primer A as sequencing primer.

[0495] For data analysis, a virtual library was first built of all possible donor-acceptor sequences by in silico concatenating all acceptor sequences with all donor sequences. These reads were then aligned to the virtual library of DSL donors-acceptors sequences, using NOVOALIGN (-t 248 -r None), and the number of reads that are mapped to every interaction with no mismatches were counted, by using custom Perl scripts. Next, from each sample the counts given by the ligations of donors to acceptors on the identical restriction sites (the "spot" ligations) was subtracted, and the counts of the interactions were normalized to the remaining number of total reads, after subtraction. Finally, from each interaction, the counts that are present in unligated controls were subtracted, after normalization.

[0496] 3D-DSL plots were generated using Matlabs where a 10 kb window was set to bundle the interaction intensities, except for a 20 kb window for NRIP1. The interactions were plotted using a Bezier curve between the two positions with the third point in the middle of the positions with the y-axis corresponding to the log 10 intensity. The peak locations were then added on the bottom of the plot.

[0497] Two E.sub.2-regulated transcription units, P2RY2 and KCNK5, were first examined. In the case of the P2RY2 transcription unit, E.sub.2 caused an increase in the specific promoter enhancer interaction approximately 1,000-fold compared to that observed in the control cultures. In addition to promoter enhancer loops, a new interaction between enhancer and gene terminator was also observed, as compared to the -E.sub.2 condition. The P2RY2 promoter locus exhibited three additional loops, one of which did not change upon E.sub.2 treatment, but two other low intensity loops disappeared upon gene activation, consistent with the dynamic nature of gene topology. Based on results of ER.alpha. knock-down, it was noted that even after 3 days in stripped-serum medium, MCF-7 cells still exhibited some ER.alpha.-dependent basal activation of a significant cohort of coding gene targets, and hence both siRNAs and LNAs against enhancer eRNAs caused a decrease in basal transcription for some E.sub.2-regulated genes, in addition to their more robust effects on E.sub.2-stimulated gene expression. Similarly, for the KCNK5 gene locus, E.sub.2 treatment caused a >300-fold increase in promoter: enhancer interactions. Two more loops arising from enhancer in response to E.sub.2 were detected, one of them around the terminator region of gene and the other around that might represent enhancer-specific loops. These observations indicate that a major effect of the ligand was to enhance specific promoter enhancer interactions and, in some cases, enhancer: gene terminator interactions, in parallel to induction of eRNA. For some loci, new enhancer: promoter interactions were actually established and additional interactions were also observed. This raised the question whether the induced eRNAs exert any roles in the dynamic regulation of short-range and long-range induced interactions.

Example 11: Investigation of the Regulation of E.sub.2-Induced Enhancer: Promoter Interactions by Specific eRNAs

[0498] The effect of loss of specific eRNAs on E2-induced enhancer: promoter interactions was investigated.

[0499] LNAs that were highly effective against two estrogen-regulated enhancers, NRIP1 and GREB1, were characterized. Chromatin conformation capture (3C) was performed. Briefly, 25.times.10.sup.6 MCF-7 cells were fixed by adding 1% formaldehyde at room temperature for 10 min, and the reaction stopped by adding glycine. Lysis buffer (500 .mu.l 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% IGEPAL CA-630, protease inhibitors [Sigma]) was added and cells were incubated on ice. Next, cells were lysed with a Dounce homogenizer, and the suspension spun down at 5,000 rpm at 4.degree. C. The supernatant was discarded and the pellet was washed twice with 500 .mu.l ice-cold 1.times.NEBuffer 2 (NEB, Ipswich, Mass.). The pellet was then resuspended in 1.times. NEBuffer 2 and split into five separate 50 .mu.l aliquots. The extracted chromatin was then digested overnight by adding 400 units HindIII (NEB). Each digested chromatin mixture was ligated by adding T4 DNA Ligase (800 units) in 20 times of initial volume for 4 hrs at 16.degree. C. The ligase step was omitted in one chromatin aliquot from the five mentioned above to use the sample as the unligated control. After incubation at 16.degree. C., the chromatin was de-cross-linked overnight at 65.degree. C. and purified twice with phenol and then with phenol: chloroform: IAA (25:24:1). DNA was precipitated and pellets were air-dried before re-suspending in 250 .mu.l 1.times.TE buffer. To degrade any carryover RNA, 1 .mu.l RNAse A (1 mg/ml) was added to each tube and incubated at 37.degree. C. for 15 min. DNA was further purified using Phenol: Chloroform: IAA and precipitated. This enriched fraction was used for the DSL part of the protocol or subjected to PCR using unique primers, and was electrophoresed on Agarose gel.

[0500] In the case of the NRIP1 locus, which was shown to exhibit a ligand-induced enhancer: promoter loop by conventional 3C assay, treatment with LNA against eRNA caused a .about.200-fold inhibition of the coding gene expression and also inhibited both enhancer: promoter and enhancer: terminator interactions in E.sub.2-treated MCF-7 cells, assessed using conventional 3C assays and 3D-DSL. An interaction between a different upstream ER.alpha.-binding site and the promoter was not affected. In the case of the GREB1 locus, siRNA-mediated eRNA knockdown inhibited GREB1 coding gene induction and also inhibited the specific enhancer: promoter interaction induced by E.sub.2. The enhancer: terminator loop was also reduced upon siRNA-mediated knockdown of eRNA, suggesting that eRNAs are required for interactions between enhancer and adjacent regulatory elements. Additional interactions of the GREB1 promoter with a non-enhancer region were also diminished by eRNA knockdown, indicating that these loops were altered by interruption of enhancer: promoter interaction, licensing other interactions.

[0501] Together these experiments indicate that estrogen causes quantitative, as well as some qualitative, alterations in the interactions between enhancers and coding gene promoters and even between enhancers, terminators and other ER.alpha.-bound regions, which are highly diminished or abolished with down-regulation of the targeted eRNAs. For these gene targets, the eRNA was, therefore, of functional importance for robust enhancer: promoter interactions, with knock-down by either siRNA or LNA treatment invariantly diminishing, or even abolishing, these putatively activating enhancer: promoter interactions, consistent with eRNA function, even under unstimulated conditions.

Example 12: Investigation of Enhancer and Promoter Interactions

[0502] The interaction between an enhancer and promoter that spans a distance of >250 kb permitting FISH analysis, was investigated.

[0503] Cells were processed for DNA ImmunoFISH. BAC probes were commercially obtained from Empire Genomics and are listed in Table 23.

TABLE-US-00024 TABLE 23 BAS probes Gene Coordinates (including eRNA/s) FISH BAC FOXC1 chr6: 1,548,833-1,590,281 RP11-13F18 P2RY2 chr11: 72,568,712-72,653,158 RP11-352115 STARD10 chr11: 71,986,844-72,178,371 RP11-610C10 NRIP1 chr21: 15,228,005-15,513,603 RP11-22D1 GREB1 chr2: 11,530,334-11,710,942 RP11-50E1

[0504] MCF-7 cells were grown onto acid-washed polylysine-coated coverslips. Cells were treated with vehicle (EtOH) or E2 for 1 hour, washed with PBS and immediately fixed with freshly made 4% paraformaldehyde/PBS for 10 min. Permeabilization was achieved by incubating in ice-cold cytoskeletal buffer (10 mM PIPES, pH 6.8; 300 mM sucrose; 100 mM NaCl; 3 mM MgCl.sub.2; 1 mM EGTA; 20 mM vanadyl ribonucleoside complex and 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride) containing 0.5% Triton X-100 for 10 min. FISH pre-hybridization treatments included incubating the coverslips in 0.1N HCl for 5 min at room temperature, followed by digestion with 0.01N HCl/0.002% pepsin for 5 min at 37.degree. C., stopped by 50 mM MgCl.sub.2/PBS and equilibrated in 50% formamide/2.times.SSC 2 hrs prior to hybridization. Five microliters of probe/hybridization buffer mix (Empire Genomics) was used per coverslip, with a hybridization program of 76.degree. C. for 3 min followed by overnight hybridization at 37.degree. C. in a humidified dark chamber. The coverslips were then washed with pre-warmed WS1 (0.4.times.SSC/0.3% NP-40) buffer to 72.degree. C. for 2 min, and then transferred to WS2 (2.times.SSC/0.1% NP-40) buffer at room temperature for 1 min. A final wash with 1.times.PBS was performed, excess liquid was aspirated and the coverslips were then mounted with prolong gold-DAPI antifade mounting reagent (Invitrogen).

[0505] One known interaction suitable for study was chosen: the interaction between two E.sub.2-regulated P2RY2 and STARD10-transcription units at a distance of .about.428 kb (Fullwood, M. J. et al., Nature. 2009. 462: 58-64). Knockdown of the P2RY2 enhancer by specific siRNAs caused downregulation of the two E.sub.r regulated target-coding genes, P2RY2 and STARD10, both of which are upregulated following E.sub.2 treatment. These two genomic loci exhibited E.sub.2-dependent enhanced colocalization when treated with E.sub.2, for 1 hr prior to fixation and examination using FISH. When cells were transfected with siRNAs against P2RY2e, the P2RY2e knockdown blocked the previously observed P2RY2:STARD10 co-localization. Therefore, together, these data suggest that ligand-dependent induction of eRNAs initiates processes, including enhancer: promoter looping, in concert with the requirement for the resultant coding gene activation observed after hormone treatment.

[0506] Additional data supporting the suggestion that eRNAs are required for tight control of ER.alpha.-regulated genes was provided by evidence of its role in the "switch" from association with corepressors to coactivators. The methylation status of Pc2 present on growth control gene regulatory regions regulates its association with two abundant ncRNAs, TUG1 and NEAT2, located primarily with markers of distinct subnuclear individual structures-polycomb bodies (PcGs) and interchromatin granules (ICGs), respectively (Yang, L. et al., Cell. 2011. 147: 773-88). Studies were conducted to test if similar interactions might be regulated by eRNAs induced by liganded ER.alpha.. ImmunoFISH was performed using antibodies against RING1a (H-110, Santa Cruz Biotechnology), and the interchromatin granule marker, SC35 (ab88720, Abcam), and with specific BAC probes for three genomic loci interrogated (NRIP1, FOXC1, and SIAH2). This revealed that there was a reproducible E.sub.2-dependent switch in the predominant colocalization of each transcription unit from RING1a- to SC35-stained structures. When LNA transfections were used to deplete eRNA transcripts for two genes, FOXC1 and NRIP1, immunoFISH data from each locus showed a clear inhibition of their ligand-dependent-enhanced association with SC35, directly implicating the eRNA transcripts in these altered target gene co-localization events. Similar effects were observed for siRNA-mediated knock-down of the GREB-1 eRNA.

[0507] While it is quite likely that a series of complexes combinatorially contribute to these interactions, several studies have established a role for Cohesin in enhancer: promoter looping events (Hadjur, S. et al., Nature. 2009. 460: 410-3; Mishiro, T. et al., EMBO J. 2009. 28: 1234-45; Nativio, R. et al., PLoS Genet. 2009. 5: e1000739; Hou, C. et al., Proc. Natl. Acad. Sci. USA 2010. 107: 3651-6), and protein: protein interactions between Cohesin and the mediator complex protein, Med12, have been reported (Kagey, M. H. et al., Nature. 2010. 467: 430-5). Therefore, the levels of Cohesin recruitment to regulated ER.alpha.-regulated enhancers were assessed after ligand addition. An increased occupancy was observed of the Cohesin subunit Rad21 on ER.alpha. target genes enhancers adjacent to up-regulated coding genes after E.sub.2 treatment, as studied by conventional ChIP and metanalysis of ChIP-seq data. Based on fractionation studies, Rad21 association with chromatin was diminished following RNaseA treatment. Depletion of specific NRIP1e eRNA by siRNA or LNA or FOXC1e eRNA by LNA, resulted in a decrease of Cohesin recruitment to enhancers in response to E.sub.2, with essentially no significant alteration of the H3K4me enhancer mark, or in ligand-dependent increase of ER.alpha. recruitment. Therefore, eRNAs may help to stabilize the ER.alpha.-dependent recruitment of Cohesin to the regulatory enhancers, perhaps by recruiting some common or related complexes that contribute to this event.

[0508] Possible interactions between the Cohesin complex and regulated eRNAs were explored using RNA immunoprecipitation (RIP) with an anti-Rad21-specific antibody (ab922, Abcam). RNA immunopreciptations were performed as described previously (Chen, J. et al., Mol. Endocrinol. 2006. 20: 1-13). Briefly, cells were washed in PBS, cross-linked by 1% paraformaldehyde for 10 min at room temperature, and 125 mM glycine was added to quench the cross-linking. The cell pellet was then lysed in Buffer A and then Buffer B. IP was performed overnight using 1-5 .mu.g antibody. Thirty microliters of Dynabeads were used per IP or in a beads-alone reaction. IP complexes were washed and de-cross-linked at 65.degree. C. DNase I treatment was given to get rid of genomic DNA. RNA was isolated and cDNA synthesis was performed.

[0509] This assay revealed that the eRNAs of FOXC1, PGR and TFF1, while highly divergent in primary sequence, exhibited interactions with Rad21 that were further enhanced after ligand treatment. siRNA-mediated depletion of Rad21 caused loss of enhancer: promoter interactions, both basal and E.sub.2-induced, when assessed by 3C assay for the NRIP1 and GREB1 gene loci. In addition, these data suggest that eRNAs might be required for effective additional recruitment of the Cohesin complex to the enhancer in response to E.sub.2, and their subsequent role in stabilizing enhancer: promoter interactions. This implies that the specific eRNA sequences participate in coding gene activation events.

