WO2020171889A1 - Blocking lipid accumulation or inflammation in thyroid eye disease - Google Patents

Abstract

This invention relates to compositions and methods for blocking lipid accumulation or inhibiting inflammation in cells or subjects, such as those with thyroid eye disease.

Description

Blocking Lipid Accumulation or Inflammation in Thyroid Eye Disease

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/807,302 filed on February 19, 2019. The content of the application is incorporated herein by reference in its entirety.

GOVERNMENT INTERESTS

This invention was made with government support under EY027308 awarded by National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to compositions and methods for blocking lipid accumulation and/or inflammation in cells of subjects with thyroid eye disease.

BACKGROUND OF THE INVENTION

Thyroid Eye Disease (TED) occurs in nearly half of patients with Graves’ disease, a common autoimmune disease involving the thyroid gland and stimulatory antibodies to the thyroid stimulating hormone receptor (TSHR). In TED, inflammation of the orbit leads to the accumulation of fat and scar tissue resulting from stimulation of resident orbital fibroblasts. Orbital fibroblasts can differentiate into either fat-forming adipocytes or scar-forming myofibroblasts. Other than invasive surgery or steroid treatment, few treatments can ameliorate TED symptoms. Furthermore, to date, there are no effective targeted therapies for this devastating disease. There is a need for compositions and methods for blocking lipid accumulation and/or inflammation in cells of subjects with TED and thereby treating TED.

SUMMARY OF INVENTION

This invention addresses the need mentioned above in a number of aspects.

In one aspect, the invention provides an inhibitory nucleic acid comprising a sequence that is (i) complementary to a contiguous sequence having at least 5 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, 20, 21, or 22) nucleotides present in microRNA hsa-miR-130a- 3p or microRNA hsa-miR-130b-3p, and (ii) chemically modified on at least one nucleotide. The sequence can be 5 to 200 (e.g., 10 to 100, 10 to 50, 13 to 50, 15 to 30, 20 to 25) nucleotides in length. The contiguous sequence can be at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1 or 2. In one example, the inhibitory nucleic acid is an antagomir, which can have the sequence of SEQ ID NO: 3 or 4. The inhibitory nucleic acid can have chemical modification such as 2'-0-methyl or N,N- diethyl-4-(4-nitronaphthalen-l-ylazo)-phenylamine (ZEN). One exemplar inhibitory nucleic acid with such chemical modifications comprises the sequence of SEQ ID NO: 5.

The invention also provides a pharmaceutical composition comprising the nucleic acid described above and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is suitable for ophthalmic use. The pharmaceutical com position can further comprise an additional therapeutic agent.

The invention also provides a method of decreasing lipid accumulation or inhibiting inflammation in a cell in a subject in need thereof. The method includes administering to the subject an inhibitory nucleic acid comprising a sequence that is complementary to a contiguous sequence having at least 5 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, 20, 21, or 22) nucleotides present in microRNA hsa-miR-130a-3p or micro RNA hsa-miR- 130b-3p.

The invention further provides a method of decreasing lipid accumulation or inhibiting an inflammation marker in a cell. The method includes contacting the cell with an inhibitory nucleic acid described above comprising a sequence that is complementary to a contiguous sequence having at least 5 (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 28, 19, 20, 21, or 22) nucleotides present in microRNA hsa-miR-130a-3p or microRNA hsa-miR- 130b-3p.

In each of the methods described herein, the sequence can be 5-200 (e.g., 10 to 100, 10 to 50, 13 to 50, 15 to 30, and 20 to 25) nucleotides in length. The contiguous sequence can be at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) identical to SEQ ID NO: 1 or 2. In one example, the inhibitory nucleic acid is an antagomir, which can have the sequence of SEQ ID NO: 3 or 4. The inhibitory nucleic acid can have chemical modification such as 2'-0-methyl or N,N-diethyl-4-(4-nitronaphthalen-l-ylazo)-phenylamine (ZEN). One exemplar inhibitory nucleic acid with such chemical modifications comprises the sequence of SEQ ID NO: 5. The cell can be a fibroblast, an adipocyte, or myofibroblast. The subject can be one having Graves’ disease or TED or one having a risk of having Graves’ disease or TED. The method described above can further comprise before or after the administering or contacting step (i) obtaining an expression level of miR-130a-3p or miR- 130b-3p in a sample from the subject and (ii) comparing the expression level with a predetermined reference value. In a further aspect, the invention provides a method for determining whether a subject has or is at risk of having TED. The method comprises (i) obtaining an expression level of miR-130a-3p or miR-130b-3p in a sample from the subject, and (ii) comparing the expression level with a predetermined reference value. The subject is determined to have, or to be at risk of having, TED if the expression level is above a predetermined reference value. The predetermined reference value can be obtained from a control subject or a control group. In one embodiment, the control subject does not have TED. In another, the control subject is a TED patient. Examples of the sample include a body fluid sample, such as blood, serum, and plasma.

The details of one or more embodiments of the invention are set forth in the description below. Other features, objectives, and advantages of the invention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGs. 1A and IB are diagrams showing that inhibition of miR-130a with an antagomir (Antagomir 130a) blocked triglyceride accumulation and IL-6 production. TED orbital fibroblasts were treated with control antagomir (non-specific miRNA) or miR-130a antagomir. After 4 days of culture, cells and cell culture supernatant were collected for analysis of triglyceride accumulation (A) and IL-6 secretion (B). (* =p< 0.05, **= p < 0.01, Student’s T Test). Data shown are using a second-generation antagomir/inhibitor from Table 1. A first generation antagomir/inhibitor did not significantly decrease triglycerides or IL-6 levels compared to control.

FIG. 2 is a diagram showing that Graves orbital fibroblasts (also called TED fibroblasts) expressed higher levels of miR-130a and miR-130b than normal (non- TED) orbital fibroblasts. Total RNA was isolated and analyzed for expression of miR-130a, miR- 130b and U6 snRNA (control) by RT-qPCR.

FIG. 3 is a set of photographs showing that AMP activated protein kinase (AMPK) is a target of miR-130a in TED fibroblasts.

FIG. 4 is a diagram showing that AMPK activity blocked lipid accumulation in TED orbital fibroblasts.

FIG. 5 is a diagram showing that a MiR-130 family member, miR-130b, is significantly higher in TED patient’s plasmas compared to control subjects’ plasmas. Data are from 4 control and 8 TED female age-matched patients. ## p < 0.01, T-Test. FIG. 6 is a set of photographs showing that MiR-130a controls TED orbital fibroblast AMPK expression and activity to promote fatty acid synthesis and lipid accumulation. Western blot showing AMPK subunits (alpha and beta) are downregulated by a miR-130a mimic. Importantly, Acetyl-CoA synthase (ACC), a rate-limiting enzyme for fatty acid synthesis, is also blocked by miR-130a mimic. Normally, AMPK phosphorylates ACC to block lipid accumulation. However, when miR-130a is highly expressed, AMPK levels are decreased allowing ACC to maintain activation and lead to synthesis of fatty acids for lipid storage.

FIG. 7 A and 7B are set of photographs and a diagram showing a novel link between Thyl, miR-130a and TSHR. (FIG. 7 A) Western blot showing Thyl is downregulated by a miR-130a mimic and induced by an antagomir-130 (a miRNA inhibitor that blocks functions of miR-130a) in two GOF strains. (FIG. 7B) TSHR activation by thyroid stimulatory hormone (TSH) (10 mU/mL) increases endogenous miR-130a expression and addition of a miR-130a inhibitor blocks TSH-induced miR-130a expression.

FIGs. 8 A, 8B, and 8C are a set of diagram showing that miR-130a controls inflammatory signaling in TED orbital fibroblasts. (FIG. 8A) Orbital fibroblasts were treated with control, miR-130a mimic or the antagomir-130. After two days, culture media was isolated and analyzed for inflammatory cytokines. miR-130a mimic expression increased both IL-6 and IL-8 while inhibition of miR-130a reduced expression of these inflammatory cytokines. (FIG. 8B) Orbital fibroblasts were treated with control or antagomir-miR-130a and treated with 10 mU/mL TSH to induce inflammatory signaling. After 2 days, cells were isolated and expression of inflammatory cytokines were measured by qPCR. TSH induced expression of both IL6 and IL8, however, addition of antagomir-miR-130a attenuated expression of the inflammatory mediators. (FIG. 8C) Orbital fibroblasts were treated with control or antagomir-miR-130a and treated with 5ng/mL interleukin- 1 beta (IL-1B) to induce inflammatory signaling. After 2 days, cells were isolated and expression of inflammatory microRNAs (miR-146a and miR-155) were measured by qPCR. IL-1B induced expression of both miR-146a and miR-155, however, addition of the antagomir-miR-130a attenuated expression of the inflammatory miRNAs. Mean + Std. Dev. *p < 0.01 by Student’s t-test.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compositions and methods for blocking lipid accumulation and/or inflammation in cells of subjects with thyroid eye disease. Certain aspects of this invention are based, at least in part, on an unexpected discovery that: (i) two closely related microRNAs, miR-130a and miR-130b, are upregulated in TED orbital fibroblasts compared to normal orbital fibroblasts; (ii) miR-130a and miR-130b decrease AMPK activity to increase lipid accumulation and activity of the pro-inflammatory transcription factor, NF-kB; and (iii) blocking miR-1 30a or miR-130b leads to a significant reduction in triglyceride accumulation (the intracellular storage form of fatty acids); and Thus, inhibiting miR-130a and miR-130b (and thereby regulating the key target AMPK) with stabilized and potent miR- 130 inhibitors is a novel therapy for TED. The inhibitors are useful in treating other inflammatory disorders of the eye including keratitis, uveitis, and dry eye.

MicroRNAs (miRNAs) are endogenous, small RNAs that serve to regulate up to 90% of all human genes by suppressing target mRNA translation and/or increasing target mRNA degradation. Additionally, miRNAs are essential regulators of inflammation and cellular differentiation, two processes that play a critical role in TED pathophysiology. As disclosed herein, blocking miR-130a function using a novel, stabilized miR-130a inhibitor can prevent lipid accumulation and inflammatory mediator production (see, e.g., Fig 1). Shown in the table below are some exemplary sequences.