[0510] The effects of E.sub.2-dependent coding gene stimulation in MCF-7 cells treated with SMC3 (Cohesin subunit) siRNA (chosen for its more effective knock-down compared to Rad21 siRNA) vs. control siRNA was also assessed. Inhibition of most of the E.sub.2-induced coding gene transcriptional program, with loss of 50% of coding gene activation program by E.sub.2, was observed with significant inactivation for the remaining E.sub.2-activated transcription units. Using SMC3-specific siRNAs, inhibition of ER.alpha. target genes was confirmed by Q-PCR. Indeed, analysis of GRO-Seq data and MA analyses further confirmed the broad inhibition of E.sub.2-dependent activating transcriptional effects following SMC3 knockdown in MCF-7 cells.

Example 13: Design of Antisense Oligonucleotides Targeting Murine Mmp9 eRNA

[0511] Antisense oligonucleotides were designed targeting a Mmp9 eRNA. The newly designed oligonucleotides in Table 24 were designed as uniform deoxy oligonucleotides with phosphate backbones, or as oligonucleotides containing deoxy, MOE and (S)-cEt units and a phosphorothioate backbone. "Start site" indicates the 5'-most nucleoside to which the oligonucleotide is targeted in the murine eRNA sequence. "Stop site" indicates the 3'-most nucleoside to which the oligonucleotide is targeted murine eRNA sequence. Each gapmer listed in Table 24 is targeted to SEQ ID NO: 202 (GENBANK Accession number NT_039207.7 truncated from 105809967 to 105810417). The Chemistry column describes the chemistry of each oligonucleotide, where `d` indicates 2' deoxyribose; `o` indicates phosphate ester; `e` indicates 2'-O-methyoxyethyl ribose (MOE); `s` indicates thioate ester; `m` indicates 5' methyl group; `k` indicates (S)-cEt. The SEQ ID NO for each antisense oligonucleotide is associated with its sequence and is not intended to require the chemistry indicated in Table 24.

TABLE-US-00025 TABLE 24 Antisense oligonucleotides targeted targeted to SEQ ID NO: 202 (GENBANK Accession number NT_039207.7 truncated from 105809967 to 105810417). SEQ ISIS Start Stop ID No Site Site Sequence Chemistry Notation NO 566188 14 29 TCCATTGCCACCCCCA Tes mCes mCks Ads Tds Tds Gds mCds 124 mCds Ads mCds mCds mCds mCks mCks Ae 566189 17 32 TCCTCCATTGCCACCC Tes mCes mCks Tds mCds mCds Ads 125 Tds Tds Gds mCds mCds Ads mCks mCks mCe 566190 20 35 ACTTCCTCCATTGCCA Aes mCes Tks Tds mCds mCds Tds 126 mCds mCds Ads Tds Tds Gds mCks mCks Ae 566191 23 38 CCAACTTCCTCCATTG mCes mCes Aks Ads mCds Tds Tds 127 mCds mCds Tds mCds mCds Ads Tks Tks Ge 566192 41 56 ACACTTCTCTCCCTAC Aes mCes Aks mCds Tds Tds mCds Tds 128 mCds Tds mCds mCds mCds Tks Aks mCe 566193 65 80 GGCTTGGAATCCATCA Ges Ges mCks Tds Tds Gds Gds Ads 129 Ads Tds mCds mCds Ads Tks mCks Ae 566194 91 106 CAACTGCGGAGGGAGG mCes Aes Aks mCds Tds Gds mCds Gds 130 Gds Ads Gds Gds Gds Aks Gks Ge 566195 94 109 TACCAACTGCGGAGGG Tes Aes mCks mCds Ads Ads mCds Tds 131 Gds mCds Gds Gds Ads Gks Gks Ge 566196 97 112 TCTTACCAACTGCGGA Tes mCes Tks Tds Ads mCds mCds Ads 132 Ads mCds Tds Gds mCds Gks Gks Ae 566197 100 115 CTTTCTTACCAACTGC mCes Tes Tks Tds mCds Tds Tds Ads 133 mCds mCds Ads Ads mCds Tks Gks mCe 566198 103 118 GTTCTTTCTTACCAAC Ges Tes Tks mCds Tds Tds Tds mCds 134 Tds Tds Ads mCds mCds Aks Aks mCe 566199 114 129 GATTGCTGCTGGTTCT Ges Aes Tks Tds Gds mCds Tds Gds 135 mCds Tds Gds Gds Tds Tks mCks Te 566200 117 132 CAGGATTGCTGCTGGT mCes Aes Gks Gds Ads Tds Tds Gds 136 mCds Tds Gds mCds Tds Gks Gks Te 566201 120 135 CTTCAGGATTGCTGCT mCes Tes Tks mCds Ads Gds Gds Ads 137 Tds Tds Gds mCds Tds Gks mCks Te 566202 123 138 CGCCTTCAGGATTGCT mCes Ges mCks mCds Tds Tds mCds 138 Ads Gds Gds Ads Tds Tds Gks mCks Te 562914 126 147 CACTTCACTCGCCTTCAGGATT Cdo Ado Cdo Tdo Tdo Cdo Ado Cdo 139 Tdo Cdo Gdo Cdo Cdo Tdo Tdo Cdo Ado Gdo Gdo Ado Tdo Td 566203 126 141 ACTCGCCTTCAGGATT Aes mCes Tks mCds Gds mCds mCds 140 Tds Tds mCds Ads Gds Gds Aks Tks Te 566204 129 144 TTCACTCGCCTTCAGG Tes Tes mCks Ads mCds Tds mCds Gds 141 mCds mCds Tds Tds mCds Aks Gks Ge 566205 132 147 CACTTCACTCGCCTTC mCes Aes mCks Tds Tds mCds Ads 142 mCds Tds mCds Gds mCds mCds Tks Tks mCe 566206 135 150 TACCACTTCACTCGCC Tes Aes mCks mCds Ads mCds Tds Tds 143 mCds Ads mCds Tds mCds Gks mCks mCe 566207 138 153 GGGTACCACTTCACTC Ges Ges Gks Tds Ads mCds mCds Ads 144 mCds Tds Tds mCds Ads mCks Tks mCe 566208 141 156 AGTGGGTACCACTTCA Aes Ges Tks Gds Gds Gds Tds Ads 145 mCds mCds Ads mCds Tds Tks mCks Ae 566209 144 159 AGCAGTGGGTACCACT Aes Ges mCks Ads Gds Tds Gds Gds 146 Gds Tds Ads mCds mCds Aks mCks Te 566210 147 162 GTAAGCAGTGGGTACC Ges Tes Aks Ads Gds mCds Ads Gds 147 Tds Gds Gds Gds Tds Aks mCks mCe 566211 152 167 AGTGGGTAAGCAGTGG Aes Ges Tks Gds Gds Gds Tds Ads Ads 148 Gds mCds Ads Gds Tks Gks Ge 566212 155 170 AACAGTGGGTAAGCAG Aes Aes mCks Ads Gds Tds Gds Gds 149 Gds Tds Ads Ads Gds mCks Aks Ge 566213 159 174 CTGGAACAGTGGGTAA mCes Tes Gks Gds Ads Ads mCds Ads 150 Gds Tds Gds Gds Gds Tks Aks Ae 566214 162 177 TGCCTGGAACAGTGGG Tes Ges mCks mCds Tds Gds Gds Ads 151 Ads mCds Ads Gds Tds Gks Gks Ge 566215 165 180 AGGTGCCTGGAACAGT Aes Ges Gks Tds Gds mCds mCds Tds 152 Gds Gds Ads Ads mCds Aks Gks Te 566216 171 186 TTACAGAGGTGCCTGG Tes Tes Aks mCds Ads Gds Ads Gds 153 Gds Tds Gds mCds mCds Tks Gks Ge 566217 174 189 CTGTTACAGAGGTGCC mCes Tes Gks Tds Tds Ads mCds Ads 154 Gds Ads Gds Gds Tds Gks mCks mCe 566218 177 192 GGCCTGTTACAGAGGT Ges Ges mCks mCds Tds Gds Tds Tds 155 Ads mCds Ads Gds Ads Gks Gks Te 566219 180 195 GTGGGCCTGTTACAGA Ges Tes Gks Gds Gds mCds mCds Tds 156 Gds Tds Tds Ads mCds Aks Gks Ae 566220 183 198 CATGTGGGCCTGTTAC mCes Aes Tks Gds Tds Gds Gds Gds 157 mCds mCds Tds Gds Tds Tks Aks mCe 566221 186 201 TCCCATGTGGGCCTGT Tes mCes mCks mCds Ads Tds Gds Tds 158 Gds Gds Gds mCds mCds Tks Gks Te 566222 189 204 GTTTCCCATGTGGGCC Ges Tes Tks Tds mCds mCds mCds Ads 159 Tds Gds Tds Gds Gds Gks mCks mCe 566223 192 207 AGTGTTTCCCATGTGG Aes Ges Tks Gds Tds Tds Tds mCds 160 mCds mCds Ads Tds Gds Tks Gks Ge 566224 201 216 GCTAATAACAGTGTTT Ges mCes Tks Ads Ads Tds Ads Ads 161 mCds Ads Gds Tds Gds Tks Tks Te 566225 204 219 AGTGCTAATAACAGTG Aes Ges Tks Gds mCds Tds Ads Ads 162 Tds Ads Ads mCds Ads Gks Tks Ge 566226 207 222 ACAAGTGCTAATAACA Aes mCes Aks Ads Gds Tds Gds mCds 163 Tds Ads Ads Tds Ads Aks mCks Ae 562911 218 241 CATTTCCCCCATCTTAAAAACAAG Cdo Ado Tdo Tdo Tdo Cdo Cdo Cdo 164 Cdo Cdo Ado Tdo Cdo Tdo Tdo Ado Ado Ado Ado Ado Cdo Ado Ado Gd 566227 224 239 TTTCCCCCATCTTAAA Tes Tes Tks mCds mCds mCds mCds 165 mCds Ads Tds mCds Tds Tds Aks Aks Ae 566228 231 246 CCTACCATTTCCCCCA mCes mCes Tks Ads mCds mCds Ads 166 Tds Tds Tds mCds mCds mCds mCks mCks Ae 566229 234 249 CAACCTACCATTTCCC mCes Aes Aks mCds mCds Tds Ads 167 mCds mCds Ads Tds Tds Tds mCks mCks mCe 566230 237 252 ACACAACCTACCATTT Aes mCes Aks mCds Ads Ads mCds 168 mCds Tds Ads mCds mCds Ads Tks Tks Te 566231 240 255 TCGACACAACCTACCA Tes mCes Gks Ads mCds Ads mCds Ads 169 Ads mCds mCds Tds Ads mCks mCks Ae 566232 243 258 CTATCGACACAACCTA mCes Tes Aks Tds mCds Gds Ads mCds 170 Ads mCds Ads Ads mCds mCks Tks Ae 566233 246 261 CAGCTATCGACACAAC mCes Aes Gks mCds Tds Ads Tds mCds 171 Gds Ads mCds Ads mCds Aks Aks mCe 566234 249 264 CCCCAGCTATCGACAC mCes mCes mCks mCds Ads Gds mCds 172 Tds Ads Tds mCds Gds Ads mCks Aks mCe 566235 252 267 TGACCCCAGCTATCGA Tes Ges Aks mCds mCds mCds mCds 173 Ads Gds mCds Tds Ads Tds mCks Gks Ae 566236 255 270 GTGTGACCCCAGCTAT Ges Tes Gks Tds Gds Ads mCds mCds 174 mCds mCds Ads Gds mCds Tks Aks Te 566237 258 273 ATTGTGTGACCCCAGC Aes Tes Tks Gds Tds Gds Tds Gds Ads 2 mCds mCds mCds mCds Aks Gks mCe 566238 261 276 CTCATTGTGTGACCCC mCes Tes mCks Ads Tds Tds Gds Tds 175 Gds Tds Gds Ads mCds mCks mCks mCe 566239 264 279 CAGCTCATTGTGTGAC mCes Aes Gks mCds Tds mCds Ads Tds 176 Tds Gds Tds Gds Tds Gks Aks mCe 566240 268 283 GCTTCAGCTCATTGTG Ges mCes Tks Tds mCds Ads Gds mCds 177 Tds mCds Ads Tds Tds Gks Tks Ge 566241 271 286 CAAGCTTCAGCTCATT mCes Aes Aks Gds mCds Tds Tds mCds 3 Ads Gds mCds Tds mCds Aks Tks Te 566242 274 289 ATCCAAGCTTCAGCTC Aes Tes mCks mCds Ads Ads Gds mCds 178 Tds Tds mCds Ads Gds mCks Tks mCe 566243 280 295 GCCGAAATCCAAGCTT Ges mCes mCks Gds Ads Ads Ads Tds 179 mCds mCds Ads Ads Gds mCks Tks Te 566244 283 298 ACTGCCGAAATCCAAG Aes mCes Tks Gds mCds mCds Gds Ads 180 Ads Ads Tds mCds mCds Aks Aks Ge 566245 286 301 TGGACTGCCGAAATCC Tes Ges Gks Ads mCds Tds Gds mCds 181 mCds Gds Ads Ads Ads Tks mCks mCe 566246 289 304 GATTGGACTGCCGAAA Ges Aes Tks Tds Gds Gds Ads mCds 182 Tds Gds mCds mCds Gds Aks Aks Ae 566247 292 307 TGGGATTGGACTGCCG Tes Ges Gks Gds Ads Tds Tds Gds Gds 183 Ads mCds Tds Gds mCks mCks Ge 566248 295 310 CTCTGGGATTGGACTG mCes Tes mCks Tds Gds Gds Gds Ads 184 Tds Tds Gds Gds Ads mCks Tks Ge 566249 298 313 CCACTCTGGGATTGGA mCes mCes Aks mCds Tds mCds Tds 185 Gds Gds Gds Ads Tds Tds Gks Gks Ae 566250 302 317 GCTCCCACTCTGGGAT Ges mCes Tks mCds mCds mCds Ads mCds Tds mCds Tds Gds Gds Gks Aks 186 Te 566251 319 334 TGAGTAGGCCAATGGG Tes Ges Aks Gds Tds Ads Gds Gds 187 mCds mCds Ads Ads Tds Gks Gks Ge 566252 322 337 GAGTGAGTAGGCCAAT Ges Aes Gks Tds Gds Ads Gds Tds Ads 188 Gds Gds mCds mCds Aks Aks Te 566253 325 340 GCAGAGTGAGTAGGCC Ges mCes Aks Gds Ads Gds Tds Gds 189 Ads Gds Tds Ads Gds Gks mCks mCe 566254 343 358 GAGGCCAGGACTTGGC Ges Aes Gks Gds mCds mCds Ads Gds 190 Gds Ads mCds Tds Tds Gks Gks mCe 566255 353 368 AAGTTCAGCAGAGGCC Aes Aes Gks Tds Tds mCds Ads Gds 191 mCds Ads Gds Ads Gds Gks mCks mCe 566256 356 371 AACAAGTTCAGCAGAG Aes Aes mCks Ads Ads Gds Tds Tds 192 mCds Ads Gds mCds Ads Gks Aks Ge 566257 365 380 AGATGTGGAAACAAGT Aes Ges Aks Tds Gds Tds Gds Gds Ads 193 Ads Ads mCds Ads Aks Gks Te 566258 387 402 ATTCTAATTTCCTTCG Aes Tes Tks mCds Tds Ads Ads Tds Tds 194 Tds mCds mCds Tds Tks mCks Ge