Table 1

In the table, the underlined bases in miR-130b denote differences from miR-130a, while“m” indicates 2’-0 methyl group added to increase binding affinity to target miRNA and prevent endonuclease-mediated degradation. “ZEN” indicates a modification by the compound, N,N-diethyl-4-(4-nitronaphthalen-l-ylazo)-phenylamine, which prevents exonuclease activity to increase RNA stability. Shown below is the structure of the ZEN compound (Lennox, et al, Nucleic Acids (2013) 2, ell7;):

Inhibitory Nucleic Acids

The present invention encompasses inhibitory nucleic acids that decrease the expression or activity of any of the microRNAs ( e.g ., mature micro RNA or precursor microRNA) listed in Table 1 (e.g., hsa-miR-130a or hsa-miR-130b), or decrease the expression or activity of an inflammatory marker (e.g., IL-6 and other inflammation cytokines).

An inflammatory marker refers to a factor or protein whose expression level increases during the initiation and progression of inflammation· Examples of positive acute phase inflammatory markers include, but are not limited to, c-reactive protein, serum amyloid A, serum amyloid P component, complement proteins such C2, C3, C4, C5, C9, B, Cl inhibitor and C4 binding protein, fibrinogen, von Willebrand factor, al -antitrypsin, al- antichymo trypsin, a2-antiplasmin, heparin co factor II, plasminogen activator inhibitor I, haptoglobin, haemopexin, ceruloplasmin, manganese superoxide dismutase, al-acid glycoprotein, haeme oxygenase, mannose-binding protein, leukocyte protein I, lipoporotein (a), lipopolysaccharide-binding protein, and interleukins such as IL-1, IL-2, IL-6, IL-10 and receptors thereof. Additional inflammatory markers are described in e.g. , Ballantyne CM et al, Markers of inflammation and their clinical significance. Atheroscler Suppl. 2005 May;6(2):21-9, which is herein incorporated by reference.

Inhibitory nucleic acids useful in the present methods and compositions contains a sequence that hybridizes to at least a portion of a target nucleic acid and modulate its function. Examples include antagomirs, antisense oligonucleotides (RNA, DNA, or a chimeric thereof), ribozymes, external guide sequence (EGS) oligonucleotides, short interfering RNA (siRNA) compounds, micro interfering RNA (miRNA) compounds, small temporal RNA (stRNA) compounds, short hairpin RNA (shRNA) compounds, small RNA- induced gene activation (RNAa) compounds, small activating RNAs (saRNAs), peptide nucleic acids (PNAs), oligomeric compounds, or oligonucleotide mimetics or combinations thereof. Representative documents describing such compounds include, e.g., WO 2010/040112 and US 20140235697, each of which is herein incorporated by reference. In preferred embodiments, the inhibitory nucleic acids include antagomirs.

In some embodiments, an inhibitory nucleic acid can be an oligonucleotide that is about 5 to 200 (e.g., 10 to 100, 10 to 50, 13 to 50, 15 to 30, 20 to 25) nucleotides in length. One having ordinary skill in the art can appreciate that this embodies oligonucleotides having antisense portions of 5, 6, 7, 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, or 50 nucleotides in length, or any range therewithin. In some embodiments, the sequence that hybridizes to at least a portion of a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, or any range therewithin.

In some embodiments, the inhibitory nucleic acids can be chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference.

In some embodiments, the inhibitory nucleic acid comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-0-alkyl, 2'-0-alkyl-0-alkyl or 2'- fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2'- fluoro, 2'-amino, or/and 2' O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3' end of the RNA. Such modifications are incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (/._<?., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown to make an oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. The modified oligos can survive intact for a longer time than unmodified oligonucleotides. In one example, nucleotide and nucleoside modifications include one or more modification by the above-mentioned ZEN compound, N,N-diethyl-4-(4- nitronaphthalen-l-ylazo)-phenylamine, Lennox, et al, Nucleic Acids (2013) 2, ell7, which is incorporated by reference.

Other examples of suitable modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short-chain alkyl or cycloalkyl intersugar linkages, or short-chain heteroatomic or heterocyclic intersugar linkages. More preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2— NH— O— CH2, CH, ~N(CH3) -0-CH2 (known as a methylene(methylimino) or MMI backbone], CH2-0— N (CH3)-CH2, CH2-N(CH3)-N(CH3)-CH2 and 0-N(CH3)-CH2-CH2 backbones, wherein the native phosphodiester backbone is represented as O— P-O— CHI); amide backbones (see De Mesmaeker et al, Ace. Chem. Res. 28:366-374, 1995); morpholino backbone structures (see U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 254: 1497, 1991). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5 -2. See U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301 ; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361 ; and 5,625,050, each of which is incorporated by reference.

Morpholino-based oligomeric compounds are described in Braasch et al., Biochemistry 41(14):4503-4510, 2002; Genesis, volume 30, issue 3, 2001 ; Heasman, J., Dev. Biol., 243:209-214, 2002; Nasevicius et al., Nat. Genet. 26: 216-220, 2000; Lacerra et al., Proc. Natl. Acad. Sci. U.S.A. 97:9591-9596, 2000; and U.S. Pat. No. 5,034,506. Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc. 122, 8595-8602, 2000.

Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short- chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. See U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

One or more substituted sugar moieties can also be included, e.g., one of the following at the 2’ position: OH, SH, SCH₃, F, OCN, OCH₃, 0CH₃-0-(CH₂)n CH₃, 0(CH₂) n NH₂ or 0(CH₂) n CH₃, where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O— , S— , or N- alkyl; O— , S--, or N- alkenyl; SOCH3; S02 CH3; ON02; N02; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2'-methoxyethoxy[2'-0— CH₂CH₂OCH₃, also known as 2'-0-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta 78:486, 1995). Other preferred modifications include 2'- methoxy(2'-0— CH₃), 2'-propoxy(2'-OCH₂ CH₂CH₃) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.

Inhibitory nucleic acids can also include, additionally or alternatively, nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5- Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC, and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2 -thio thymine, 5-bromouracil, 5- hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6- diaminopurine. See Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-77; and Gebeyehu et al., Nucl. Acids Res. 15:4513, 1987, each of which is herein incorporated by reference. A "universal" base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2<0>C (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are presently preferred base substitutions.

In some examples, modified nucleobases comprise synthetic and natural nucleobases, such as 5-methylcytosine, 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 -thio thymine, and 2- thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo -uracil), 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-methylquanine and 7- methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, and 7-deazaadenine, and 3- deazaguanine and 3-deazaadenine.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact, more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at within a single nucleoside within an oligonucleotide.

In some embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al, Science 254:1497-1500, 1991.

In some embodiments, the inhibitory nucleic acids are chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the inhibitory nucleic acids. Such moieties comprise but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. 4:1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad. Sci. 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Lett. 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res. 20, 533-538, 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett. 259:327-330, 1990; Svinarchuk et al., Biochimie 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. 36:3651-3654, 1995; Shea et al., Nucl. Acids Res. 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett. 36:3651- 3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta 1264: 229-237, 1995), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. 277:923-937, 1996). See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;

5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;

5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;

4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481 ; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941 (each of which is herein incorporated by reference).

These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, and polyethers, groups that enhance the pharmacodynamic properties of nucleic acids, and groups that enhance the pharmacokinetic properties of nucleic acids. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence- specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism, or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No. PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, which are incorporated herein by reference. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethylammonium l,2-di-0-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,

5,580,731; 5,580,731 ; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;

5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;

4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;

5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;

5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;

5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481 ; 5,587,371; 5,595,726; 5,597,696;

5,599,923; 5,599,928 and 5,688,941 (each of which is incorporated by reference).

The inhibitory nucleic acids useful in the present methods are sufficiently complementary to the target miRNA, i.e. , hybridize sufficiently well and with sufficient specificity, to give the desired effect. "Complementary" refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miRNA, then the bases are considered complementary to each other at that position. In some embodiments, 100% complementarity is not required. In some embodiments, 100% complementarity is preferred. Routine methods can be used to design an inhibitory nucleic acid that binds to the target sequence with sufficient specificity.

While the specific sequences of certain exemplary target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure. Target segments of 5, 6, 7, 8, 9, 10 or more nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides within the seed sequence or immediately adjacent thereto, are considered suitable for targeting as well. In some embodiments, target segments can include sequences that comprise at least the 5 consecutive nucleotides from the 5'- terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately upstream of the 5'-terminus of the seed sequence and continuing until the inhibitory nucleic acid contains about 5 to about 30 nucleotides). In some embodiments, target segments are represented by RNA sequences that comprise at least the 5 consecutive nucleotides from the 3'-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same miRNA beginning immediately downstream of the 3'-terminus of the target segment and continuing until the inhibitory nucleic acid contains about 5 to about 30 nucleotides). One having skill in the art armed with the sequences provided herein will be able, without undue experimentation, to identify further preferred regions to target. In some embodiments, an inhibitory nucleic acid contain a sequence that is complementary to at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 contiguous nucleotides present in the target such as the target miRNA (e.g., mature or precursor hsa-miR-130a or hsa-miR-130b, or the target mRNA (e.g., IL-6).

Once one or more target regions, segments or sites have been identified, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i. e. , do not substantially bind to other non- target RNAs), to give the desired effect. In the context of this invention, hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Complementary, as used herein, refers to the capacity for precise pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a miRNA molecule or an mRNA molecule, then the inhibitory nucleic acid and the miRNA or mRNA are considered complementary to each other at that position. The inhibitory nucleic acids and the miRNA or mRNA are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the inhibitory nucleic acid and the miRNA target. For example, if a base at one position of an inhibitory nucleic acid is capable of hydrogen bonding with a base at the corresponding position of a miRNA or an mRNA, then the bases are considered complementary to each other at that position. A complementarity of 100% is not required.

It is understood in the art that a complementary nucleic acid sequence need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. A complementary nucleic acid sequence for purposes of the present methods is specifically hybridisable when binding of the sequence to the target miRNA or mRNA molecule interferes with the normal function of the target miRNA or mRNA to cause a loss of expression or activity, and there is a sufficient degree of complementarity to avoid non specific binding of the sequence to non-target RNA sequences under conditions in which specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed under suitable conditions of stringency. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30 °C, more preferably of at least about 37 °C, and most preferably of at least about 42 °C Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred embodiment, hybridization will occur at 30 °C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37 °C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 pg/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42 °C In 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, more preferably of at least about 42 °C, and even more preferably of at least about 68 C. In a preferred embodiment, wash steps will occur at 25 °C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68 °C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci. U.S.A. 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

In general, the inhibitory nucleic acids useful in the methods described herein have at least 80% sequence complementarity to a target region within the target nucleic acid, e.g., 90%, 95%, or 100% sequence complementarity to the target region within a miRNA. For example, an antisense compound in which 18 of 20 nucleobases of the antisense oligonucleotide are complementary, and would therefore specifically hybridize, to a target region would represent 90 percent complementarity. Percent complementarity of an inhibitory nucleic acid with a region of a target nucleic acid can be determined routinely using basic local alignment search tools (BLAST programs) (Altschul et al., J. Mol. Biol. 215:403-410, 1990; Zhang and Madden, Genome Res. 7:649-656, 1997).