566259 392 407 CATCTATTCTAATTTC mCes Aes Tks mCds Tds Ads Tds Tds 195 mCds Tds Ads Ads Tds Tks Tks mCe 566260 395 410 CCCCATCTATTCTAAT mCes mCes mCks mCds Ads Tds mCds 196 Tds Ads Tds Tds mCds Tds Aks Aks Te 566261 398 413 TGTCCCCATCTATTCT Tes Ges Tks mCds mCds mCds mCds 197 Ads Tds mCds Tds Ads Tds Tks mCks Te 566262 401 416 AGGTGTCCCCATCTAT Aes Ges Gks Tds Gds Tds mCds mCds 198 mCds mCds Ads Tds mCds Tks Aks Te 566263 404 419 TGGAGGTGTCCCCATC Tes Ges Gks Ads Gds Gds Tds Gds Tds 199 mCds mCds mCds mCds Aks Tks mCe 566264 410 425 CACACATGGAGGTGTC mCes Aes mCks Ads mCds Ads Tds Gds 200 Gds Ads Gds Gds Tds Gks Tks mCe 566265 432 447 GAGTCAACAGAAATAC Ges Aes Gks Tds mCds Ads Ads mCds 201 Ads Gds Ads Ads Ads Tks Aks mCe

Sequence CWU 1

1

2031338DNAMus musculus 1tgtgggggtg gcaatggagg aagttggggg tagggagaga agtgttcttg ctttgatgga 60ttccaagccc tgggttctcc ctccctccgc agttggtaag aaagaaccag cagcaatcct 120gaaggcgagt gaagtggtac ccactgctta cccactgttc caggcacctc tgtaacaggc 180ccacatggga aacactgtta ttagcacttg tttttaagat gggggaaatg gtaggttgtg 240tcgatagctg gggtcacaca atgagctgaa gcttggattt cggcagtcca atcccagagt 300gggagccccc attggcctac tcactctgcc tgccaagt 338216DNAArtificial SequenceSynthetic oligonucleotide 2attgtgtgac cccagc 16316DNAArtificial SequenceSynthetic oligonucleotide 3caagcttcag ctcatt 16427001DNAMus musculus 4tccttccttc cttccttcct tccttccttt cttccttcct tcccacctgt tctctctctc 60tctctctctc tctctctctc tgttattgtt cttttggttt tgttttgggg tttttttggt 120ttttttggtt ttttggtttt ttggtttttt ttttttttgg tttttcaaga cagggcttct 180ctgtatatcc ctggctgtcc tggaactcac tctgtagacc aggctggcct cgaactcaga 240aatccgcctg cctctgcctc ccaagtgcta ggattaaagg tgtgtgccac cacacccagc 300tttttgtttc attcttttga gacacagttt ctctgtgatt ctgtgtagct ttgactgtcc 360tggaactcac tatgtacaca ggctagcctg gagctcagag gtccacccgt ctctgcctcc 420taagtgctgg gatcacaggc gggtgccacc acctcccatc ctaagtacat tcgtttcttg 480tttttaaatt tattttttac actccatatt ctatccccca ccccaccccc atgcaccctc 540cgactgctct acatcccaca cctccacccc accccacccc gcttccaagt ggatgttccc 600aacccccaag cccacctgac ctaatctccc tggggcctcc agtctcttga gggttagatg 660catcatctct tactaaagta cgttctcaaa tgcataaatg gttgttcttt aggaagccat 720gggaaatcct cactttgtca atagcaaatc tagagatgta cattgggaat ctgactggtc 780ccaggtcctc tgggccctga gttcacttct gctccctaat gtgtggcaca cctgagaatg 840tgcagctcat aagctgatgg gtggagccca gatgacaccc cggcccgcag gtgcacaggg 900cagacttaga ttgtctggtt ctcagctctg gctctttgac ctgctgccca gcctgtctca 960gcccaggcct caggcagcag ttcacctcat tgcctcaaga gggagctcca aggttgccaa 1020gataggaaat tgttggaagc gggtgactga ccaggccagc cttttcctgt ccccagctgc 1080agctgccccc ttggagaagc agtggtggcc acttactctg cctgtgggcc ctgagggcac 1140ctgctctggg ctctagaacc ttacagaagc caccttgttt ccaaggccag atgttgcaca 1200tgtaaatctc tgatgtgagt ggcaggtagc ctcaagttgt ccctttaact tccttcagct 1260tcagtctcct cacctgaaag gaaagactga aaattcaaac acaggtgcca accagggaga 1320aaggaggaga ggcagggagg gtgggggatg ggatgggaac ctgagtaaga tacaaagata 1380caacaaaaac tccaaaccgt tcttctgagt gggcaaaccg ccatacacgt aacctcagca 1440cttgggaggt ggaaacggga ggatcaggag tcaaggccat cctcagctcc atactgagct 1500cctggataac ctgataaatt aacatctgac tttatatctc aatctccagg gctactgtga 1560taactgtgac aatctggggc ctttgttttg tttctgatga ttttggagcc tggaagacca 1620agctccaagt gtcagtggat tcgttctgag gattcaatct gtgggttgca ggcagccatg 1680gctcactgtg tcctctgtgt ccccgtcccc cccccccgcc cccaggttgg tctcaaactt 1740ggatgagctt ggagtcctcg tcctccacct ctgagtgctg ggatggtagt tgtgcctcgc 1800ctcactggct tgcaggtgca ggagatcaac ccagagcaag cactgtactt accaagccac 1860accccaagtc atcattttca gaggagaccc tcacatgtga gtgaaggcta agtgtagctt 1920gggagagagg gtggactgtg aggggggcag ggggggcagc ctttggaaca ccagcagctc 1980tgtccttggt ctggtgccgt ttccctgttc tggtaaattc tgacttcact actgcttcct 2040aacatagggc acaactgaga acataacaga tgcccaacct ggaccatttc cccttattta 2100tctacgtaaa actgagtctc ggtacattcg agttgcagga tccaccttag cctctgggta 2160gtggggagag ggcacacagc tgggtgctcc cccattagaa tttaagatga tttttttttc 2220ctagtaaaca gagctttcag aaaagcagag agagaagcct gtctgacagg gttgggtggt 2280aatggggatg ggctgcagag aatgcggaga gttctaaagg accctggatg tcagcaaagc 2340ccagggaagg gggcagctct acttggctcc ctgacagtga agcatgttct ctgccacctg 2400gtccctctcc tggagccgtc cctgcaaaag tgaagcctgg atccctaggc agacattgct 2460ccttcttctg tgagccccca gctttttaat ctcttactta tcctgcctgg gtagaaggtg 2520tttttgccac tgctacaact tacaccaacc tggcctcaac tatgtcgggg ttgttgctgc 2580tgtttcgaga taagacctcc ctctgtagct caggctggcc tcctagtcgc agcaatcctc 2640tggcctccaa gtcgcagcaa tcctctggcc tccaagtcac agccgtcctc tggcctcggc 2700ctcccaagtg ctgggatccc agggatgagt catatcaccc tccaaggggg cagtgattag 2760tggtggtgat caataattat taaatagtta agggctgtgg tggtaggaat ggaaatggct 2820atcccacagc tcatgtgttg gttaactgtt tggaaagaat tggtgggtgt ggccttgttg 2880gagggggtgt gtcactgggg atttaaaaaa ccattcccag tgtgtctctc tgcttccttc 2940ttttggatca agatgaggat tctcagctgt tccttctgcc gtgcctttac tccaccacca 3000tgggccctaa ctctctgaag ccataagctc aacatttcct tcttttataa gttgccttgg 3060gtttagctga gtatcacagc gtgttgtcct gggagcttgt cctggggtaa cagaagagct 3120cagaagttct agcttggagg gctgaggtga gaacttagat ctagaatagg ctggccacag 3180agcaagagcc tgtctcagaa gcaagggaaa ccttaggcaa catctgatgg tttattttgg 3240aataaaaaag aacttttgct tccatttgtt ttgtttattt tgggtttttt tttggggggg 3300ggttcttcat tatttagaga atactttatt agtttttgta atcaaaccca cgtagataag 3360accttacata tttaacacag tgcattgccc ctgtacaaat ggggaaaaaa ttaagtccaa 3420catttctaga ccaatatggc tgtccatttc tgttcaatgc caactcaaca cagtaaactg 3480gatacttttt tccaaagttg acagcacttt tgcttccatt tgtaatgcac cgtagttcag 3540ccacctccaa atgctactgc ttagaaatac tttaattggg agctcggtag ttaaaagcac 3600tggctgctct ggaagagggc ctggatttga ttcccgcccc actatgtgac tcacaaccct 3660agtctcaggg gacctgggat gccctcttct ggcctctttg ggtccaagca tgcatgtggc 3720agtggccata cagacaaaca cttatatcca taaaataaca aaaataggtc tcgaaagaaa 3780tagtttaata ggaggctgga aagatggctc agtggttagg agtgctcact gccttctaga 3840ggtcagtgtg ggtcccagca cccacgtcag atggctcaca cccacatata gctccagagc 3900cagggatcag tgctctattc tggacttcat ttatgctata tagactgtac acacacacac 3960acatgcacac acatacatgc ccacacacac ttgcacacac acatgtgcat acatacatgc 4020ccacacatac atgcacacac acttgtgcac acatacatgc ccacacacac ttgcacacat 4080acatacccac acatacatac ccacacacac ttgcacacac atgcacagat ggatgaggta 4140cagtctgtgg ccttcaggtc catagagcag gcttgctgcc tcctcaccct gaaatgggat 4200gtcctgcaag ctcaggcgag ctcagtgccc tcggtgtgtg cagccgtcca tctgggaaca 4260aggataccag cgtgagttac tgacactcgt cacagagctc acctgctgcg gcatccatga 4320gcaggacagg ctggcttgtc agcccacagc aggtgaggca gggaggaaag gcatgcacag 4380gccaggggat gtgtgtgctg gagaagagga agcggataga atgcagtggg aagtaaaaag 4440aagccatggt gctgagaaca tgctcaccag acagaagaga ggagcctccg agagtgctaa 4500gtctgtgatg ctacgactcg gggtcctctc tggcacatgg gtagatggcc ggatggtgag 4560atgaataaga tggatgggaa gggagtggtg ggcacagggt accctcaggc cccagctgaa 4620ggtggagttg tgtgggaaac agacacggat gagattggtt gacagcaaca gtaggagatt 4680ctcagccaga ttccagacag gaacacgagg aagagcttga atgccctggg gatacagctc 4740agatggcaaa atgttagact agcctgagtt ccatccccag cccctcataa acggtggagc 4800atcccagact tgggcagtgg aggcaggagg atgaaaggct caaggtcttt ttaaccccat 4860agctatttaa agtccagccc aggatgtgtg ggacctatct caacaacaaa acaaacaaaa 4920acaaagccag ggggctgaag aaatggctcg gtggctaaga gcacacactg ctcttgcaga 4980ggacacaagt tcgactccca gcacctatag tgatgggcaa ctcacgactg ccacagggag 5040atctgagttc tctagcctcc ctgggcacat atacataatt aaaaagaaag ctcgaaaaca 5100aaaagtttag cagaaatgaa taaggacacg gagtcggtga gctggaatag gatgggggtg 5160gttctgggag gagaggggta gaggagggat tgtgatcgaa atacatgtga agttcccaag 5220gaattcatac aaataaaagc caaaaaaaca agagctgaag tggggacagg atcggggtga 5280ggagctgagc cccaggggaa gctgagtagc ccctgactta gctgctgggg caacgggaaa 5340gggccaatgt ctgcgagcct gtgaggatct gctggctctc tgtgaatctc tcctgcaggg 5400aaagaagaac agacagggag gccagaagga aggagaaagg gaactaaccg aggcttcctc 5460aaatgtgaac agctggagag caactggagc ttttagttcc tacctcactg gacaaagcag 5520gctcctgaga actcccaaga gttgtgatat ggtcagtggt cagcgaacct gggcccagtg 5580ctgcacaggg ggtaccagag gcttcctaag aacactggaa agcagagggg ccctcacagc 5640ttgcctgaga cccctattaa ggagagcttg agacctgact tgtgtgctgc tggactccga 5700ggctacagac tgccatctca ctaggaataa ataatatcca tccaccccca ggtctgccaa 5760cccacagccc cctgtaaacc ttaggtctca gtaattccta ggtcagccac cacacagtct 5820ccagcaatcc ccaggtctgc cacccaacag tccccagtag tccccagcag tccccagcaa 5880tctccaggtt tgccccagtc tcaagtcccc tagatttgaa atgcaggctc tcttgcggtc 5940agctggttcc cagcctggag cgttgagccc gttgagcttg cttgtcacag catctaaaga 6000tagggtacca cacaggaaac cagatgctat tggctaactt cgaaaataaa gatgccctca 6060tagagaggga agcacttgcc tctggtggag tctgcgtgag actgggtgag tgactggcac 6120ttcctgcaga agttcccttc ccatctgctc aggtaagtac cacggactcc agactcctcc 6180catacctggg cgtggggtca atcaggcaca gtcacagggt ttctgatgct gtttgccttc 6240cgtgtctatt ttcctgagga tactgcagat aaatcaggag agtaaaggac cttttgcacg 6300ctcgtaactt gaggagttag taattacatt ttagggatgt gaaaaaaaag gtttatatta 6360gatgttacca aaaaaggcct tgatataaaa tcacatctgg gtaattggaa atattgatat 6420tcttaaagat cacaattcag attgccagtc agactttttt gatccatgcc tctaatccca 6480gcacttggga aggtgaagca ggaggatcga tacaagtttg agaccagcct aggctagcta 6540atgagctcta ggccagttta ggcttctgtg tgagaccgcc ttaaacaaac agcaccaacc 6600ataaaaacta gtgattgtgg ttttcatttc tttttaaaag gagcttttat cttcagagac 6660tcatacttga atgtctatag aaacagaata atctaattca gccatgaaat aggacataga 6720tgaaatatta actgggagtt gataattcct ggagctgagt ggcgggtgcc aggtgtttcc 6780tttgatttcc cccccccccc ccccggtttt ttgagacagg gtttttctgt gtaactgtga 6840ctatcctgga actcattctg tagaccaggc ttgccttgaa cttatagaga tccacctgcc 6900tctgccccct cagtactggg attaaaggcg tgcgcctcca ttgcctggct cccctgattg 6960tatataggct tgtgaatata aagagaataa tgttatataa atagagttgc ttgcctagca 7020cacaaggagc cctgggccct gggcacaatc ttcagcatct aataaaattg gacttggtgg 7080cacacactgt gatcacagca ccctggaggt aaaaggcggg aggtcggaag tgcaagactc 7140ttctcagtga agcagaattt tgaggccagc ctggacttta tgggatcctg cctctaaaaa 7200caacaataat gatgatggtg acggtggtgg tgatgacggt ggtggtggta gtggtggtgc 7260tggtggtggt ggtggtggtg gtggtggtgg tggtgataaa tgttcaaaac gatgttagca 7320tttttactat aaatataaat ttatgtgtca aaaaagctaa gagtgctatg ctaaacctgc 7380agagttggat agttttaagg attcctcttc ccctccccct cttttaaatg cttttttggt 7440accatcttca atagtcataa tgaacattta ttacatttat aataaacttg tcacaaactc 7500attcttcagt tttataaatg ctcagaacca tggctcagac acttgggcac agacatctga 7560gctaggagtc gtggtcttag ggaggaaacg ataagaacag gccaggaggg ctggagaggt 7620ggctcagtgg ttaagagcac cgactgctct tccgaaggtc ctgagttcaa atcccagcag 7680ccacatgatg gctcacgacc atccgtaatg agatccgacg ccctcttctg gtgtgtctga 7740agacagctac agtgtactta gatataataa ataaatcttt aaaaaaatat ttaaaaaaaa 7800aaaacgaaca ggccaggagg agatacaccc ctgcttgaca cagacaagcc cctattcaga 7860ggaccctgca gcctcgctca ctctgtgcct gatcatttat aaccatgtgc gagaaaagct 7920gggaacttgg gattctcctg ccttctgtct ttccaattct gccatttttt ttcttttaat 7980tttttcccaa ctgcttgaaa tacaatactt gaggtaggaa gaaattcagt caaatgtgaa 8040gatacgcagg tcataagaag accctgttca ggcccaggtc ataagagggc cccagcctac 8100tgagaggaga caagctcaga gtgagacctg gcctgctgag ccttgggaac ctatgagggt 8160cagcagggta ggtgcagtgg ctgaactgga atggggtttt cctggacaac agaaatggcc 8220ccaggaagct gttcggtggc ttttcccaga gaaatgtgaa ccaagttatc caccaacgct 8280atgactcctg gggcaagcag gaacgacccc acacccaagt ttaggctgga gaatcagcaa 8340gtcttttggg gttactcata gcaacatgcc ctagggttag tcacagctta tctcagtggg 8400gtgcccactc atgagagctg tgttcctggg actccttagc ctcctgagag ggaaccttgt 8460aagttagctg aatcctgtga gcctctatcc cctttcctgg aggccatcgt ggcttcttta 8520ccctctatcc tgttcacctt tgtttttatc aaaggatgct ggagcagaaa atagtcccag 8580cccagttcag tctggctagc ctggcccttc acacacccct ttttttctgg gaagaagaac 8640ccgaccaatg ctaatctctt tgaaatatat cctccgccct ttgcttctgc cgaccccatt 8700gcacagggag ggctggaggg tggtgggaga tgctgttcaa cttagggaca cagtacacac 8760tgcccctgcc aacacttctg gcaaagtcag cttcctgctt ttcctatggc agtaggcttc 8820ggagtgcacc gttagagtgt ggtatgcaaa gtcccaaaca cacattggac gtctttagtg 8880cctaaaacaa tacgggccag caaagtggat tcaggcacct gttgctgatg tgttagaatg 8940gcggtcgcat ctcagcagca ctctgggcca aggcagttcc acgtggaaga ggttttattg 9000gggaaagaga gagaaggtgg ctgaataaac agaacaggca aggagagaga gtgcgagcag 9060tgttctcctt atttatatgg agaatgacat aacgcaggta aaggtggaag gtgagccaag 9120tggatgttgg gaatatggtg cctgttgcct tggcaacctg tctgcaggtc gcaagtcatg 9180ctgttgccta tgtgatgtcc tgatgccaac agttgccaag cttgaccacc tgagtttgat 9240ccctgggaca tgcgcagtag aaggacagaa ctgactccct caagatgtaa tctgaccacg 9300tgttgtggca tgtgcgaaag tgtgtgtggg gaggagcgtg ggcccccccc cacacacaca 9360cacacacaaa tgatcaaggt gtactctaac ctgcatcgtg gctagtccaa gtgtgtgcac 9420acatacatat aaataaatgt aattttaaaa accatgcatg tctattactt cagggaaagt 9480taagctctgt gaccttaggg ctttaaaatg acagcctgct ttcagaggtt actaaggatc 9540ccaggtcgtg ggaacgggag cgaagaaggc atgctagggc tgatccagat caaaaccaac 9600tctagttcct tcttctcccg agccagctgt gcaggggtgg ccatgtcagg catggcctgg 9660ggactgcatg agctgcaggg ggggaactca ggctgttgcg ggggggagga gggggtcggg 9720gtggggagac cctcgctgac tttgatgagc cacagctcag gagctctcag aagctgggac 9780cttgtgtgac tcaggtcatt gcctgccaag gaaaaccaaa ggaaggctaa ggcttgtgac 9840caaatctgtg gtctgccttt cctcctggag gttcagggca aactgccctg agggtggaat 9900ctccctgaac tctgaatgga gagcgcttta accaactcat ctggtcaaat tgttttgaat 9960tatgagtatt aatagaaaaa agcaaatctg tattgctttt ccttaaatgc ctttccagaa 10020cattgctaag ctaaacctac tgactatgta gcacagtacg ctgagagaca tgaaaggtca 10080tcagcaatat aggccaggct attggagaga ggcaggagga agtcagtgag tttgaggcca 10140tcttggagac ccaggacaac ataaagagac cctgtctcaa caacaacaac aaaaaacttc 10200attatataaa accttgcaaa cagttttttt aaatctcaca tcagaattaa tcagctccaa 10260tgtcaatgca ccataatgaa cgtatagttt gaattggcat ttgaatttcc tccaggctct 10320ctggggtgga gtctgcagct ctcctgtccc cctcttctgc tacacagcca gcttggctct 10380ctgtaaacca ggctgcccct gaaatctccc aaacaagtag gctcttcccc acccccaccc 10440ccacatcccc tcttgtctgc tgttttgggt actttctctc acatcctctc tacttgacca 10500ggaaacccca gactcagccc ctctgagcct acctcacctc attccctgat acctccctct 10560gtctgcaagt ttcctcccac ctttaaaagt ttcttttaaa atatttattt atttttagtt 10620tacatgcatt gatgttttgt ctgcatgtat gtctgtgtga gggtgtcaga tcccctggaa 10680ctggagtcac agactgtcgt gagctgccat gtgggtgctg ggaattgaac ccaggtcttc 10740tgggagtgct agtagccagt gctcttagtt gctgagccat ctctccagac ctaaaggtga 10800ctttttatga caccctttta tgcccctcct tcagtcctaa aggagacaga aatagagacc 10860caagcctgag tctattattt aaccttccct gtatttggtg ataaacattt atagagctgc 10920ttctgtgttt ctttctcagc aggttcttgt acagcactca tccagtacag tacttgtctt 10980agttagggtt ttactgctgt gaacagacac catgaccaag gcaagtctta taaaaaacaa 11040catttaactg gggctgcctt acaggttcag aggttcagtc cattatcatc aaggtgggag 11100catggcagta tccaggcagg catggtgcag gcagagctga gagttctaca tcttcatcca 11160aaggctgcta gtggaagact gacttccagg caactagtgt gaggatctta tacccacacc 11220cacagtgaca cacccattcc aactaggtca cacctattcc aacaaagcca caccttcaga 11280tggtgccact ccctggtcca aggatataca aaccatcaca ctccactccc tggtccccac 11340agacttgttc aaaaacatga gtctatgggg gccatactta aacatagcat aatgcaaaat 11400gcatttagtc caactttcaa agtcctcata gtctatagca gtcgcaacaa tgttaaaagt 11460ccaaagttca agggctcttc tgagattcat ccaactaatt aactgtaatc cccaaagcaa 11520ggcaggaaac cagctgtgca aactccaaac tctgcatctc catggctgat gtcaaagcgg 11580tcttcagatc tcctctcctt tttcatcttt gttgactgca acaaactgct ttctcctggg 11640ctggttccac tccctgttag cagctttcct cagcatgtat cccatggctc tggtatcttt 11700aacatctttg agtctccaag gcaatttcaa tgttacagct tcttgtttca gtgtctggga 11760ttctacttga tcttttggac tcctccatca cttttccagc tctgccctct gtagcactct 11820aagctcaggt tgttcactcc actacggctg ctgttctctt ggtgatcatc ttactgacat 11880ctccaataca ctggggtgtt ccactccaac taagcttcac caatagcctc tcataggctc 11940tcttcagggt gccaagcctc aaatcctttg catgacccct tcagtcctgg gccatcaact 12000acagctgagg cttcaccttc tccagtggcc ttccctggcc tctcacagtg ctaagcctca 12060gcttctctcc atgatccctt cattccttca caagcatact gcctgagtca cctgagtgac 12120tcttacacat taccaagtcc agtcgcagca tgaggtacaa ccttgggtat ctctggaaca 12180cagctacttt gtgctctcag aaaacaattt ccagaagatt ttacctcagt gatgctggta 12240tcttcttaat cactactaat tccttagctc cagctaacca gcatcaattg ccccagtagt 12300tccttccatt cttaactcta gagccagagc ctcatggctg aagctgccga gttctgctgc 12360ttacaggaac tagaacatga tccccttgta ctattattac attatcacta gctttatgtt 12420ttctaaatcc ttcactgcct aagcttggct atcctggttc ttgctctgta gattgacctt 12480gaatgcagag atcagcatgc ctgtctcatg ggattaaagg tgtataccac catgcctggt 12540tctaaattca gctgggtagg gtcttgcccc aaggtcccac tcccttaatc tgttatttcc 12600tagaacacag gatttttctt catttcactt cctggtatgc ctttaatact cgaaccatat 12660attttatatt ttgcctttct aagcttgcta tgcttgttca aacttctctt tgtgagactt 12720aaccagagaa caaagtctct gctgggcttt tttgaaactt cctttgtcag tgcaattaat 12780gcaattaatc caagtctctt caccttagcc tcaggcagac ttttcagaca agggtaaaaa 12840gtaaccacat tcttcaccaa aataccacaa aaacagtctt tatgccacat tctgaaattc 12900ttctcctttc ttgggccagg tcaatacagt tcaaattact cccagcaaca aagtcttcca 12960taatcctact aggatgacca attaaacccc atttaaagca ttccactgct tcccaaatcc 13020aaagtcccaa aatccacatt ctttcaaata aagcatggtc aggcctatca cagcaatacc 13080ccactcctgg taccaacttt tgtcttagtt agggttttac tgctgtgaac agtaaaccca 13140tgaccaaggc aagtcttcta aaaaaacaac atttaattgg ggctgcctta caggttcaga 13200ggttcaatcc attatcatca aggtgggagc atggcagtat ccaggcaggc atggcgcagg 13260aggagctgag agttctatat cttcatctaa aggctgctag tggaagactg acttccaggc 13320aactagggtg aggatcttat acccacactc acagtgacac acccattcca accaggtcac 13380acctattcca acaaggccat accttcagat ggtgccactc cctggtccaa agatataaaa 13440accatcacag tactctagag cagaatgaaa atcaaaagct tcttgtagca cagaatcttc 13500ccggacagag atcaaagaga aaggaagccc tccccttacc tgctcaataa accagagtgt 13560gttatcctag acaatctact aggaatctga gacaggaatc cttcctccct tcccatttca 13620catacaatca aatgcatcag tccttaaggt gatcaacaac tgtgcatgaa gcaagaggac 13680ctgacagcgt atacatacat acatgcatgt gtgagagcat gtgtgcacgt gtgtatatgt 13740gtgtgtgtat gcatgcatga gtgcatgcct gcatttgtga atgtatgtgt gtatgcatgc 13800atgcttgtgt gtgtgcatgt gtgtatacat gagtgtgtgt gcatgtgtgt gtgtgtgtgt 13860gtgtgtgttt acgtgtgtaa aaccaacatc cagtgccctt ctcagtcatt tccaccttgt 13920cttttcagag agggtctctt ctttttcttt agaactcacc catttggttg ggtcaactgg 13980tgagagagct ccaaagatcc ctctgcattt tccaaatgct ggggttatat catgcctagc 14040atcttatatt tttggttgtg agcctagtct ttaataactg agccatctct ccagccccat 14100gtttagcttc cttttttttt tctttctttt tttttttttt tttttttttg gtttttcgag 14160acagggtttc tttatatagc cctggctgtc ctgaaactca ctctgtagac caggctggcc 14220tcagactcag aaatccacct gcctctgcct cctaggtgct ggaattaaag gcatgcgcca 14280ccactgccca gcccatgttt agcttcttaa tatgggttca gaggctagaa ctctcatctt 14340ggagtttgca aaataagtaa tttgctgact gagccattcc acccacccaa gacccctgga 14400tgttggtttt acacacaaaa acgagtgggc ttggtggctc