Antisense and other compounds of the invention that hybridize to a miRNA or an mRNA can be identified through routine experimentation. In general, the inhibitory nucleic acids must retain specificity for their target, /._<?. , must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. For further disclosure regarding inhibitory nucleic acids, see US2010/0317718 (antisense oligos); US2010/0249052 (double- stranded ribonucleic acid (dsRNA)); US2009/0181914 and US2010/0234451 (LNAs); US2007/0191294 (siRNA analogues); US2008/0249039 (modified siRNA); and WO2010/129746 and W02010/040112 (inhibitory nucleic acids), each of which is herein incorporated by reference.

Antagomirs

In some embodiments, the inhibitory nucleic acid is an antagomir. Antagomirs are chemically modified antisense oligonucleotides that target a micro RNA (e.g., hsa-miR-130a or hsa-miR-130b). For example, an antagomir for use in the methods described herein can include a nucleotide sequence sufficiently complementary to hybridize to a miRNA target sequence of about 12 to 25 nucleotides, preferably about 15 to 22 nucleotides.

Preferably, an antagomir of a miRNA molecule is from 7 to 30 nucleotides in length, preferably 10 to 30 nucleotides in length, preferably 12 to 28 nucleotides in length, more preferably 20-22 nucleotides in length. Said molecule can have a length of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more.

The chemical structure of the nucleotides of an antagomir of a miRNA molecule or equivalent or source thereof may be modified to increase stability, binding affinity and/or specificity. The antagomir may comprise or consists of a RNA molecule or preferably a modified RNA molecule. A preferred modified RNA molecule comprises a modified sugar. One example of such modification is the introduction of a 2'-0-methyl or 2'-0-methoxyethyl group or 2' fluoride group on the nucleic acid to improve nuclease resistance and binding affinity to RNA. Another example of such modification is the introduction of a ZEN moiety, e.g. , at the 3'-end. In some embodiments, antagomirs have various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake. For example, in addition to the modifications discussed above for antisense oligos, an antagomir can have one or more of complete or partial 2'-0-methylation of sugar and/or a phosphorothioate backbone. Phosphorothioate modifications provide protection against RNase activity and their lipophilicity contributes to enhanced tissue uptake. In some embodiments, the antagomir can include one or more phosphorothioate backbone modifications. See, e.g., Krutzfeldt et al., Nature 438:685-689, 2005; Czech, N. Engl. J. Med. 354:1194-1195, 2006; Robertson et al., Silence 1 :10, 2010; Marquez and McCaffrey, Human Gene Ther. 19(1):27- 38, 2008; van Rooij et al., Circ. Res. 103(9):919-928, 2008; and Liu et al., Int. J. Mol. Sci. 9:978-999, 2008.

Antagomirs useful in the present methods can also be modified with respect to their length or otherwise the number of nucleotides making up the antagomir. In general, the antagomirs are about 20-22 nucleotides in length for optimal function, as this size matches the size of most mature microRNAs. The antagomirs must retain specificity for their target, i.e., must not directly bind to, or directly significantly affect expression levels of, transcripts other than the intended target. In some embodiments, the inhibitory nucleic acid can be locked and includes a cholesterol moiety (e.g., a locked antagomir).

Pharmaceutical Compositions

The present invention provides pharmaceutical compositions and formulations comprising any one or more of the inhibitory nucleic acids described above (e.g., one or more inhibitory nucleic acids targeting hsa-miR-130a and/or hsa-miR-130b or inflammatory marker proteins). The pharmaceutical compositions and formulations can be administered in any suitable way, including parenterally, topically, orally or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

The inhibitory nucleic acids can be administered alone or as a component of a pharmaceutical composition. These active agents may be formulated for administration, in any suitable way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants can also be present in the compositions. In some embodiments, one or more cationic lipids, cationic polymers, or nanoparticles can be included in compositions containing the one or more inhibitory nucleic acids (e.g. , compositions containing one or more inhibitory nucleic acids targeting hsa-miR-130a).

Ophthalmic Formulation

In some embodiments, the inhibitory nucleic acid or composition of the invention is used for treating various eye disorders or eye diseases, such as TED and others characterized by lipid accumulation and/or inflammation· Accordingly, compositions of the invention can be an ophthalmic formulation. Such ophthalmic formulations can be homogeneous or heterogeneous formulations. In one example, the one or more active agents of the invention can be administered topically to the eye, and a preferred embodiment of the formulation is a topical pharmaceutical composition suitable for application to the eye. Topical pharmaceutical compositions suitable for application to the eye in general include solutions, suspensions, dispersions, drops, gels, hydrogels and ointments. See, e.g., U.S. Pat. No. 5,407,926 and PCT applications WO 2004/058289, WO 01/30337 and WO 01/68053, the disclosures of all of which are incorporated herein by reference.

Topical formulations suitable for application to the eye comprise one or more active agents of the invention in an aqueous or nonaqueous base. The topical formulations can also include absorption enhancers, permeation enhancers, thickening agents, viscosity enhancers, agents for adjusting and/or maintaining the pH, agents to adjust the osmotic pressure, preservatives, surfactants, buffers, salts (preferably sodium chloride), suspending agents, dispersing agents, solubilizing agents, stabilizers and/or tonicity agents.

An absorption or permeation enhancer can promote absorption or permeation of the one or more active agents of the invention into the eye, while a thickening agent or viscosity enhancer is capable of increasing the residence time of one or more active agents of the invention in the eye. See PCT applications WO 2004/058289, WO 01/30337 and WO 01/68053, the contents of which are incorporated herein by reference. Exemplary absorption/permeation enhancers include methylsulfonylmethane, alone or in combination with dimethylsulfoxide, carboxylic acids and surfactants. Exemplary thickening agents and viscosity enhancers include dextrans, polyethylene glycols, polyvinylpyrrolidone, polysaccharide gels, GELRITE, cellulosic polymers (such as hydroxypropyl methylcellulose), carboxyl-containing polymers (such as polymers or copolymers of acrylic acid), polyvinyl alcohol and hyaluronic acid or a salt thereof.

Liquid dosage forms (e.g., solutions, suspensions, dispersions and drops) suitable for treatment of the eye can be prepared, for example, by dissolving, dispersing, suspending, etc. one or more active agents of the invention in a vehicle, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, to form a solution, dispersion or suspension. If desired, the pharmaceutical formulation may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH buffering agents and the like, for example sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.

Aqueous solutions and suspensions suitable for treatment of the eye can include preservatives, surfactants, buffers, salts (preferably sodium chloride), tonicity agents and water. If suspensions are used, the particle sizes can be less than 10 pm to minimize eye irritation. If solutions or suspensions are used, the amount delivered to the eye should not exceed 50 pi to avoid excessive spillage from the eye.

Colloidal suspensions suitable for treatment of the eye are generally formed from microparticles (/._<?., microspheres, nanospheres, microcapsules or nanocapsules, where microspheres and nanospheres are generally monolithic particles of a polymer matrix in which the formulation is trapped, adsorbed, or otherwise contained, while with microcapsules and nanocapsules the formulation is actually encapsulated). The upper limit for the size of these microparticles can be about 5 pm to about 10 pm.

Ophthalmic ointments suitable for treatment of the eye include one or more active agents of the invention in an appropriate base, such as mineral oil, liquid lanolin, white petrolatum, a combination of two or all three of the foregoing, or polyethylene-mineral oil gel. A preservative may optionally be included.

Ophthalmic gels suitable for treatment of the eye include one or more active agents of the invention suspended in a hydrophilic base, such as Carpobol-940 or a combination of ethanol, water and propylene glycol (e.g., in a ratio of 40:40:20). A gelling agent, such as hydroxylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, or ammoniated glycyrrhizinate, is used. A preservative and/or a tonicity agent may optionally be included.

Hydrogels suitable for treatment of the eye are formed by incorporation of a swellable, gel-forming polymer, such as those listed above as thickening agents or viscosity enhancers, except that a formulation referred to in the art as a "hydrogel" typically has a higher viscosity than a formulation referred to as a "thickened" solution or suspension. In contrast to such preformed hydrogels, a formulation may also be prepared so to form a hydrogel in situ following application to the eye. Such gels are liquid at room temperature but gel at higher temperatures (and thus are termed "thermoreversible" hydrogels), such as when placed in contact with body fluids. Biocompatible polymers that impart this property include acrylic acid polymers and copolymers, N-isopropylacrylamide derivatives and ABA block copolymers of ethylene oxide and propylene oxide (conventionally referred to as "poloxamers" and available under the PLURONIC tradename).

Preferred dispersions are liposomal, in which case the formulation is enclosed within liposomes (microscopic vesicles composed of alternating aqueous compartments and lipid bilayers).

Various vehicles can be used in the ophthalmic formulations of the present invention. These vehicles include, but are not limited to, purified water (water), polyvinyl alcohol, povidone, hydroxypropyl methyl cellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulose, cyclodextrin and a mixture of two or more thereof. The vehicle can be used in the formulation in amounts as needed to provide the concentration of the active agents or compounds disclosed herein. In one particular embodiment, the vehicle comprises water.

In some embodiments, the formulated composition contains an oil or a fatty acid ester. A fatty acid ester has the meaning commonly understood in the art, being an ester formed between an alcohol and a fatty acid. Exemplary fatty acid esters that are useful in formulations of the invention include, but are not limited to, triglyceride esters commonly known as vegetable oils, mono and diglyceride esters of fatty acids, fatty acid methyl esters, as well as other fatty acid esters that are known to one skilled in the art. It should be appreciated the fatty acid ester can be a mixture of several chemical compounds or an essentially pure compound. Typically, the fatty acid ester is a vegetable oil. Particular examples of vegetable oils that can be used include, but are not limited to, castor oil, sesame oil, soybean oil, cottonseed oil, olive oil, peanut oil, safflower oil, sunflower oil, palm oil, palm kernel oil, canola oil, and MIGLYOL OIL. In one particular embodiment, the fatty acid ester is castor oil.