atatttttgg ttgtgagcct 14460agcctttaac ggctgagcca tctctccagc ccggtggctc atatttttaa tcacagtact 14520gaggaggctc aagcagtgcg atttcagtga atttgaggct agcctaggct aacctagaga 14580agtctgggtt agagatgggc aatagcatga gatctaatct caaaaacccc tacagtgact 14640agaattctaa tattaaaaaa gtccttctgt ttctccatta cttaaaaaaa agtaataaaa 14700catttgcttg ttcacagttt gtttgtatag aaataaacaa atgccacaaa gtgctttaaa 14760gaggaagagg gagggagagg tatcaaacct cagataatgg tgtgctttag aaaggaggta 14820actctgttac ccacagggag gttcctatag cttgggtttg ggtttctctt agcattcatt 14880gtttttttgg cttggtggtg tgcctttggt ctgggctaaa ccctgctgcg caacactctc 14940actctacttt acctgagctc atagctatag gaacagaggg tccgctaact tgcacaggaa 15000actgggcgga aattgtaaaa ataaacagaa aaatagatag atagatagat agatagatag 15060atagatagat agatagatag atagatagat agatagatag atagacagat aggactgtca 15120gacactggag acatcgctcc gtcagtaaag tgtttataat acctggagac cagagctctg 15180tcctcagaat gaactaaaag gctgtgctca gcaactcagg agaatgaaaa cctaaaggca 15240gacaccaagt ttagaaatca ataagcagaa tctggagcca aaggagaggg ggaaacacca 15300taatgaggat gccaacacga ggagaagcca tcttagaaga agggaagtaa agaaacagga 15360agctggctgg aagctgtggg caacggtgcc ccatccctgg tacaaaagaa aggagtggac 15420cccaaaaaag gaagtggcca gaggaagtgg accaggatgg gcagtgagag tgaggaggga 15480attttataaa gtgggtcaca caggagtcat ttctgtttgg atttagaaca agtgtttctc 15540aatcttccta atgctgcaac ccttaaatac agttcctcct gctgtggtga ctcccaacca 15600tccaattact tcattactac ttcgtaactg tgatcttgct actgttgtga atagtaatgc 15660acccgtctgt gttttctgat ggtcttagat gacccctgcc aaaaggctac tgggctccca 15720aagggtcagg acccacaggt tgagaaccgc tgatgtagac cattccaatc tctgcttcta 15780ggttcaactc agattcccca gctgaatatc agttagtgga accacaagct aatgctggag 15840aatggagacc tgagagataa gggaggtttc cgtggacata gttgggtgga agtaggaagg 15900gtatttgtgc ctacatgagt gtgcgtgcgt gtgtgtgtgt gtaggtgcag ctgtgcacac 15960atgtatgtgt atgcgtgtgt gcttgtgcag gtgtacctgt gcacacatgt gcatgcatgt 16020gtgtgtgcaa gtgcacacgt gtatgtgcat gtgtgtgtgt ataggtacag ctgtgcaaac 16080atgtattgca tgcatgtgtg tgtgtgtgtg tgtgcgtgtg tgtgcaggtg caggtgcagc 16140tgtgcacaca tgtatgtgta tgcatgtgga ggccagaggt caaccttatg tgtcatttct 16200caggtacccc ccacattatg tttgtagact gtctctcaat gacatctgag gcttatggtt 16260cagatgcaag ccagtgagcc ttccgtgtgg ttccggatac cttcatgtct tcacagcagg 16320catctcactg actgggtcca tcaggatttg tgttctcccc tgagctatgt aatgcacagc 16380tgccactgtt ggctgatggt tcaatgccca cacaaaccag cccccagata tgcttcctgg 16440aagcccccct aagagtgaaa gacagctgag ggagtcctgc agagagattc cttgtcagat 16500ttttaccttt ccttttcgag tgtcctctgc ttcggtagaa tgccagcact ggggcaggtc 16560aatgatctca ggctgcctca ctatgtagcc caggctagcc ccaaactaaa gattaccctg 16620gattcctggc tgctctccat ctgctgccag gaaggaagcc ctgcctttag gttcatttcc 16680cagtctaaac atctcagagt acccagagat caaggcttgc tcaggtcaga ccagaaagtt 16740catttgctgg gcttatcggg ttggaggacg tcttgggagg gagcacgctt tgctgacctg 16800aggtttctaa ctctagtgga atccccgtgc tctggggctc atcgatgatg ctgggtttga 16860accaacacag cgtgctagca tcttccaagt agatataaac cttgtgtcat tctcactcac 16920agcggaggca gtgtggtttc tcagcatcgg gctagaagac tcggcacaaa ttgctggcac 16980aggtgtctgt ctgtccgcca gtagccatgg ccagtgtgat gggtgagaat gcatgctgag 17040atgatgacga gtctttcccg cactggtttc tgattgtgga gccacggtga gctcctactg 17100ggaccccacg tgtgagactt atcttcccct gccaggtctt acttttaccc agtcactcac 17160tcatttagca cagcagatac ctgcagagct gtcaagtgta agggcccagg gcccacagcc 17220ataaaagata tgtgcacacc ctgtgcagct ctccaggaag aggccgcgcg gaaggtgtca 17280ccatcacttt ctcggaacct gtcattgcat tgctatttat tttagttttc catcaaaatt 17340catatctttt tcttgatttt taaaattgaa aaatccaaca attaatagat tttccaaatg 17400gagaatactt acgtggctaa gaaagaatcc aagcaaatat agtaaaacta aacctccttt 17460tctgggtcct tcggtcctca gaatcgccct ccggcactgt tgctcccagg gattcagata 17520cacacacaca cacacacaca cacacacaca cacacatgca cacatacaca tacacacaca 17580cacatatata cacacataca cacacacaca tacacacaca cacatacaca cacatacaca 17640cacatacaca catacacaca catacacaca tacacacaca tacacacaca cacacacaga 17700aagagaggga gagggagagg gagacggagg gagagagaga accttttcca accttctctc 17760aaatggaagc ggtctatgtc catgggacct cacacgggcc ctgtggtttt gccttgaagc 17820gtttagtgtt ctgtcattca gattcacagt agttagctct gtcaagactg atcagagctc 17880aataacgggc atcatactct cctccacttt caaaataagc caagaaaggt acatttcttg 17940cgtcagtgga aaaaaaaata ccatctttac tatgcaaatt ttccacagtg ctcagggtcc 18000agtaaaacga gttccacggg ctgtctaaca gtcagacggt ctctgcaagg gcctgctgta 18060acccacaggt taacagatgg tctctgcaag agcctgctgt aacccatggg ctgtctaaca 18120gtcagacggt ctctgcaagg gcctgctgta atccatgggc tgtctaacag tcagacggtc 18180tctgcaaggg cctgctgtaa cccatgggct gtctaacagt cagacggtct ctgcaaaggt 18240ctgctgtaac ccacgggctg tctaacagtc agacagtctc tgcaagggcc tgctgtaacc 18300ttggactagg ctctctcctt agactagctc gtggtcaatc aggagactga accccagact 18360tgcagaggga cccagcaagc ccaaagtctc ctagctcatt ctctggctag agggctccat 18420cctccacaga agtccacaga agctgttgga ctggaggaca gatccccagc caaagagctt 18480ttcctagctt tgacctcctc ttgggggtca gcactgctaa ctcttcacac tgggagaagg 18540aggaagactg tggctaccct ggggtctccc acttatgacc tccacaggtc atattgctgc 18600atggcatgtc ttcccgcacc tgggaagata atccactctg catacaagtc tctcaggata 18660agcaacaatt cctgtttgct cgtctgtcaa ctatagatat atgaaatatc acattttagg 18720ggatacttat ttatttactt atttatttgt aagacaaagt ctgaaatatc ccaaattgga 18780ctgaagcttg ctgaagcttg ctgtgtagcc aagaatgagg taaccttgaa ctctgccagt 18840tctcctgtct ccacctcctg attgctggga ttacacatgc atactaatat gcctggtttg 18900tgtgacgctg aggtccaagc ccaaggcttc atgctcccta ggcaagtact cacaccgcag 18960ttacatcctc agccctgaat gacacccgtt gggtccagcg agtgctgtca gccagcagtc 19020tggtactgac cagcttgaac gctcctgagt aagatcacca caaaggccct ctgcagagaa 19080aggggccccg tggcctgaag cagagactgc ctatgcaatc cccgcagcct gaactccatc 19140acagcccgag gtggtccaga gcagacagcc tcagcccagt ccactagggg agagattggc 19200tggctggctg gctgacatgc aaattcacag acaggcaatg caggaagaag ccatggctgg 19260gtgggtgggc tgcggtgaga ccattgtgag ctgggtgcgc tgggtgtgga aactccctgt 19320taaaacccgt gagctttgct ttccattgca atggggaagg aggcaaaggc agccaggaag 19380tgcactctga gatgacttta gacttctgaa gggtctgtct gtggtttcaa gaggcagagg 19440aggaagccca gagcagcttg acagagtggc caaccaacac atcctgcctg gagcgtgagg 19500ggaaggggac aggtgttatt acccaacttg tggggaaaga atagggatag gctaggggtg 19560agaatatccc aatagatgag ccactgaaag caagagcagc aggctggctg agcccatcct 19620ctgcctggtg tcagctgatg cttctctggc tacagagccc atgctacaac aatgcttcct 19680ttccacctag agctgccaga tggccaaccc agtgcgattg ggatggagga actttccaca 19740cagaattaac cctgacaaaa tcctgggtac tatgagacaa gagtgtaggg actacactgg 19800ttattttcct cacaccagtg accaaattcc aggctagaag caactaagga tgaaagaagt 19860tacagcttct tctatgatag cctgggctga ggctgaggct gaggctctgg accacctcgg 19920gccatgatgg aggtcaggct gcaggacaag catagcagtc tttgcttcag gctgcaggac 19980cttctttttt ttttaattag gtattttctt catttacatt tcaaatgcta tcccaaaagt 20040ccccccgtac tgcccccccc cattccccta cccacccact cccacttctt ggccctggcg 20100ttcccctgta ctgaggcata taaagtttac aagaccaagg ggcctctctt cccaatgatg 20160gcctactaag ccatcttctg atacatatgc agctagagac atgaactcgg ggggtactgg 20220ttagttcata ctgttgttcc acctataggg ttgcagttcc cttcagctcc ttgggtactt 20280tctctagctc ctccattggg gaccctgtgt tccatccaat agctgactgt gagcatccac 20340ttctgtgttt gctaggcccc agcatagtct cacaagagac agctatatca gggtcctttc 20400agcaaaatct tgcttgtgta tgcaatggtg tcagtgtttg gaggctgatt atgggatgga 20460tccccgggtg tggcagtctc tagatggtcc atccttttgt ctcagctcca aactttgtct 20520ctgtaactcc ttccatgggt gttttgttcc caattctaag aaggggcaag gtgtccacac 20580tttggtcttc cttcttcttg agtttcatgt gttttgcaaa ttgtatcttg tatcttgggt 20640attctaaagg accttcttct ctgagcacct ctgctgtgat cttagtaagc tggtcagtaa 20700gctgcctcac agctggtgtg tccttcctcc ccgaaaggaa gtggcatact gactggtggg 20760atcactctgg tgtgaagtgg tgtgaagtcc tggcaggcgt gggaggcagc aggttcctat 20820gtgcctgtaa atgggaaagc tcactcactc cttttctagg aggtttggga ccctaaccta 20880tgcaatggtt ccatccacgt ttagagtgtt ttggttttgt ttttgtttta ccaagacaga 20940atttcactgt gtagccctgg atggcctgta actcactctt gtagaccagg ctggcctcag 21000actcagaaat ctgccagcct ctgcctacca agtgctaaga ttaaaggcgt gtactaccag 21060gcccctggct acagtgggtc tccctacctt attaactcaa tatttaaaat ccctcccatg 21120gggctggaga gatggctcag gagtttagag cattagctgc tcttccagag gacttaggtt 21180caattcccag aacctacatg gtggctccca accatctgtt ttcaaaggtt ccaacaccct 21240cttctggtct gggcaccagg cacaaccatg gtgctggcca gtcatgcaca catgtaaata 21300aataaatctt tgtaaaaatt ataaataaat taggcccctc acagatgtgc tgagatgtgc 21360atttccatgg agattctaaa ttccatcaaa gagctgagag atgcaaatgc tattctcaaa 21420gaggaatcag tccgggtggc tcccatgtca gggtggcccc catgtcaggg tggcctacaa 21480acacctgtaa gtccaactct aggggatcca ttgccccctt caggactcaa caaacacctg 21540cactccatgt gccttcccac acacaaaccc acatgcacat aattttaaat aaaatcatat 21600ttaaaaaaaa aagaaccagc gtagtatggg ctcatatctt tgatcccagc attcaggaga 21660cagagacaga cagatctctg tgagttgcag accaacaggg ttccaagcaa gtcagggttt 21720cacaatgaga ccctgttgca aaaataaaaa taaaaacaga aatgttttaa aagataggat 21780gagtgaagac aaaatctagt tccaattgtt caccctttca gtgttttctc ccgcttgctg 21840catgcagcca gtgagaaccg cgatcctcta agactcacgt gatctggttt gctgcataca 21900gccagtgaga actgcgatcc tctaagactc acgtggacct gcttactgca tgcagccagt 21960gagaactaca atcctttaag gctcacgtga tctggtttct ccttcccctc caggacctca 22020ccatgtccac ctccttccct gaactggatc tagagaattt tgagtatgac gattctgctg 22080aggcctgtta tttgggcgac attgtggcct ttggaaccat cttcctgtcc gtcttctacg 22140ccctcgtctt cacgttcggt ctggtgggaa atctgttggt ggtcctcgct ctcaccaaca 22200gccggaagcc caagagcatc actgacatct acctcctgaa cctggccttg agcgacctgc 22260tctttgtggc caccttgccc ttctggactc actacctcat cagccatgag ggcctccaca 22320atgccatgtg caagctcacg actgccttct tcttcattgg cttctttggg ggcatattct 22380tcatcaccgt catcagcatc gaccggtacc ttgccatcgt cctggccgcc aactccatga 22440acaaccggac agtgcagcac ggtgtcacca ttagtctggg cgtctgggcg gcggccatct 22500tagtggcgtc accccagttc atgttcacaa agagaaagga caacgagtgt ctgggtgact 22560accccgaggt cctgcaggaa atgtggcccg tgctccgcaa ctcggaagtc aacatcctgg 22620gcttcgccct gcccttgctt atcatgagct tttgctactt ccgcatcatc cagacgctgt 22680tttcctgcaa gaatcgcaag aaggccagag ccgtcagact catcctcctg gtggtctttg 22740ccttcttcct cttctggaca ccatacaaca tcatgatttt cctggagact ctcaagttct 22800acaacttctt ccccagttgt gacatgaaga gggacctgag gttggccctc agtgtgacgg 22860agacagtggc gttcagccac tgttgcctca acccctttat ctacgccttt gccggggaaa 22920agttcagaag atacctggga cacctgtata ggaagtgcct ggccgtcctg tgcggtcatc 22980ctgtccacac cggcttctcg ccagagtccc agaggagcag gcaggacagc attctgagca 23040gtttcactca ctacacaagc gagggagatg ggtctctcct gctctgaagg ggtctccccg 23100accctagctc cactaggaac ccagagttct tgcatcagat ttccctgccg ctccccctgc 23160atcttatgtg caagaaatat ggaccagatg cctgcaaacc aaccccgtgg tgtttttttg 23220aaaaatttat gttcaatgtg tgaaaaacac acgtatctct tactgcaaat gttgaacatt 23280ggggcttact ggtgacaaaa attctaacca gattagtgca attacaaagg ggtttggtga 23340gtcctggttg catgatcatg tgataaagga caactaagtc ctcagactga gtggaaacca 23400aggcttggct ccaatgtccc ctctctgacc ttcagatcct tcatagtgac agatcatcca 23460ggttctatca tcagagaagg accacatctc tctgatttca aaattggtat tcctagggaa 23520cacctccgtt ggccgagtgt gtcgggtgtc cattccttga ctaggtggtg ttattgaaat 23580agaagggata cctaagatgc tgttggaatc tcaaggttag cggttgagag agacacatct 23640ctcagaagct ggggggtggg agctactctg acagcaagaa ctgctgactc gccttaccat 23700ggagctcatt caggctcccc ttcagtaact agcagtatct gttgctagct tctttaatct 23760tctgttgaga atgtcctgaa ctctccaagg gttagaattt gggttactgc tcacagcatc 23820aaattcaatc ccaaggccct gtcctccaag accaggaaga taggatgcag ttctaacaag 23880agactccacg ctgactcctc attccacagg actccgtcca cccagttggc catgtccctt 23940tttcttgtct tgacccacat ccctccactc ttctcagccc aggggaagaa atagaaagag 24000ccatgcccca gagtgaaggg atcgattcat atcccaactc tgttgcttgc acattgggga 24060gactggagcc aacagagctt cagtcccttc aatatagaaa ggggactgtg gtgccttcat 24120ccattctagc atagtacctc agcaaggact ttagcattcc ctggatcagg aactgttacc 24180ctgcctctga gaaatggagc tggtggagtg ggaggaacct gcaggctttg ctgcaggaca 24240cagccagaca agaggagacc tattcctaca gcatccccca acatgtgcct agatctacaa 24300agccattccc atgtccccgc tgtcagggca agacccaggc cacgagagca gcttgctttc 24360tcccctgacc ctgcaagcat cacgtaggat gtagccacaa gaatgtcttc atgcattcac 24420tgactccggt ctcatttgca ggcttacatt gaaagttcca gttctgcaac ttgcttttta 24480aaaaacactg gatttcaggg gctggagaga tggctcagtg gttaagagca cagtctgctc 24540ctacagaggt cccaagttca attcccatgt catgtctgaa gacaaggaca gtgtagtcaa 24600aaaaaacact ggaattcaaa tagctccctg ttcctgtgtt ggttgtgata accatttcag 24660aagtctctcc cagcctgttg ctcacggcgg catgtctgat atctccttgg cagtctgtat 24720gtttgtgtcg aggatgacag acagactgag tcatatcccc cacaggcata tccaaccctt 24780tgttattgca acaccatgct gtcatattca aaggtcacaa atgagaacct caaaattgag 24840ttaccaaagt gggagcctgg agagatggct cagtggttaa tagcactggc tgctcttcca 24900gaggtctggg gttggattcc cagcaagcat agtagcttac aatggtctat aatataactc 24960tagttccagg tgacccaaca ccctcttcta atttgtgagg gtagcagaca tgtgcattgt 25020gaacattcag gcaaacaccc atacatacaa aataagtaaa aaagaaaaag aaaggaagga 25080atggagagag agaaagaggg ggagagagag agggagaggg agagggagag ggagagggag 25140ggagggagag ggagggaggg agggagggag ggagggaggg agggagggaa aggaaagaaa 25200gaaagaaaga aaggaaggaa ggaaggaagg aaggaaggaa ggaagaaaga aagaaagagg 25260gagagagaga aagagagaaa gagagaaaga gaaagagaga aagagagaaa gagaaagaga 25320gaaagagaga aagaagaaag aaagacagaa aattggtttt tcaagatagg ttctctctat 25380gtagcctcgg ctgtcctgaa actctctgtg tagaccaggc tggcctagag ctcaaagaaa 25440tccacctgcc tctgcctcct gagtactggg atcgaaggtc tgtggcacca tggcaggctt 25500agtcaataaa tgttttattt tcaaaagtat cctataatgt tttaagtaag tttatgcttt 25560ggtgttggtc tgtatttccc gctgtctcgg gtcacatggt taagcgtgcc tagagtgtgt 25620ctatcccact tgtaattctg tcaataaaca ttggtttcct tccagctctt ggcaagtacg 25680agtctcaggg aactttaatc tgtgcacgtt tcctctccct gctcacttat ttccatcaga 25740ttaattcttt gccatggggg tagctgggtc aaagggcaca ggaatgtttc agctctgttc 25800tccagtacat acaattttat caaaaagcaa agcctggcca ggcatgctaa cctacatcct 25860taacgacagc attcacgagg cagagggagg tatctctgcc agcccaagac cagcctggta 25920acattgtaag actgaaaaaa aaacctagga aaacaaaaaa ccaaaaaaac cctaaagctc 25980tctagggaag gccagccgct cctcagcacc taaagcatcg ttgtcttgac caacctggaa 26040gtaatggggc tgacaaggct gtcacaggca gccatcttgg cagggctgct ttgactgcca 26100agctcagtaa gatccagtgc agcttgcttg aaaattttta atattgattg atccgttgtt 26160tgtacatgta tgtgtgtgta tgcacatgta tgtatgttta tgcatgtgtg tatgtgtgtg 26220tgtgtttatg catgtgtgta catgtgtgta tatatgcatg tgtatatatg tgtgcatgtg 26280tatgtgtgca catgagtgta tgcatgtgca tgcacatgaa cacacatgaa gaaactcaga 26340ggacaaattg cagaagtcag ggctcccttt ccaccacata ggtcccagga tcaaactccg 26400gttgaggggc ttggtggcaa ttgtctttac ctgatgaacc atctctccag gcccgcttta 26460gtttttgcat acacatgacc ttcatttgat aatgacaatt tgttacaagt ctttacgaag 26520cacatatcct agcgggctcc agagatgttt ccacagcagt gagaccacac ctctgttctc 26580agaggttttg atttattttt tgagatagag tctcactttg tagaccaggc tgccctggaa 26640ctcggaaatc tgcctgcctc ttcccctgga gtgttgggat taaaggtgtg cgccaccacc 26700ctgggcttga tatctgtgga cagaaaaggc ctggctgtgt ttaacttgct tgctgtaaga 26760cagagcagag gtaataagat ctgacccaag acttcaggga tatgagcttc tccaggagaa 26820gcaggggctg tttgctagga ggcagggatg tggaattcgt gtgagagagc acgaggttcc 26880ttcagtgtca gtccctcatt ccaactgatg agcctgagag ttgtcatcaa aggttaatct 26940gttcctggac tcccatgtag agaagaaaga cttctggcct gagaagcaaa catagggaag 27000c 27001520DNAArtificial SequenceSynthetic oligonucleotide 5tagtgcggac ctacccacga 20620DNAArtificial SequenceSynthetic oligonucleotide 6gcacctttcc ctcggatggg 20764DNAArtificial SequenceSynthetic oligonucleotide 7gatcgtcgga ctgtagaact ctcaagcaga agacggcata cgattttttt tttttttttt 60tttn 64818DNAArtificial SequenceSynthetic oligonucleotide 8caagcagaag acggcata 18946DNAArtificial SequenceSynthetic oligonucleotide 9aatgatacgg cgaccaccga caggttcaga gttctacagt ccgacg 461032DNAArtificial SequenceSynthetic oligonucleotide 10cgacaggttc agagttctac agtccgacga tc 321125RNAArtificial SequenceSynthetic oligonucleotide 11cagagucaga gagucagaga gagau 251225RNAArtificial SequenceSynthetic oligonucleotide 12aucucucucu gacucucuga cucug 251325RNAArtificial SequenceSynthetic oligonucleotide 13gaguuuggac cugugaccuu ccuaa 251425RNAArtificial SequenceSynthetic oligonucleotide 14uuaggaaggu cacaggucca aacuc 251525RNAArtificial SequenceSynthetic oligonucleotide 15aaucuccugg gaggaugaag cuguu 251625RNAArtificial SequenceSynthetic oligonucleotide 16aacagcuuca uccucccagg agauu 251725RNAArtificial SequenceSynthetic oligonucleotide 17accacuguuu cugacugcuu ucuca 251825RNAArtificial SequenceSynthetic oligonucleotide 18ugagaaagca gucagaaaca guggu 251925RNAArtificial SequenceSynthetic oligonucleotide 19ggauugagag ugaccaggac auuua 252025RNAArtificial SequenceSynthetic oligonucleotide 20uaaauguccu ggucacucuc aaucc 252125RNAArtificial SequenceSynthetic oligonucleotide 21cgcccagccu aauuguagua cuuua 252225RNAArtificial SequenceSynthetic oligonucleotide 22uaaaguacua caauuaggcu gggcg 252319RNAArtificial SequenceSynthetic oligonucleotide 23gcaaauucuu ucaugacaa 192419RNAArtificial SequenceSynthetic oligonucleotide 24uugucaugaa agaauuugc 192519RNAArtificial SequenceSynthetic oligonucleotide 25gcaaagaugg