In some embodiments of this invention, an emulsion-stabilizing polymer is used. While not intending to limit the scope of the invention, emulsion-stabilizing polymers generally contain hydrophilic groups such as cellulose, sugars, ethylene oxide, hydroxide, carboxylic acids or other polyelectrolytes. Without being bound by any theory, it is believed that these polymers help to stabilize emulsions by increasing the viscosity of the formulation as well as by reducing the interfacial tension. Some examples of emulsion stabilizing polymers useful in this invention include, but are not limited to, carbomers, PEMULEN, sodium carboxymethylcellulose, hydroxypropylmethylcellulose, povidone, polyvinyl alcohol, polyethylene glycol and a mixture of two or more thereof.

In another embodiment of this invention, the ophthalmic formulation comprises a surfactant. Without being bound by any theory, a surfactant is used to help facilitate the formation of the emulsion and improve its stability. Any type of surfactant can be used including, anionic, cationic, amphoteric, zwitterionic, nonionic, as well as a mixture of two or more thereof. In one particular embodiment, the formulation of the invention comprises a nonionic surfactant. Exemplary nonionic surfactants include, but are not limited to, polysorbates, poloxamers, alcohol ethoxylates, ethylene glycol-propylene glycol block copolymers, fatty acid amides, alkylphenol ethoxylates, phospholipids, and two or mixture thereof. In one particular embodiment, the surfactant is Polysorbate 80 (ICI Americas, Inc., Wilmington, Del.).

Various buffers and means for adjusting pH can be used so long as the resulting preparation is ophthalmically acceptable. Accordingly, useful buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers and borate buffers. In one particular embodiment, a buffering agent is used to maintain the pH in the therapeutically useful range of pH 4-10, typically about pH 5-8, often a pH range of 6.5-8.0, more often a pH range of 7.0-8.0, and most often a pH range of 1 2 1 6 It should be appreciated, however, that the scope of the invention is not limited to these particular pH ranges. In general, any pH range that provides suitable penetration of the active ingredient(s) through the eye can be used. Typically, a buffering agent known to those skilled in the art is used including, but not limited to, acetate, borate, tris, carbonate, citrate, histidine, succinate, and phosphate. In one particular embodiment, the buffering agent comprises boric acid. In another embodiment, the buffering agent comprises sodium citrate.

In another embodiment, a tonicity agent (or tonicity-adjusting agent) can be used to adjust the composition of the formulation to the desired isotonic range. The tonicity adjusting agent can be a polyol or a disaccharide including non-reducing disaccharides. Such tonicity agents are known to one skilled in the art, and include, but are not limited to, glycerin, mannitol, sorbitol, trehalose, xylitol, sodium chloride, and other electrolytes. In one particular embodiment, the tonicity agent is glycerin. In some embodiments, the formulations are preservative-free. In other embodiments, a preservative is used. Preservatives are used to prevent bacterial contamination in multiple- use ophthalmic preparations. Exemplary preservatives include, but are not limited to, benzalkonium chloride, stabilized oxychloro complexes (otherwise known as PURITE), phenylmercuric acetate, chlorobutanol, benzyl alcohol, parabens, and thimerosal.

Other excipient components or ingredients that can also be included in the ophthalmic formulations of the present invention are chelating agents and antibiotics. Suitable chelating agents are known in the art. Particular examples of useful chelating agents include, but are not limited to, edetate salts like edetate disodium, edetate calcium disodium, edetate sodium, edetate trisodium, and edetate dipotassium. In one particular embodiment, the chelating agent is edentate disodium. It should be appreciated that other chelating agents may also be used in place of or in conjunction with edentate disodium. Some examples of antibiotics that can be included in formulations of the invention include, but are not limited to, trimethoprim sulfate/polymyxin B sulfate, gatifloxacin, moxifloxacin hydrochloride, tobramycin, teicoplanin, vancomycin, azithromycin, clarithromycin, amoxicillin, penicillin, ampicillin, carbenicillin, ciprofloxacin, levofloxacin, amikacin, gentamicin, kanamycin, neomycin and streptomycin.

The formulations of the present invention can be packaged in various package forms known in the field of topical ophthalmics. In one embodiment, the formulation is packaged in sterile, preservative- free single-use packs or vials or containers (/._<?., the unit dose vials). Each vial may be made of low-density polyethylene to contain a small quantity of the formulation for a single use. This way, where the pharmaceutical composition is sterilized and contained in disposable single-dose containers for topical use in drop form, multiple vials in the form of a set of 30 vials, 60 vials and so on can be packaged in a tray with a lid, for example, a polypropylene tray with an aluminum peelable lid. The entire contents of each tray can be dispensed intact, and one vial or pack is used each time and immediately discarded after each use. For example, plastic ampules or vials or containers can be manufactured using blow-fill-seal (BFS) technology. The BFS processes may involve plastic extrusion, molding, aseptic filling, and hermetic sealing in one sequential operation and those processes are known in the art. In another embodiment, the formulation is packaged in multi dose vials such that the materials can be dispensed as sterile at each time using specialized container/closure maintaining the sterility integrity. In yet another embodiment, the formulation is packed in conventional vials/containers as sterile product. In some embodiments, the dosage form of the invention is eye drops of solution or suspension. Eye drops typically may contain aqueous/oily suspensions of the active ingredients in pharmaceutically acceptable carriers and/or excipients. Eye drops can be formulated with an aqueous or nonaqueous base comprising one or more dispersing agents, solubilizing agents or suspending agents. Drops can be delivered by means of a simple eye dropper-capped bottle or by means of a plastic bottle adapted to deliver liquid contents dropwise by means of a specially shaped closure.

The active agents of the invention can be administered via intraocular injection. Pharmaceutical formulations for intraocular injection can include solutions, emulsions, suspensions, particles, capsules, microspheres, liposomes, matrices, etc. See, e.g., U.S. Pat. No. 6,060,463, U.S. Patent Application Publication No. 2005/0101582, and PCT application WO 2004/043480, which are hereby incorporated by reference in their entirety.

The active agents of the invention can also be administered surgically as an ocular implant. For instance, a reservoir container having a diffusible wall of polyvinyl alcohol or polyvinyl acetate and containing one or more active agents of the invention can be implanted in or on the sclera. As another example, one or more active agents of the invention can be incorporated into a polymeric matrix made of a polymer, such as polycaprolactone, poly(glycolic) acid, poly(lactic) acid, poly( anhydride), or a lipid, such as sebacic acid, and may be implanted on the sclera or in the eye. This can be accomplished with a subject receiving a topical or local anesthetic and using a small incision made behind the cornea. The matrix is then inserted through the incision and sutured to the sclera.

Other Formulations

In addition to ophthalmic compositions, the compositions of the invention include those suitable for intradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g. , nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Pharmaceutical formulations of this invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such formulations can contain sweetening agents, flavoring agents, coloring agents, and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients that are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc., and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

The pharmaceutical composition can be administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi, J. Clin. Pharmacol. 35:1187-1193, 1995; Tjwa, Ann. Allergy Asthma Immunol. 75:107-111, 1995). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols. In some embodiments, the pharmaceutical compounds can be delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

In some embodiments, the pharmaceutical composition can be delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug that slowly release subcutaneously; see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gel formulations, see, e.g., Gao, Pharm. Res. 12:857-863, 1995; or, as microspheres for oral administration, see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997.

In some embodiments, the pharmaceutical composition can be parenterally administered, such as by intravenous (IV) administration or administration into a body cavity, a lumen of an organ, or into the cranium (e.g., intracranial injection or infusion) or the cerebrospinal fluid of a subject. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well-known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity-adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally acceptable diluent or solvent, such as a solution of 1,3- butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time.

In some embodiments, the pharmaceutical compositions and formulations can be lyophilized. Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g. , mannitol, trehalose, raffinose, and sucrose, or mixtures thereof.

The compositions and formulations can be delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al- Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm. 46:1576-1587, 1989.

Additional Active Agents

The inhibitory nucleic acids can be preferably administered in the form of pharmaceutical formulation that includes one or more of the inhibitory nucleic acids, alone or in combination with one or more additional active agents, together with a pharmaceutically acceptable carrier. As disclosed herein compositions of the invention are useful for treatment of various eye disorders or eye diseases that are characterized by lipid accumulation and/or inflammation· The compositions may contain one or more additional active agents for inhibiting lipid accumulation or inflammation or for treating various related eye disorders (such as keratitis, uveitis, and dry eye). Examples of these additional active agents include an anti-inflammatory compound (e.g., non-steroidal anti-inflammatory drug), an alpha 2 adrenergic receptor agonist, a beta- adrenergic receptor agonist, an immunosuppressant, a calcineurin inhibitor (e.g., cyclosporine), a lymphocyte associated antigen antagonist, a beta-blocker, a prostaglandin analog, a histamine receptor antagonist, a carbonic anhydrase inhibitor; and an antibiotic.

Treatment Methods

The inhibitory nucleic acid or composition of the invention is useful for treatment of various eye disorders or eye diseases, such as TED and others characterized by lipid accumulation and/or inflammation· Examples of the eye diseases include, but not limited to, dry eye syndrome (keratoconjunctivitis sicca), Sjogren's syndrome, congenital alacrima, xerophthalmia (dry eye from vitamin A deficiency), keratomalacia, ocular rosacea, eyelid disorders, meibomian gland disease, meibomian gland dysfunction, ectropion, blepharitis, blepharochalasis, sarcoidosis, stye, hordeolum, chalazion, ptosis, pterygium, eyelid edema, eyelid dermatitis, trichiasis, madarosis, dacryoadenitis, stevens-johnson syndrome, ocular graft versus host disease, dacryocystitis, conjunctivitis, keratoconjunctivitis, blepharoconjunctivitis, blepharokeratoconjunctivitis, allergic conjunctivitis, vernal conjunctivitis, conjunctival suffusion, conjunctivochalasis, subconjunctival hemorrhage, pterygium, pinguecula, chemosis, iritis, iridocyclitis, anterior uveitis, glaucoma, ocular hypertension, red eye, keratitis, scleritis, episcleritis, peripheral ulcerative keratitis, neurotrophic keratitis, neurotrophic eye disease, corneal ulcer, ulcerative keratitis, corneal abrasion, photokeratitis, ultraviolet keratitis, exposure keratitis, superficial punctuate keratitis, thygeson's superficial punctuate keratopathy, herpes zoster keratitis, acne rosacea, corneal neovascularization, corneal dystrophy, epithelial basement membrane dystrophy, fuch's dystrophy, posterior polymorphous corneal dystrophy, macular corneal dystrophy, cyclitis, uveitis, iritis, post-operative inflammation following ocular surgery (/._<?., eyelid surgery, cataract surgery, corneal surgery, refractive surgery including photorefractive keratectomy, glaucoma surgery, lacrimal gland surgery, conjunctival surgery, eye muscle surgery), ocular surface conditions caused by chemical burns, thermal burns or physical trauma. Additional examples include ocular conditions caused by the following autoimmune or vascular disorders: rheumatoid arthritis, juvenile rheumatoid arthritis, ankulosing spondylitis, reiter's syndrome, enteropathic arthritis, psoriatic arthritis, discoid and systemic lupus erythematosus, multiple sclerosis, graves' disease, antiphospholipid syndrome, sarcoidosis, wegner's granulomatosis, behcet's syndrome, polyarteritis nodosa, takayasu's arteritis, dermatomyositis, psoriasis, relapsing polychondritis, vasculitis, sickle cell-anemia, type II diabetes, diabetic retinopathy, and a combination thereof.

A use of the invention preferably comprises the step of administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising an inhibitory nucleic acid molecule, an equivalent or a source thereof as defined herein. The formulations of the invention can be administered for prophylactic and/or therapeutic treatments.

In some embodiments, for therapeutic applications, compositions are administered to a subject who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications. For example, in some embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to reduce the number of symptoms or reduce the severity, duration, or frequency of one or more symptoms of an eye disorder in a subject.

The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, /._<?., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, /._<?., the active agents' rate of absorption, bio availability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones, J. Steroid Biochem. Mol. Biol. 58:611- 617, 1996; Groning, Pharmazie 51:337-341, 1996; Fotherby, Contraception 54:59-69, 1996; Johnson, J. Pharm. Sci. 84:1144-1146, 1995; Rohatagi, Pharmazie 50:610-613, 1995; Brophy, Eur. J. Clin. Pharmacol. 24:103-108, 1983; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent, and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, /._<?. , dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate.

Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases, or symptoms. In alternative embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray, or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005. In some embodiments, the methods described herein can include co-administration with other drugs or pharmaceuticals, e.g., any of the treatments of an eye disorder described herein.

In some other embodiments, a use of the invention comprises the step of administering to a subject a therapeutically effective amount of a pharmaceutical composition comprising a construct to express an inhibitory nucleic acid for decreasing the activity or steady state level of miRNA-130a or 130b or equivalent as defined herein. A nucleic acid construct may be an expression construct as further specified herein. Preferably, an expression construct can be a viral gene therapy vector selected from gene therapy vectors based on, e.g., an adenovirus, an adeno-associated virus (AAV), a herpes virus, a pox virus, an oncolytic virus vector and a retrovirus. A preferred viral gene therapy vector is an AAV or Lentiviral vector.

In this case, an RNA molecule may be encoded by a nucleic acid molecule comprised in a vector. The term "vector" is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be "exogenous," which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, lentivirus, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al, 1989 and Ausubel et al, 1996, both incorporated herein by reference. In addition to encoding a modified polypeptide such as modified gelonin, a vector may encode non-modified polypeptide sequences such as a tag or targeting molecule.

The term "expression vector" refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described.

It is to be understood that the delivery of oligonucleotides and/or expression vectors to a target cell can be accomplished using different methods. In certain embodiments, a transfection agent can be used. In general, a transfection agent (e.g., a transfection reagent and/or delivery vehicle) can be a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and enhances their entry into cells. Non-limiting examples of useful transfection reagents include cationic liposomes and lipids, polyamines, calcium phosphate precipitates, polycations, histone proteins, polyethylenimine, polylysine, and polyampholyte complexes. Another delivery method can include electroporating RNAs into a cell without inducing significant cell death. In addition, miRNAs can be transfected at different concentrations.

Non- limiting examples of useful reagents for delivery of a nucleic acid (e.g., miRNA, anti-miRNA, and expression vectors) include protein and polymer complexes (polyp lexes), lipids and liposomes (lipoplexes), combinations of polymers and lipids (lipopolyp lexes), and multilayered and recharged particles. Transfection agents may also condense nucleic acids. Transfection agents may also be used to associate functional groups with a polynucleotide. Functional groups can include cell targeting moieties, cell receptor ligands, nuclear localization signals, compounds that enhance release of contents from endosomes or other intracellular vesicles (such as membrane active compounds), and other compounds that alter the behavior or interactions of the compound or complex to which they are attached (interaction modifiers).

In certain embodiments, an inhibitory nucleic acid and a transfection reagent can be delivered systematically such as by injection. In other embodiments, they may be injected into particular areas comprising target cells, such as particular organs, or an inflamed tissue. A skilled artisan will be able to select and use an appropriate system for delivering inhibitory nucleic acid or an expression vector to target cells in vivo, ex vivo and/or in vitro without undue experimentation.

There are a number of ways in which expression vectors may be introduced into cells. In certain embodiments of the invention, the expression vector comprises a virus or engineered vector derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells.

The retroviruses are a group of single- stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells; they can also be used as vectors. Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984), lentivirus (WO 2008/071959, WO 2004/054512), Hemaglutinating Virus of Japan (WO 2004/035779), Baculovirus (WO 2006/048662) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988; Horwich et al, 1990).

Other suitable methods for nucleic acid delivery to affect expression of compositions of the present invention include any method by which a nucleic acid (e.g., DNA, including viral and nonviral vectors) can be introduced into an organelle, a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harlan and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); by electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al, 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al, 1987; Wong et al, 1980; Kaneda et al., 1989; Kato et al., 1991); by photochemical internalization (WO 2008/007073); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al, 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference). Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transfected or transformed. Biomarkers and Related Methods

As disclosed herein, miR-130a or b was identified as a bio marker for TED based on its altered expression patterns in TED patients and healthy subjects. The markers, related kits, reagents and systems disclosed herein can be used in determining whether a subject has or is at risk of having TED. Alternatively, they can be used for determining a prognosis of such a disorder in a subject.

Diagnosis Methods

In one aspect, the invention provides qualitative and quantitative information to determine whether a subject has or is predisposed to TED. A subject having TED or prone to it can be determined based on the expression levels, patterns, or profiles of the above- described miR- both in a test sample from the subject. In other words,

the RNAs can be used as markers to indicate the presence or absence of the disorder. Diagnostic and prognostic assays of the invention include methods for assessing the expression level of the markers. The methods and kits allow one to detect TED. For example, a relative increase in the expression level(s) of miR-130a or miR-130b or both from the blood is indicative of presence the disorder. Conversely, a lower expression level or a lack of the expression is indicative lack of the disorder.

The presence, level, or absence of miR-130a or miR-130b or both in a test sample can be evaluated by obtaining a test sample from a test subject and contacting the test sample with a compound or an agent capable of detecting the nucleic acid (e.g., RNA or DNA probe). The test sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The level of expression of a marker of interest can be measured in a number of ways, including measuring its RNA.

Expressed RNA samples can be isolated from biological samples using any of a number of well-known procedures. For example, biological samples can be lysed in a guanidinium-based lysis buffer, optionally containing additional components to stabilize the RNA. In some embodiments, the lysis buffer can contain purified RNAs as controls to monitor recovery and stability of RNA from cell cultures. Lysates may be used immediately or stored frozen at, e.g. , -80 °C. Optionally, total RNA can be purified from cell lysates (or other types of samples) using silica-based isolation in an automation-compatible, 96-well format, such as the RNEASY purification platform (QIAGEN, Inc.). Other RNA isolation methods are contemplated, such as extraction with silica-coated beads or guanidinium. Further methods for RNA isolation and preparation can be devised by one skilled in the art. The methods of the present invention can be performed using crude samples (e.g., blood, serum, plasma, or cell lysates). RNAse inhibitors are optionally added to the crude samples. When using crude cellular lysates, it should be noted that genomic DNA can contribute one or more copies of a target sequence, e.g., a gene, depending on the sample. In situations in which the target sequence is derived from one or more highly expressed genes, the signal arising from genomic DNA may not be significant. But for genes expressed at low levels, the background can be eliminated by treating the samples with DNAse, or by using primers that target splice junctions for subsequent priming of cDNA or amplification products.

The level of RNA corresponding to a marker or gene can be determined both in situ and in vitro. RNA isolated from a test sample or cDNA prepared from it can be used in sequencing, hybridization or amplification assays that include, Southern or Northern analyses, PCR analyses, and probe arrays. An exemplary diagnostic method for the detection of RNA levels involves contacting the isolated RNA or cDNA or cRNA with a nucleic acid probe that can hybridize to the RNA encoded by the gene. The probe can be a full-length nucleic acid or a portion thereof, such as an oligonucleotide of at least 10 nucleotides in length and sufficient to specifically hybridize under stringent conditions to the RNA.

In one format, RNA (or cDNA prepared from it) is immobilized on a surface and contacted with the probes, for example, by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In another format, the probes are immobilized on a surface and the RNA (or cDNA or cRNA) is contacted with the probes, for example, in a nucleic acid chip array. A skilled artisan can adapt known RNA detection methods for detecting the level of RNA (or cDNA or cRNA prepared from it).

The level of RNA (or cDNA prepared from it) in a sample encoded by a gene to be examined can be evaluated with nucleic acid amplification, e.g. , by standard PCR (U.S. Patent No. 4,683,202), RT-PCR (Bustin S. J Mol Endocrinol. 25:169-93, 2000), quantitative PCR (Ong Y. et al, Hematology. 7:59-67, 2002), real time PCR (Ginzinger D. Exp Hematol. 30:503-12, 2002), and in situ PCR (Thaker V. Methods Mol Biol. 115:379-402, 1999), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art.

In another embodiment, the methods of the invention further include contacting a control sample with a compound or agent capable of detecting the RNA of a gene and comparing the presence of the RNA in the control sample with the presence of the RNA in the test sample. The above-described methods and markers can be used to assess the risk of a subject for developing TED. In particular, the invention can be applied to those in high-risk cohort who already have certain risks to gain critical insight into early detection.

A change in levels of the markers can be detected prior to, or in the early stages of, the development of TED. The invention therefore also provides a method for screening a subject who is at risk of developing TED, comprising evaluating the level of the miR-130a or miR-130b or both in a sample (e.g. , the blood or a fraction thereof). Accordingly, an increased level of miR-130a or b or both in the biological sample as compared to the level in a control sample, is indicative of the subject being at risk for TED. Subjects with a change in the level of the maker(s) are candidates for farther monitoring and testing. Such further testing can comprise histological examination of tissue samples or other techniques within the skill in the art.