auagagaua 192619RNAArtificial SequenceSynthetic oligonucleotide 26uaucucuauc caucuuugc 192719RNAArtificial SequenceSynthetic oligonucleotide 27gcacauaccu cauuagaga 192819RNAArtificial SequenceSynthetic oligonucleotide 28ucucuaauga gguaugugc 192919RNAArtificial SequenceSynthetic oligonucleotide 29gguauuaaua gcucugaaa 193019RNAArtificial SequenceSynthetic oligonucleotide 30uuucagagcu auuaauacc 193119RNAArtificial SequenceSynthetic oligonucleotide 31gggagagggu cuacaauua 193219RNAArtificial SequenceSynthetic oligonucleotide 32uaauugucga cccucuccc 193319RNAArtificial SequenceSynthetic oligonucleotide 33ggccagaucu ccugugaua 193419RNAArtificial SequenceSynthetic oligonucleotide 34uaucacagga gaucuggcc 193519RNAArtificial SequenceSynthetic oligonucleotide 35gcuccauucu gcugcucaa 193619RNAArtificial SequenceSynthetic oligonucleotide 36uugagcagca gaauggagc 193719RNAArtificial SequenceSynthetic oligonucleotide 37cuaacgugac agugacaua 193819RNAArtificial SequenceSynthetic oligonucleotide 38uaugucacug ucacguuag 193919RNAArtificial SequenceSynthetic oligonucleotide 39gcaaaaggua ggaggguuu 194019RNAArtificial SequenceSynthetic oligonucleotide 40aaacccuccu accuuuugc 194119RNAArtificial SequenceSynthetic oligonucleotide 41ggagaugaau ugauagaga 194219RNAArtificial SequenceSynthetic oligonucleotide 42ucucuaucaa uucaucucc 194319RNAArtificial SequenceSynthetic oligonucleotide 43cagaagagcu auuugguau 194419RNAArtificial SequenceSynthetic oligonucleotide 44auaccaaaua gcucuucug 194519RNAArtificial SequenceSynthetic oligonucleotide 45gaguggacuu cacaagaaa 194619RNAArtificial SequenceSynthetic oligonucleotide 46uuucuuguga aguccacuc 194719RNAArtificial SequenceSynthetic oligonucleotide 47agagaagaau gaaggugaa 194819RNAArtificial SequenceSynthetic oligonucleotide 48uucaccuuca uucuucucu 194919RNAArtificial SequenceSynthetic oligonucleotide 49ccacagguga gcagaaauu 195019RNAArtificial SequenceSynthetic oligonucleotide 50aauuucugcu caccugugg 195119RNAArtificial SequenceSynthetic oligonucleotide 51ccuaauuccc agaagcaga 195219RNAArtificial SequenceSynthetic oligonucleotide 52ucugcuucug ggaauuagg 195319RNAArtificial SequenceSynthetic oligonucleotide 53cgaaauggcc uaaagauga 195419RNAArtificial SequenceSynthetic oligonucleotide 54ucaucuuuag gccauuucg 195519RNAArtificial SequenceSynthetic oligonucleotide 55acacaaaggu ggaaggaaa 195619RNAArtificial SequenceSynthetic oligonucleotide 56uuuccuucca ccuuugugu 195719RNAArtificial SequenceSynthetic oligonucleotide 57ggaagaaccu gcagagaug 195819RNAArtificial SequenceSynthetic oligonucleotide 58caucucugca gguucuucc 195916DNAArtificial SequenceSynthetic oligonucleotide 59cacgtctata caccac 166016DNAArtificial SequenceSynthetic oligonucleotide 60gaattaacgc ctgagg 166116DNAArtificial SequenceSynthetic oligonucleotide 61gaactgacaa aggtgg 166216DNAArtificial SequenceSynthetic oligonucleotide 62atctccccac tcaagg 166316DNAArtificial SequenceSynthetic oligonucleotide 63catttttctg ctgacc 166416DNAArtificial SequenceSynthetic oligonucleotide 64acaagacaga ggcaga 166516DNAArtificial SequenceSynthetic oligonucleotide 65tcagttggag gacagt 166616DNAArtificial SequenceSynthetic oligonucleotide 66gaaggagcag gtgaaa 166716DNAArtificial SequenceSynthetic oligonucleotide 67ggtatttccg cttcac 166816DNAArtificial SequenceSynthetic oligonucleotide 68aggataccag gacaca 166916DNAArtificial SequenceSynthetic oligonucleotide 69tgataaaagc agggtc 167021DNAArtificial SequenceSynthetic oligonucleotide 70tcagttccca gcattctcat c 217121DNAArtificial SequenceSynthetic oligonucleotide 71ttgagccttg gagacagaaa g 217220DNAArtificial SequenceSynthetic oligonucleotide 72tccaaagcat cccattcctg 207321DNAArtificial SequenceSynthetic oligonucleotide 73tgagcaaaac aagacaaacc g 217424DNAArtificial SequenceSynthetic oligonucleotide 74ttatgttgct cttgatagac tccc 247521DNAArtificial SequenceSynthetic oligonucleotide 75gctaggtgct gtctgagatt c 217625DNAArtificial SequenceSynthetic oligonucleotide 76ttcaagcaaa gattatagcc atgtg 257722DNAArtificial SequenceSynthetic oligonucleotide 77atccagtgca gagtaacatc ag 227821DNAArtificial SequenceSynthetic oligonucleotide 78agatgcctct gcatactggt t 217920DNAArtificial SequenceSynthetic oligonucleotide 79cagaccatat tgggccacag 208020DNAArtificial SequenceSynthetic oligonucleotide 80ccacagcaga aaaccactga 208120DNAArtificial SequenceSynthetic oligonucleotide 81ttccctctgc actgactcct 208220DNAArtificial SequenceSynthetic oligonucleotide 82cgtcttttcc cactgacaca 208320DNAArtificial SequenceSynthetic oligonucleotide 83cccctcccca gaagaaaata 208421DNAArtificial SequenceSynthetic oligonucleotide 84ctgaggaaca caagactagc c 218522DNAArtificial SequenceSynthetic oligonucleotide 85actggactca ttttgggaca tc 228620DNAArtificial SequenceSynthetic oligonucleotide 86attgtgcatg gctcttaccc 208720DNAArtificial SequenceSynthetic oligonucleotide 87cttggtgcat gtgagcttgt 208820DNAArtificial SequenceSynthetic oligonucleotide 88agcttctggt tccaaggtca 208920DNAArtificial SequenceSynthetic oligonucleotide 89catgtgctgt tgttgctgtg 209020DNAArtificial SequenceSynthetic oligonucleotide 90tgaaagggaa gacgcagatg 209121DNAArtificial SequenceSynthetic oligonucleotide 91ttgtatcctt tgactgggca g 219220DNAArtificial SequenceSynthetic oligonucleotide 92aaagaaggca ggggaacaat 209320DNAArtificial SequenceSynthetic oligonucleotide 93cacttgggca atccagaaat 209421DNAArtificial SequenceSynthetic oligonucleotide 94tcacctgtgg aaagagacaa c 219521DNAArtificial SequenceSynthetic oligonucleotide 95agaacctttt gctccctagt g 219620DNAArtificial SequenceSynthetic oligonucleotide 96ttaaacgagc ctggagttgg 209720DNAArtificial SequenceSynthetic oligonucleotide 97aaattcctca gagcccagtg 209818DNAArtificial SequenceSynthetic oligonucleotide 98ggctcagaga ggccaaaa 189924DNAArtificial SequenceSynthetic oligonucleotide 99tggaccctat catctccttt aact 2410020DNAArtificial SequenceSynthetic oligonucleotide 100ggaaaggaat tgctggatca 2010120DNAArtificial SequenceSynthetic oligonucleotide 101gtgcaaccac ttgggaaact 2010221DNAArtificial SequenceSynthetic oligonucleotide 102cagagatgag gaaaggtttg c 2110320DNAArtificial SequenceSynthetic oligonucleotide 103atctgcttca cggtctcatg 2010421DNAArtificial SequenceSynthetic oligonucleotide 104agtagctgtc aaatggcctt c 2110520DNAArtificial SequenceSynthetic oligonucleotide 105ttagttcggc tttgagggtg 2010620DNAArtificial SequenceSynthetic oligonucleotide 106ggggacctgt ttttcctgtt 2010720DNAArtificial SequenceSynthetic oligonucleotide 107gacttggatc tggacctgga 2010820DNAArtificial SequenceSynthetic oligonucleotide 108tctgtctgcc aacaagcagt 2010920DNAArtificial SequenceSynthetic oligonucleotide 109gcactgtagc gagactggag 2011020DNAArtificial SequenceSynthetic oligonucleotide 110tcctgctgtg caaagtgttc 2011120DNAArtificial SequenceSynthetic oligonucleotide 111aaatccatcg ggtatctgga 2011220DNAArtificial SequenceSynthetic oligonucleotide 112tgccaagaga ctagggcagt 2011320DNAArtificial SequenceSynthetic oligonucleotide 113gaatacgaag ggtgggatca 2011420DNAArtificial SequenceSynthetic oligonucleotide 114catcacaggg aaccagacct 2011520DNAArtificial SequenceSynthetic oligonucleotide 115caccccgaag agaccataga 2011620DNAArtificial SequenceSynthetic oligonucleotide 116tcaggaacct ggctatggag 2011720DNAArtificial SequenceSynthetic oligonucleotide 117ggcaggagta gggacggtat 2011820DNAArtificial SequenceSynthetic oligonucleotide 118gccagaagat gcacacttga 2011920DNAArtificial SequenceSynthetic oligonucleotide 119caagctctga gcctctgctt 2012020DNAArtificial SequenceSynthetic oligonucleotide 120caccatggag aacaaggtga 2012120DNAArtificial SequenceSynthetic oligonucleotide 121tgacaccagg aaaaccacaa 2012218DNAArtificial SequenceSynthetic oligonucleotide 122ggcaggacca gcttctga 1812318DNAArtificial SequenceSynthetic oligonucleotide 123ctgttcccac caccttgg 1812416DNAArtificial SequenceSynthetic oligonucleotide 124tccattgcca ccccca 1612516DNAArtificial SequenceSynthetic oligonucleotide 125tcctccattg ccaccc 1612616DNAArtificial SequenceSynthetic oligonucleotide 126acttcctcca ttgcca 1612716DNAArtificial SequenceSynthetic oligonucleotide 127ccaacttcct ccattg 1612816DNAArtificial SequenceSynthetic oligonucleotide 128acacttctct ccctac 1612916DNAArtificial SequenceSynthetic oligonucleotide 129ggcttggaat ccatca 1613016DNAArtificial SequenceSynthetic oligonucleotide 130caactgcgga gggagg 1613116DNAArtificial SequenceSynthetic oligonucleotide 131taccaactgc ggaggg 1613216DNAArtificial SequenceSynthetic oligonucleotide 132tcttaccaac tgcgga 1613316DNAArtificial SequenceSynthetic oligonucleotide 133ctttcttacc aactgc 1613416DNAArtificial SequenceSynthetic oligonucleotide 134gttctttctt accaac 1613516DNAArtificial SequenceSynthetic oligonucleotide 135gattgctgct ggttct 1613616DNAArtificial SequenceSynthetic oligonucleotide 136caggattgct gctggt 1613716DNAArtificial SequenceSynthetic oligonucleotide 137cttcaggatt gctgct 1613816DNAArtificial SequenceSynthetic oligonucleotide 138cgccttcagg attgct 1613922DNAArtificial SequenceSynthetic oligonucleotide 139cacttcactc gccttcagga tt 2214016DNAArtificial SequenceSynthetic oligonucleotide 140actcgccttc aggatt 1614116DNAArtificial SequenceSynthetic oligonucleotide 141ttcactcgcc ttcagg 1614216DNAArtificial SequenceSynthetic oligonucleotide 142cacttcactc gccttc 1614316DNAArtificial SequenceSynthetic oligonucleotide 143taccacttca ctcgcc 1614416DNAArtificial SequenceSynthetic oligonucleotide 144gggtaccact tcactc 1614516DNAArtificial SequenceSynthetic oligonucleotide 145agtgggtacc acttca 1614616DNAArtificial SequenceSynthetic oligonucleotide 146agcagtgggt accact 1614716DNAArtificial SequenceSynthetic oligonucleotide 147gtaagcagtg ggtacc 1614816DNAArtificial SequenceSynthetic oligonucleotide 148agtgggtaag cagtgg 1614916DNAArtificial SequenceSynthetic oligonucleotide 149aacagtgggt aagcag 1615016DNAArtificial SequenceSynthetic oligonucleotide 150ctggaacagt gggtaa 1615116DNAArtificial SequenceSynthetic oligonucleotide 151tgcctggaac agtggg 1615216DNAArtificial SequenceSynthetic oligonucleotide 152aggtgcctgg