Prognosis Methods

The diagnostic methods described above can identify subjects having or at risk of developing TED. In addition, changes in expression levels and/or trends of the above- mentioned markers in a biological sample, e.g., blood, serum, or plasma, can provide an early indication of recovery or lack thereof. For example, a further increase or persistently increased expression level(s) of miR- both indicate a poor prognosis,

i.e., lack of improvement or health decline. Accordingly, these markers allow one to assess post-treatment recovery of TED. The analysis of this select group of markers or a subset thereof indicates outcomes of the conditions.

The prognostic assays described herein can be used to determine whether a subject is suitable to be administered with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat TED. For example, such assays can be used to determine whether a subject can be administered with an antagomir described herein.

Thus, also provided by this invention is a method of monitoring a treatment for TED in a subject. For this purpose, expression levels of the markers disclosed herein can be determined for test samples from a subject before, during, or after undergoing a treatment. The magnitudes of the changes in the levels as compared to a baseline level are then assessed. A decrease of the magnitudes of the changes after the treatment indicates that the subject can be further treated by the same treatment. For example, a relative decrease in the expression level of one or more of miR-130a and miR-130b, is indicative of recovery from the disorder. Conversely, further increase or persistent high expression levels of one or more of miR-130a and miR-130b is indicate lack of improvement.

Information obtained from practice of the above assays is useful in prognostication, identifying progression of, and clinical management of diseases and other deleterious conditions affecting an individual subject’s health status. In preferred embodiments, the foregoing diagnostic assays provide information useful in prognostication, identifying progression of and management of TED. The information more specifically assists the clinician in designing treatment regimens to treat such condition.

Kits

This invention further includes reagent kits and diagnostic systems containing reagents for performing the above-described methods, including methods for nucleic acid amplification, copying, primer extension, detection, identification, and/or quantification. To that end, one or more of the reaction components for the methods disclosed herein can be supplied in the form of a kit for use in the detection of a target nucleic acid. In such a kit, an appropriate amount of one or more reaction components is provided in one or more containers or held on a substrate (e.g., by electrostatic interactions or covalent bonding).

The kit described herein can include one or more primers for primer extension or PCR. The kit can also contain additional materials for practicing the above-described methods. In some embodiments, the kit contains some or all of the reagents, materials for performing a method according to the invention. The kit thus may comprise some or all of the reagents for performing a PCR reaction using the primers. Some or all of the components of the kits can be provided in containers separate from the container(s) containing the primers. Examples of additional components of the kits include one or more different polymerases, one or more primers that are specific for a control nucleic acid or for a target nucleic acid, one or more probes that are specific for a control nucleic acid or for a target nucleic acid, buffers for polymerization reactions (in IX or concentrated forms), and one or more dyes or fluorescent molecules for detecting polymerization products. The kit may also include one or more of the following components: supports, terminating, modifying or digestion reagents, osmolytes, and an apparatus for detecting a detection probe.

The reaction components used in an amplification and/or detection process may be provided in a variety of forms. For example, the components (e.g., enzymes, nucleotide triphosphates, probes and/or primers) can be suspended in an aqueous solution or as a freeze- dried or lyophilized powder, pellet, or bead. In the latter case, the components, when reconstituted, form a complete mixture of components for use in an assay.

A kit or system may contain, in an amount sufficient for at least one assay, any combination of the components described herein, and may further include instructions recorded in a tangible form for use of the components. In some applications, one or more reaction components may be provided in pre-measured single use amounts in individual, typically disposable, tubes or equivalent containers. With such an arrangement, the sample to be tested for the presence of a target nucleic acid can be added to the individual tubes and amplification carried out directly. The amount of a component supplied in the kit can be any appropriate amount, and may depend on the target market to which the product is directed. General guidelines for determining appropriate amounts may be found in, for example, Joseph Sambrook and David W. Russell, Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, 2001; and Frederick M. Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons, 2003.

The kits of the invention can comprise any number of additional reagents or substances that are useful for practicing a method of the invention. Such substances include, but are not limited to: reagents (including buffers) for isolating cells, reagent for lysis of cells, divalent cation chelating agents or other agents that inhibit unwanted nucleases, control DNA/RNA for use in ensuring that primers, the polymerase and other components of reactions are functioning properly, RNA isolation reagents (including buffers), amplification reaction reagents (including buffers), and wash solutions. The kits of the invention can be provided at any temperature. For example, for storage of kits containing protein components or complexes thereof in a liquid, it is preferred that they are provided and maintained below 0 °C, preferably at or below -20 °C, or otherwise in a frozen state.

The container(s) in which the components are supplied can be any conventional container that is capable of holding the supplied form, for instance, microfuge tubes, ampoules, bottles, or integral testing devices, such as fluidic devices, cartridges, lateral flow, or other similar devices. The kits can include either labeled or unlabeled nucleic acid probes for use in amplification or detection of target nucleic acids. In some embodiments, the kits can further include instructions to use the components in any of the methods described herein, e.g., a method using a crude matrix without nucleic acid extraction and/or purification. The kits or system can also include packaging materials for holding the container or combination of containers. Typical packaging materials for such kits and systems include solid matrices (e.g., glass, plastic, paper, foil, micro-particles and the like) that hold the reaction components or detection probes in any of a variety of configurations (e.g., in a vial, microtiter plate well, microarray, and the like).

Definitions

A nucleic acid or polynucleotide refers to a DNA molecule (e.g., a cDNA or genomic DNA), an RNA molecule (e.g. , an mRNA), or a DNA or RNA analog. A DNA or RNA analog can be synthesized from nucleotide analogs. The nucleic acid molecule can be single- stranded or double-stranded, but preferably is double- stranded DNA. An "isolated nucleic acid" refers to a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid or to that of any fragment of a naturally occurring genomic nucleic acid. The term therefore covers, for example, (a) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule but is not flanked by both of the coding sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; (b) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by polymerase chain reaction (PCR), or a restriction fragment; and (d) a recombinant nucleotide sequence that is part of a hybrid gene, i.e., a gene encoding a fusion protein. The nucleic acid described above can be used to express the protein of this invention. For this purpose, one can operatively linked the nucleic acid to suitable regulatory sequences to generate an expression vector.

As used herein, an "inhibitory nucleic acid" is a nucleic acid (e.g., RNA, RNA interference, miRNA, siRNA, shRNA, or antisense RNA molecule, or a portion thereof, or a mimetic thereof) that when administered to a mammalian cell results in a decrease in the expression of a target sequence of gene. Typically, an inhibitory nucleic acid comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. Typically, expression of a target is reduced by 10%, 25%, 50%, 75%, or even 90-100%.

In certain embodiments, an inhibitory nucleic acid is sufficiently complementary to a portion of the miRNA or pre-miRNA sequence of a miR (e.g., hsa-miR-130a-3p or hsa-miR- 130b-3p). The inhibitory nucleic acid can have a region that is at least 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a portion of the miRNA or pre-miRNA sequence of a miRNA. As used herein nucleic acids and/or nucleic acid sequences are "homologous" when they are derived, naturally or artificially, from a common ancestral nucleic acid or nucleic acid sequence. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or sequences thereof). The precise percentage of identity between sequences that is useful in establishing homology varies with the nucleic acid and protein at issue, but as little as 25% sequence identity is routinely used to establish homology. Higher levels of sequence identity, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more can also be used to establish homology. Methods for determining sequence similarity percentages (e.g., BLASTN using default parameters) are generally available. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

As used herein, the percent homology between two amino acid or nucleotide s sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (/._<?., % homology=# of identical positions/total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non- limiting examples below.

The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm known in the art, such as that of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

MicroRNAs or miRNAs are small RNAs of 17-25 nucleotides, which function as regulators of gene expression in eukaryotes. miRNAs are initially expressed in the nucleus as part of long primary transcripts called primary miRNAs (pri-miRNAs). Inside the nucleus, pri-miRNAs are partially digested by the enzyme Drosha, to form 65-120 nucleotide-long hairpin precursor miRNAs (pre- miRNAs). The precursor miRNAs have two regions of complementarity that enables them to form a stem-loop- or fold-back- like structure. They are exported to the cytoplasm for further processing by Dicer into shorter, mature miRNAs, which are the active molecules. In animals, these short RNAs comprise a 5' proximal "seed" region (nucleotides 2 to 8) which appears to be the primary determinant of the pairing specificity of the miRNA to the 3' untranslated region (3'-UTR) of a target mRNA.

In preferred embodiment, a miRNA molecule or an equivalent or mimic or isomiR thereof comprises at least 6 of the 7 contiguous nucleotides present in a given seed sequence of miR-130a-3p or miR-130b-3p as SEQ ID NO: 1 or 2 identified in Table 1 and has at least 70% identity over the whole mature sequence. Preferably, the identity is at least 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.

An antagomir of a miRNA molecule or equivalent thereof may be a nucleic acid, preferably a RNA that is complementary to a part of the corresponding miRNA molecule or equivalent thereof. Preferred antagomir are complementary to a part of sequences of mature miRNAs or isomiR (e.g. , SEQ ID NO: 1 or 2). A part may mean at least 50% of the length of the sequence, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In a preferred embodiment, an antagomir or an equivalent thereof is complementary to a seed sequence or a part of said seed sequence of a miRNA molecule or equivalent thereof. A part may mean at least 50% of the length of the seed sequence, at least 60%, at least 70%, at least 80%, at least 90% or 100%. Preferably, an antagomir is from 8 to 30 nucleotides in length, preferably 10 to 30 nucleotides in length, preferably 12 to 28 nucleotides in length, more preferably said molecule has a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more and is complementary to a part of sequences of mature miRNAs or isomiR. A part may mean at least 50% of the length of a given sequence, at least 60%, at least 70%, at least 80%, at least 90% or 100%.

The terms "miRNA-130a" or“miR-130a” refer to micro RNA- 130a, including miR- 130a, pri-miR-130a, pre-miR-130a, mature miR-130a, miRNA-130a seed sequence, sequences comprising a miRNA- 130a seed sequence, and variants thereof.

The terms "miRNA-130b" or“miR-130b” refer to micro RNA- 130b, including miR- 130b, pri-miR-130b, pre-miR-130b, mature miR-130b, miRNA-130b seed sequence, sequences comprising a miRNA- 130b seed sequence, and variants thereof.