aacagt 1615316DNAArtificial SequenceSynthetic oligonucleotide 153ttacagaggt gcctgg 1615416DNAArtificial SequenceSynthetic oligonucleotide 154ctgttacaga ggtgcc 1615516DNAArtificial SequenceSynthetic oligonucleotide 155ggcctgttac agaggt 1615616DNAArtificial SequenceSynthetic oligonucleotide 156gtgggcctgt tacaga 1615716DNAArtificial SequenceSynthetic oligonucleotide 157catgtgggcc tgttac 1615816DNAArtificial SequenceSynthetic oligonucleotide 158tcccatgtgg gcctgt 1615916DNAArtificial SequenceSynthetic oligonucleotide 159gtttcccatg tgggcc 1616016DNAArtificial SequenceSynthetic oligonucleotide 160agtgtttccc atgtgg 1616116DNAArtificial SequenceSynthetic oligonucleotide 161gctaataaca gtgttt 1616216DNAArtificial SequenceSynthetic oligonucleotide 162agtgctaata acagtg 1616316DNAArtificial SequenceSynthetic oligonucleotide 163acaagtgcta ataaca 1616424DNAArtificial SequenceSynthetic oligonucleotide 164catttccccc atcttaaaaa caag 2416516DNAArtificial SequenceSynthetic oligonucleotide 165tttcccccat cttaaa 1616616DNAArtificial SequenceSynthetic oligonucleotide 166cctaccattt ccccca 1616716DNAArtificial SequenceSynthetic oligonucleotide 167caacctacca tttccc 1616816DNAArtificial SequenceSynthetic oligonucleotide 168acacaaccta ccattt 1616916DNAArtificial SequenceSynthetic oligonucleotide 169tcgacacaac ctacca 1617016DNAArtificial SequenceSynthetic oligonucleotide 170ctatcgacac aaccta 1617116DNAArtificial SequenceSynthetic oligonucleotide 171cagctatcga cacaac 1617216DNAArtificial SequenceSynthetic oligonucleotide 172ccccagctat cgacac 1617316DNAArtificial SequenceSynthetic oligonucleotide 173tgaccccagc tatcga 1617416DNAArtificial SequenceSynthetic oligonucleotide 174gtgtgacccc agctat 1617516DNAArtificial SequenceSynthetic oligonucleotide 175ctcattgtgt gacccc 1617616DNAArtificial SequenceSynthetic oligonucleotide 176cagctcattg tgtgac 1617716DNAArtificial SequenceSynthetic oligonucleotide 177gcttcagctc attgtg 1617816DNAArtificial SequenceSynthetic oligonucleotide 178atccaagctt cagctc 1617916DNAArtificial SequenceSynthetic oligonucleotide 179gccgaaatcc aagctt 1618016DNAArtificial SequenceSynthetic oligonucleotide 180actgccgaaa tccaag 1618116DNAArtificial SequenceSynthetic oligonucleotide 181tggactgccg aaatcc 1618216DNAArtificial SequenceSynthetic oligonucleotide 182gattggactg ccgaaa 1618316DNAArtificial SequenceSynthetic oligonucleotide 183tgggattgga ctgccg 1618416DNAArtificial SequenceSynthetic oligonucleotide 184ctctgggatt ggactg 1618516DNAArtificial SequenceSynthetic oligonucleotide 185ccactctggg attgga 1618616DNAArtificial SequenceSynthetic oligonucleotide 186gctcccactc tgggat 1618716DNAArtificial SequenceSynthetic oligonucleotide 187tgagtaggcc aatggg 1618816DNAArtificial SequenceSynthetic oligonucleotide 188gagtgagtag gccaat 1618916DNAArtificial SequenceSynthetic oligonucleotide 189gcagagtgag taggcc 1619016DNAArtificial SequenceSynthetic oligonucleotide 190gaggccagga cttggc 1619116DNAArtificial SequenceSynthetic oligonucleotide 191aagttcagca gaggcc 1619216DNAArtificial SequenceSynthetic oligonucleotide 192aacaagttca gcagag 1619316DNAArtificial SequenceSynthetic oligonucleotide 193agatgtggaa acaagt 1619416DNAArtificial SequenceSynthetic oligonucleotide 194attctaattt ccttcg 1619516DNAArtificial SequenceSynthetic oligonucleotide 195catctattct aatttc 1619616DNAArtificial SequenceSynthetic oligonucleotide 196ccccatctat tctaat 1619716DNAArtificial SequenceSynthetic oligonucleotide 197tgtccccatc tattct 1619816DNAArtificial SequenceSynthetic oligonucleotide 198aggtgtcccc atctat 1619916DNAArtificial SequenceSynthetic oligonucleotide 199tggaggtgtc cccatc 1620016DNAArtificial SequenceSynthetic oligonucleotide 200cacacatgga ggtgtc 1620116DNAArtificial SequenceSynthetic oligonucleotide 201gagtcaacag aaatac 16202457DNAMus musculus 202cagggtgtat gtgtgggggt ggcaatggag gaagttgggg gtagggagag aagtgttctt 60gctttgatgg attccaagcc ctgggttctc cctccctccg cagttggtaa gaaagaacca 120gcagcaatcc tgaaggcgag tgaagtggta cccactgctt acccactgtt ccaggcacct 180ctgtaacagg cccacatggg aaacactgtt attagcactt gtttttaaga tgggggaaat 240ggtaggttgt gtcgatagct ggggtcacac aatgagctga agcttggatt tcggcagtcc 300aatcccagag tgggagcccc cattggccta ctcactctgc ctgccaagtc ctggcctctg 360ctgaacttgt ttccacatct gcatgacgaa ggaaattaga atagatgggg acacctccat 420gtgtggtgtt tgtatttctg ttgactcttt tttttct 4572033185DNAMus musculus 203ctcaccatga gtccctggca gcccctgctc ctggctctcc tggctttcgg ctgcagctct 60gctgcccctt accagcgcca gccgactttt gtggtcttcc ccaaagacct gaaaacctcc 120aacctcacgg acacccagct ggcagaggca tacttgtacc gctatggtta cacccgggcc 180gcccagatga tgggagagaa gcagtctcta cggccggctt tgctgatgct tcagaagcag 240ctctccctgc cccagactgg tgagctggac agccagacac taaaggccat tcgaacacca 300cgctgtggtg tcccagacgt gggtcgattc caaaccttca aaggcctcaa gtgggaccat 360cataacatca catactggat ccaaaactac tctgaagact tgccgcgaga catgatcgat 420gacgccttcg cgcgcgcctt cgcggtgtgg ggcgaggtgg cacccctcac cttcacccgc 480gtgtacggac ccgaagcgga cattgtcatc cagtttggtg tcgcggagca cggagacggg 540tatcccttcg acggcaagga cggccttctg gcacacgcct ttccccctgg cgccggcgtt 600cagggagatg cccatttcga cgacgacgag ttgtggtcgc tgggcaaagg cgtcgtgatc 660cccacttact atggaaactc aaatggtgcc ccatgtcact ttcccttcac cttcgaggga 720cgctcctatt cggcctgcac cacagacggc cgcaacgacg gcacgccttg gtgtagcaca 780acagctgact acgataagga cggcaaattt ggtttctgcc ctagtgagag actctacacg 840gagcacggca acggagaagg caaaccctgt gtgttcccgt tcatctttga gggccgctcc 900tactctgcct gcaccactaa aggccgctcg gatggttacc gctggtgcgc caccacagcc 960aactatgacc aggataaact gtatggcttc tgccctaccc gagtggacgc gaccgtagtt 1020gggggcaact cggcaggaga gctgtgcgtc ttccccttcg tcttcctggg caagcagtac 1080tcttcctgta ccagcgacgg ccgcagggat gggcgcctct ggtgtgcgac cacatcgaac 1140ttcgacactg acaagaagtg gggtttctgt ccagaccaag ggtacagcct gttcctggtg 1200gcagcgcacg agttcggcca tgcactgggc ttagatcatt ccagcgtgcc ggaagcgctc 1260atgtacccgc tgtatagcta cctcgagggc ttccctctga ataaagacga catagacggc 1320atccagtatc tgtatggtcg tggctctaag cctgacccaa ggcctccagc caccaccaca 1380actgaaccac agccgacagc acctcccact atgtgtccca ctatacctcc cacggcctat 1440cccacagtgg gccccacggt tggccctaca ggcgccccct cacctggccc cacaagcagc 1500ccgtcacctg gccctacagg cgccccctca cctggcccta cagcgccccc tactgcgggc 1560tcttctgagg cctctacaga gtctttgagt ccggcagaca atccttgcaa tgtggatgtt 1620tttgatgcta ttgctgagat ccagggcgct ctgcatttct tcaaggacgg ttggtactgg 1680aagttcctga atcatagagg aagcccatta cagggcccct tccttactgc ccgcacgtgg 1740ccagccctgc ctgcaacgct ggactccgcc tttgaggatc cgcagaccaa gagggttttc 1800ttcttctctg gacgtcaaat gtgggtgtac acaggcaaga ccgtgctggg ccccaggagt 1860ctggataagt tgggtctagg cccagaggta acccacgtca gcgggcttct cccgcgtcgt 1920ctcgggaagg ctctgctgtt cagcaagggg cgtgtctgga gattcgactt gaagtctcag 1980aaggtggatc cccagagcgt cattcgcgtg gataaggagt tctctggtgt gccctggaac 2040tcacacgaca tcttccagta ccaagacaaa gcctatttct gccatggcaa attcttctgg 2100cgtgtgagtt tccaaaatga ggtgaacaag gtggaccatg aggtgaacca ggtggacgac 2160gtgggctacg tgacctacga cctcctgcag tgcccttgaa ctagggctcc ttctttgctt 2220caaccgtgca gtgcaagtct ctagagacca ccaccaccac caccacacac aaaccccatc 2280cgagggaaag gtgctagctg gccaggtaca gactggtgat ctcttctaga gactgggaag 2340gagtggaggc aggcagggct ctctctgccc accgtccttt cttgttggac tgtttctaat 2400aaacacggat ccccaacctt ttccagctac tttagtcaat cagcttatct gtagttgcag 2460atgcatccga gcaagaagac aactttgtag ggtggattct gaccttttat ttttgtgtgg 2520cgtctgagaa ttgaatcagc tggcttttgt gacaggcact tcaccggcta aaccacctct 2580cccgactcca gcccttttat ttattatgta tgaggttatg ttcacatgca tgtatttaac 2640ccacagaatg cttactgtgt gtcgggcgcg gctccaaccg ctgcataaat attaaggtat 2700tcagttgccc ctactggaag gtattatgta actatttctc tcttacattg gagaacacca 2760ccgagctatc cactcatcaa acatttattg agagcatccc tagggagcca ggctctctac 2820tgggcgttag ggacagaaat gttggttctt ccttcaagga ttgctcagag attctccgtg 2880tcctgtaaat ctgctgaaac cagaccccag actcctctct ctcccgagag tccaactcac 2940tcactgtggt tgctggcagc tgcagcatgc gtatacagca tgtgtgctag agaggtagag 3000ggggtctgtg cgttatggtt caggtcagac tgtgtcctcc aggtgagatg acccctcagc 3060tggaactgat ccaggaagga taaccaagtg tcttcctggc agtctttttt aaataaatga 3120ataaatgaat atttacttaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3180aaaaa 3185