As used herein, "hybridization", "hybridizes" or "capable of hybridizing" is understood to mean the forming of a double or triple stranded molecule or a molecule with partial double or triple stranded nature using techniques known to the skilled person such as southern blotting procedures. The term "anneal" as used herein is synonymous with "hybridize." The term "hybridization", "hybridize(s)" or "capable of hybridizing" may mean "low", "medium" or "high" hybridization conditions as defined below. Low to medium to high stringency conditions means prehybridization and hybridization at 42 °C in 5X SSPE, 0.3% SDS, 200 pg/ml sheared and denatured salmon sperm DNA, and either 25% 35% or 50% formamide for low to medium to high stringencies respectively. Subsequently, the hybridization reaction is washed three times for 30 minutes each using 2X SSC, 0.2% SDS and either 55 °C, 65 °C, or 75 °C for low to medium to high stringencies.

A vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The vector can be capable of autonomous replication or integrate into a host DNA. Examples of the vector include a plasmid, cosmid, or viral vector. The vector includes a nucleic acid in a form suitable for expression of the nucleic acid in a host cell. Preferably the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed.

A "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence, as well as tissue-specific regulatory and/or inducible sequences. The design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein or RNA desired, and the like. The expression vector can be introduced into host cells to produce a polypeptide of interest. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. A strong promoter is one which causes mRNAs to be initiated at high frequency.

The term "operably-linked" or “operably- linked” is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled to perform their usual function. Thus, a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence. For example, a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence. A flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably-linked" to the coding sequence. Each nucleotide sequence coding for a polypeptide will typically have its own operably linked promoter sequence.

"Expression cassette" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, which may include a promoter operably linked to the nucleotide sequence of interest that may be operably linked to termination signals. The coding region usually codes for a functional RNA of interest. The expression cassette including the nucleotide sequence of interest may be chimeric. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of a regulatable promoter that initiates transcription only when the host cell is exposed to some particular stimulus. In the case of a multicellular organism, the promoter can also be specific to a particular tissue or organ or stage of development.

Such expression cassettes can include a transcriptional initiation region linked to a nucleotide sequence of interest. Such an expression cassette is provided with a plurality of restriction sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.

The term "cytokines" generally refers to proteins produced by a wide variety of hematopoietic and non-hematopoietic cells that affect the behavior of other cells. Cytokines are important for both the innate and adaptive immune responses.

The term "subject" includes human and non-human animals. The preferred subject for treatment is a human. As used herein, the terms "subject" and "patient" are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms "subject" and "subjects" may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous monkey, chimpanzee, etc) and a human). In one embodiment, the subject is a human. In another embodiment, the subject is an experimental, non-human animal or animal suitable as a disease model.

As used herein,“treating” or“treatment” refers to administration of a compound or agent to a subject who has a disorder or is at risk of developing the disorder with the purpose to cure, alleviate, relieve, remedy, delay the onset of, prevent, or ameliorate the disorder, the symptom of the disorder, the disease state secondary to the disorder, or the predisposition toward the disorder. The terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition. "Ameliorating" generally refers to the reduction in the number or severity of signs or symptoms of a disease or disorder.

The terms "prevent," "preventing" and "prevention" generally refer to a decrease in the occurrence of disease or disorder in a subject. The prevention may be complete, e.g., the total absence of the disease or disorder in the subject. The prevention may also be partial, such that the occurrence of the disease or disorder in the subject is less than that which would have occurred without embodiments of the present invention. "Preventing" a disease generally refers to inhibiting the full development of a disease.

The term "therapeutically effective amount" generally refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disease or disorder. For example, with respect to the treatment of TED, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the lipid accumulation or level of an inflammatory cytokine by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

An "effective amount" or "therapeutically effective amount" of an inhibitory nucleic acid can also be an amount sufficient to produce the desired effect, e.g., inhibition of expression of a target sequence in comparison to the normal expression level detected in the absence of the inhibitory nucleic acid. Inhibition of expression of a target gene or target sequence by an inhibitory nucleic acid is achieved when the expression level of the target gene mRNA or protein is about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 0% relative to the expression level of the target gene mRNA or protein of a control sample. The desired effect of an inhibitory nucleic acid may also be measured by detecting an increase in the expression of genes down regulated by the miRNA targeted by the inhibitory nucleic acid.

The term“pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

A“pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be“acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active compound. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate.

The term“about” generally refers to plus or minus 10% of the indicated number. For example,“about 10%” may indicate a range of 9% to 11%, and“about 1” may mean from 0.9- 1.1. Other meanings of“about” may be apparent from the context, such as rounding off, so, for example“about 1” may also mean from 0.5 to 1.4.

A“sample” as used herein means any biological fluid or tissue obtained from an organism (e.g. , patient) or from components (e.g., blood) of an organism. The sample may be of any biological tissue, cell(s) or fluid. The sample may be a“clinical sample” which is a sample derived from a subject, such as a human patient or veterinary subject. The most suitable sample for use in this invention includes peripheral blood. Other useful biological samples may include, without limitation, whole blood, saliva, urine, synovial fluid, bone marrow, cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions, sputum, semen, amniotic fluid, bronchoalveolar lavage fluid, and other cellular exudates from a patient. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. A biological sample may also be referred to as a“patient sample.” A biological sample may also include a substantially purified or isolated protein, membrane preparation, or cell culture.

As used herein the term "reference value" refers to a value that statistically correlates to a particular outcome when compared to an assay result. In preferred embodiments, the reference value is determined from statistical analysis of studies that compare RNA expression with known clinical outcomes. The reference value may be a threshold score value or a cutoff score value. Typically, a reference value will be a threshold above (or below) which one outcome is more probable and below which an alternative outcome is more probable.

In one embodiment, a reference level may be one or more gene expression (e.g., in the form of RNA) levels expressed as an average of the level of the expression from samples taken from a control population of healthy (disease-free) subjects. In another embodiment, the reference level may be the level in the same subject at a different time, e.g., before the present assay, such as the level determined prior to the subject developing the disease or prior to initiating therapy. In general, samples are normalized by a common factor. For example, cell-containing samples are normalized by protein content or cell count. Nucleic acid samples may also be normalized relative to an internal control nucleic acid.

"Control" or "Control subject" as used herein refers to the source of the reference level (e.g., reference gene expression profiles) as well as the particular panel of control subjects identified in the examples below.

The terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative measurement, and include determining if a characteristic, trait, or feature is present or not. Assessing may be relative or absolute. Assessing the presence of a target includes determining the amount of the target present, as well as determining whether it is present or absent.

The term“prognosis” as used herein refers to the prediction of the probable course and outcome of a clinical condition or disease. A prognosis is usually made by evaluating factors or symptoms of a disease that are indicative of a favorable or unfavorable course or outcome of the disease.

The term“prediction” is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, The predictive methods of the present invention can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods described herein are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen.

The phrase“determining the prognosis” as used herein refers to the process by which the skilled artisan can predict the course or outcome of a condition in a patient. The term “prognosis” does not refer to the ability to predict the course or outcome of a condition with 100% accuracy instead, the skilled artisan will understand that the term“prognosis” refers to an increased probability that a certain course or outcome will occur; that is, that a course or outcome is more likely to occur in a patient exhibiting a given condition, when compared to those individuals not exhibiting the condition.

The terms“favorable prognosis” and“positive prognosis,” or“unfavorable prognosis” and“negative prognosis” as used herein are relative terms for the prediction of the probable course and/or likely outcome of a condition or a disease. A favorable or positive prognosis predicts a better outcome for a condition than an unfavorable or negative prognosis. In a general sense, a“favorable prognosis” is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition, whereas an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition.

EXAMPLES

Example 1

This example descibes evidence that blocking miR-130a function using a novel, stabilized miR-130a inhibitor (Table 1) prevented lipid accumulation and inflammatory mediator production.

Briefly, TED orbital fibroblasts were treated with a control antagomir (non-specific miRNA) or miR-130a antagomir (SEQ ID NO: 5) at 5 nM. After 4 days of culture, the cells and cell culture supernatant were collected for analysis of triglyceride accumulation and IL-6 secretion. The results are shown in FIGs. 1A and IB.

The results indicate that blocking miR-130a with the stabilized inhibitor leads to a significant reduction in triglyceride accumulation (the intracellular storage form of fatty acids) (Fig. 1A) and IL-6 (a potent inflammatory cytokine that is elevated in TED) (Fig. IB) (* =p< 0.05, **= p < 0.01, Student’s T Test). This data demonstrates the concept that targeting miR-130a using a stabilized miRNA antagomir (antagomir- 130a) is a novel and powerful treatment for the disease.

As shown in the table below, it was found that the first generation inhibitor (SEQ ID NO: 3) was not potent enough at the doses used (5 nM treatment with 1 dose per experiment) and did not significantly decrease triglycerides or IL-6 levels compared to control.

Interestingly, it was also found that miR-130a and miR-130b were both upregulated in TED orbital fibroblasts compared to normal orbital fibroblasts (FIG. 2). It is possible that the antagomir (SEQ ID NO: 5) can also block the function of miR-130b in addition to miR-130a.

Example 2

To farther examine the mechanism whereby miR-130 miRNAs could function, inventors searched for genes that may be targets of, and therefore repressed by, miR-130a and miR-130b. Analysis of putative miR-130a target genes was performed with three different prediction tools (MIRDB, PICTAR and TARGETSCAN). All three algorithms identified two candidate genes that encode subunits of 5’-AMP activated protein kinase (AMPK): AMPKA1 and AMPKB1. Further investigation revealed that AMPK maybe a critical regulator of lipid accumulation and inflammatory signaling in TED orbital fibroblasts (see below).

Briefly, two strains of TED orbital fibroblasts were treated with DMSO or 5 mM 15d- PGJ2 (an adipogenic PPARy ligand) in the presence of control miRNA or miR-130a for 8 days. Then, the cells were lysed and protein expression of AMPKA1, AMPKB1, and b-actin (as a loading control) were determined by Western blotting. As shown in FIG. 3, AMPKB1 and AMPKA1 were downregulated by both miR-130a expression and 15d-PGJ2. In particular, it was found that AMPKB1 subunit was dramatically reduced when exogenous miR-130a was introduced into TED fibroblasts. Thus, for the First time, it was shown that AMPK is regulated by miR- 130a.

Example 3

This example provides evidence that AMPK activity potently blocks lipid accumulation in TED orbital fibroblasts.

Briefly, TED orbital fibroblasts were treated with 5 pM 15d-PGJ2 for 10 days in the presence of 400 pM AICAR (an activator of AMPK) or 2 pM compound C (an inhibitor of AMPK). Lipid accumulation was measured with the ADIPORED assay. Compound C is highly specific inhibitor of AMPK activity. AICAR is a precursor to AMP and serves to activate AMPK.