Claims

1. A method of inhibiting expression of a gene in a cell comprising contacting the cell with a specific inhibitor of an enhancer RNA (eRNA), and thereby inhibiting expression of the gene in the cell; wherein the specific inhibitor is an antisense compound comprising a modified oligonucleotide complementary to the eRNA.

2. (canceled)

3. The method of claim 1, wherein the cell is mammalian.

4. The method of claim 1, wherein transcription of the eRNA is initiated from a RNA polymerase II (PolII) binding site and is capable of elongating bidirectionally.

5. (canceled)

6. The method of claim 1, wherein the eRNA is transcribed from a genomic enhancer sequence or region having a higher level of monomethylated lysine 4 of histone 3 (H3K4me1) than trimethylated lysine 4 of histone 3 (H3K4me3).

7-14. (canceled)

15. The method of claim 1, wherein the eRNA enhances transcription of chemokine receptor CX3CR1.

16. The method claim 1, wherein the transcriptional start site of the one or more genes is located on a chromosome at least 1 kilobase (kb) from the genomic enhancer sequence or region.

17. (canceled)

18. The method claim 3, wherein the cell is a hematopoietic cell.

19. (canceled)

20. The method of claim 18, wherein the hematopoietic cell is a macrophage.

21. The method of claim 1, wherein the cell is a neuron.

22. The method of claim 1, wherein the cell is a breast cell.

23. The method of claim 1, wherein the cell is a cancer cell.

24. The method of claim 1, wherein the cell contacted with a specific inhibitor of an enhancer RNA (eRNA) is in a subject.

25. (canceled)

26. The method of claim 1, wherein the antisense compound is single-stranded.

27. The method of claim 1, wherein the antisense compound is double-stranded.

28. (canceled)

29. (canceled)

30. The method of claim 29, wherein the modified oligonucleotide is 12 to 30 nucleosides in length.

31. (canceled)

32. The method of claim 1, wherein the modified oligonucleotide comprises at least one modified sugar.

33. The method of claim 32, wherein the at least one modified sugar is a bicyclic sugar.

34. The method of claim 32, wherein at least one modified sugar comprises a 2'-O-methoxyethyl group.

35. (canceled)

36. The method of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

37-87. (canceled)