The results are shown in FIG. 4. It was found that treatment of cells with the pro- adipogenic PPARy ligand 15d-PGJ2 promoted adipogenesis. In addition, activation of AMPK prevented lipid accumulation while inhibiting AMPK allowed even more lipid to accumulate.

Taken together, the results indicate that AMPK is a key target of miR-130a and miR- 130b. Thus by blocking miR-1 30a and miR- 130b, one can promote AMPK activity to decrease lipid accumulation and inflammation observed in TED.

Example 4

As disclosed herein miR-130a/b blocks AMPK to increase lipid accumulation and adipogenesis. In this example, assays were carried out to examine serum and plasma levels of miR-130a/b.

Briefly, serum and plasma samples were obtained from four control subjects and eight female age-matched TED patients. Levels of miR- 130a or b in the samples were obtained using quantitative PCR. The data were normalized to the housekeeping control small nuclear RNA, U6 snRNA. It was found that miR-130a/b levels were be detected in serum and plasma. The results are shown FIG. 5. As shown in the figure, elevated levels of miR-130b were detected in TED patients’ plasmas. More specifically, the control group had an average normalized level of miR-130b-3p of 1.0. In contrast, the TED patients had an average miR- 130b-3p level of 5.2. These results suggest that miR-130a/b levels can be as a biomarker for TED.

Example 5

Example 2 above identified that miR-130a/b targets AMPK. AMPK regulates energy homeostasis and inflammatory signaling. It is hypothesized that AMPK is a master regulator of lipid homeostasis in the orbit, where in TED, AMPK is blocked by high levels of miR- 130a/b to increase lipid accumulation and inflammation· In this example, assays were carried out to examine roles of miR-130a in controlling TED orbital fibroblast AMPK expression and activity to promote fatty acid synthesis and lipid accumulation.

Western blot assays were carried out to examine whether miR-130a controls TED orbital fibroblast AMPK expression and activity to promote fatty acid synthesis and lipid accumulation. The results are shown in FIG. 6. The Western blot results showed that AMPK subunits (alpha and beta) were downregulated by a miR-130a mimic. The miR-130a mimic is a synthetic analog of miR-130a that functions just like endogenous miR-130a. With this mimic, we show that detrimental effects of high levels of miR-130a. Importantly, Acetyl- CoA synthase, a rate-limiting enzyme for fatty acid synthesis, was also blocked by miR-130a mimic. Normally, AMPK phosphorylates ACC to block lipid accumulation. However, when miR-130a was highly expressed, AMPK levels were decreased allowing ACC to maintain activation and lead to synthesis of fatty acids for lipid storage. This is analogous to the effect of high expression of miR-130a in TED orbits which have abnormally high levels of fat tissue.

The results from these studies suggest that miR-130a/b inhibitor(s) and AMPK activators (e.g., metformin) could be novel TED therapeutics. Activation or increased expression of AMPK (e.g., by either antagomir-130a or metformin) can result in decreased inflammatory signaling and reduced lipid accumulation. This is significant since metformin is a clinically approved medication that activates AMPK and suggests a new therapeutic option for the treatment of TED. Thyl and Thyl⁺ Orbital fibroblasts (OFs) display distinct phenotypes where Thyl OFs were more prone to form lipid rich adipocytes and more prone to produce inflammatory mediators. It was found that miR-130a (miR-130a-3p) was increased 3-fold in Thyl^" cells while miR-130a (and the functionally equivalent miR-130b, data not shown) was increased 3.5 -fold in TED orbital fat tissue compared to non-TED fat.

Western blot assays here demonstrated a novel link between Thyl, miR-130a and TSHR. As shown in FIG. 7 A, Thyl was downregulated by a miR-130a mimic and induced by antagomir-130 in two fibroblast strains isolated from TED patients, referred to as GOFs (Graves’ Orbital Fibroblasts). As shown in FIG. 7B, TSHR activation by TSH (10 mU/mL) increased endogenous miR-130a expression and addition of a miR-130a inhibitor blocked TSH-induced miR-130a expression.

The results also show that miR-130a over-expression reduced Thyl expression to promote the formation of Thyl OFs (FIG. 7 A). Furthermore, TSHR (the primary autoantigen in TED) activation by its cognate agonist TSH increased endogenous miR-130a levels (FIG. 7B).

Example 6

In this example, assays were carried out to examine roles of miR-130a in controlling inflammatory signaling in TED orbital fibroblasts.

Briefly, orbital fibroblasts were treated with a control, a miR-130a mimic or an antagomir-130. After two days, the culture media were isolated and analyzed for inflammatory cytokines. As shown in FIG. 8 A, miR-130a mimic expression increased both IL-6 and IL-8 while inhibition of miR-130a reduced expression of these inflammatory cytokines.

As shown in FIG. 8B, orbital fibroblasts were treated with the control or the antagomir-miR-130a and treated with 10 mU/mL TSH to induce inflammatory signaling. After 2 days, the cells were isolated and expression of inflammatory cytokines were measured by qPCR. It was found that TSH induced expression of both IL6 and IL8, however, addition of the antagomir-miR-130a attenuated expression of the inflammatory mediators.

As shown in FIG. 8C, orbital fibroblasts were treated with the control or the antagomir-miR-130a and treated with 5ng/mL interleukin- 1 beta to induce inflammatory signaling. After 2 days, the cells were isolated and expression of inflammatory microRNAs (miR-146a and miR-155) were measured by qPCR. It was found that IL-1B induced expression of both miR-146a and miR-155, however, addition of antagomir-miR-130a attenuated expression of the inflammatory miRNAs.

These results provide further evidence that miR-130a controls inflammatory signaling in TED orbital fibroblasts. First, miR-130a mimic expression increased both IL-6 and IL-8 while inhibition of miR-130a reduced expression of these inflammatory cytokines (FIG. 8A). Second, in OFs treated with TSH to induce inflammatory signaling, antagomir-miR-130a attenuated expression of the inflammatory mediators IF6 and IF8 (FIG. 8B). Finally, in OFs were treated with interleukin- 1 beta to induce inflammatory signaling, antagomir-miR-130a attenuated expression of the inflammatory miRNAs, miR-146a and miR-155 (FIG. 8C). These results support the concept that Thyl, TSHR, and miR-130a are critical drivers of TED lipid accumulation and inflammation.

The above results indicate that blocking miR-130a using antagomir-130a can be the basis for limiting adipose-tissue remodeling and inflammation in TED. These novel interventions for TED can also be applied to other enigmatic orbital processes such as orbital inflammatory syndromes (e.g. pseudotumor of the orbit, sarcoid) and post-operative changes in the orbit such as socket contracture after enucleation. These results suggest that the antagomir- miR-130a/b and AMPK activator (e.g., metformin) are important therapeutics that could alleviate or cure TED in afflicted patients.

The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. An inhibitory nucleic acid comprising a sequence that is (i) complementary to a contiguous sequence having at least 5 nucleotides present in micro RNA hsa-miR-130a-3p or microRNA hsa-miR-130b-3p, and (ii) chemically modified on at least one nucleotide.

2. The inhibitory nucleic acid of claim 1, wherein said sequence is 5-200 nucleotides in length.

3. The inhibitory nucleic acid of any one of claims 1-2, wherein the contiguous sequence is at least 80% identical to SEQ ID NO: 1 or 2.

4. The inhibitory nucleic acid of any one of claims 1-3, wherein the inhibitory nucleic acid is an antagomir.

5. The inhibitory nucleic acid of claim 4, wherein the antagomir has a sequence of SEQ ID NO: 3 or 4.

6. The inhibitory nucleic acid of any one of claims 1-5, wherein the chemical modification comprises one or more of 2'-0-methyl or N,N-diethyl-4-(4-nitronaphthalen-l- ylazo)-phenylamine (ZEN).

7 The inhibitory nucleic acid of claim 6, comprising a sequence of SEQ ID NO: 5.

8. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-7, and a pharmaceutically acceptable carrier.

9 The pharmaceutical composition of claim 8, wherein the pharmaceutically acceptable carrier is suitable for ophthalmic use.

10. The pharmaceutical composition of any one of claims 8-9, further comprising an additional therapeutic agent.

11 A method of decreasing lipid accumulation or inhibiting inflammation in a cell in a subject in need thereof, comprising administering to the subject an inhibitory nucleic acid comprising a sequence that is complementary to a contiguous sequence having at least 5 nucleotides present in microRNA hsa-miR-130a-3p or microRNA hsa-miR-130b-3p.

12. A method of decreasing lipid accumulation or inhibiting an inflammation marker in a cell, comprising contacting the cell with an inhibitory nucleic acid comprising a sequence that is complementary to a contiguous sequence having at least 5 nucleotides present in microRNA hsa-miR-130a-3p or microRNA hsa-miR-130b-3p.

13. The method of claim 11 or 12, wherein the sequence is 5-200 nucleotides in length.

14. The method of any one of claims 11-13, wherein the contiguous sequence is at least 80% identical to SEQ ID NO: 1 or 2.

15. The method of any one of claims 11-14, wherein the inhibitory nucleic acid is an antagomir.

16. The method of claim 15, wherein the antagomir has a sequence of SEQ ID NO: 3 or 4.

17. The method of any one of claims 11-16, wherein the inhibitory nucleic acid is chemically modified on at least one nucleotide.

18. The method of claim 17, wherein the chemical modification comprises one or more of 2'-0-methyl or ZEN.

19. The method of claim 18, wherein the inhibitory nucleic acid comprises a sequence of SEQ ID NO: 5.

20. The method of any one of claims 11-19, wherein the cell is a fibroblast, an adipocyte, or myofibroblast.

21. The method of any one of claims 11 and 13-19, wherein the subject has Graves’ disease or Thyroid Eye Disease (TED) or has a risk of having Graves’ disease or TED.

22. The method of any one of claims 11 and 13-21, wherein the method further comprises (i) obtaining an expression level of miR-130a-3p or miR-130b-3p in a sample from the subject and (ii) comparing the expression level with a predetermined reference value.

23. A method for determining whether a subject has, or is at risk of having, TED, comprising

(i) obtaining an expression level of miR-130a-3p or miR-130b-3p in a sample from the subject, and

(ii) comparing the expression level with a predetermined reference value,

whereby the subject is determined to have, or to be at risk of having, TED if the expression level is above the predetermined reference value.

24. The method of claim 22 or 23 , wherein the predetermined reference value is obtained from a control subject or a control group.

25. The method of any one of claims 22-24, wherein the sample is a body fluid sample.

26. The method of claim 25 , where in the sample is selected from the group consisting of blood, serum, and plasma.