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WO2020171889A1 - Blocage de l'accumulation des lipides ou de l'inflammation dans la maladie oculaire thyroïdienne - Google Patents

Blocage de l'accumulation des lipides ou de l'inflammation dans la maladie oculaire thyroïdienne Download PDF

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WO2020171889A1
WO2020171889A1 PCT/US2019/068936 US2019068936W WO2020171889A1 WO 2020171889 A1 WO2020171889 A1 WO 2020171889A1 US 2019068936 W US2019068936 W US 2019068936W WO 2020171889 A1 WO2020171889 A1 WO 2020171889A1
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nucleic acid
sequence
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Collynn WOELLER
Steven Feldon
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University of Rochester
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/344—Position-specific modifications, e.g. on every purine, at the 3'-end
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/352—Nature of the modification linked to the nucleic acid via a carbon atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity
    • C12N2320/51—Methods for regulating/modulating their activity modulating the chemical stability, e.g. nuclease-resistance

Definitions

  • This invention relates to compositions and methods for blocking lipid accumulation and/or inflammation in cells of subjects with thyroid eye disease.
  • Thyroid Eye Disease 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).
  • TSHR thyroid stimulating hormone receptor
  • 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.
  • compositions and methods for blocking lipid accumulation and/or inflammation in cells of subjects with TED and thereby treating TED are no effective targeted therapies for this devastating disease.
  • This invention addresses the need mentioned above in a number of aspects.
  • 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.
  • 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).
  • ZEN N,N- diethyl-4-(4-nitronaphthalen-l-ylazo)-phenylamine
  • One exemplar inhibitory nucleic acid with such chemical modifications comprises the sequence of SEQ ID NO: 5.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid described above and a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • the control subject does not have TED.
  • the control subject is a TED patient.
  • the sample include a body fluid sample, such as blood, serum, and plasma.
  • 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.
  • A triglyceride accumulation
  • B IL-6 secretion
  • 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.
  • AMPK AMP activated protein kinase
  • 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.
  • ACC Acetyl-CoA synthase
  • AMPK phosphorylates ACC to block lipid accumulation.
  • 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.
  • TSH thyroid stimulatory hormone
  • 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.
  • 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.
  • 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
  • MicroRNAs 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.
  • 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;):
  • 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).
  • microRNAs e.g ., mature micro RNA or precursor microRNA listed in Table 1
  • 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 ⁇
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • beneficial properties such as increased nuclease resistance, increased uptake into cells, increased binding affinity for the target
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PNA peptide nucleic acid
  • 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.
  • 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.
  • One or more substituted sugar moieties can also be included, e.g., one of the following at the 2’ position: OH, SH, SCH 3 , F, OCN, OCH 3 , 0CH 3 -0-(CH 2 )n CH 3 , 0(CH 2 ) n NH 2 or 0(CH 2 ) n CH 3 , 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 inter
  • a preferred modification includes 2'-methoxyethoxy[2'-0— CH 2 CH 2 OCH 3 , also known as 2'-0-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta 78:486, 1995).
  • Other preferred modifications include 2'- methoxy(2'-0— CH 3 ), 2'-propoxy(2'-OCH 2 CH 2 CH 3 ) 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.
  • nucleobase often referred to in the art simply as “base” modifications or substitutions.
  • “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)aden
  • 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
  • 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.
  • an oligomeric compound an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • lipid moieties such as a cholesterol moiety, cholic acid, a thio
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • target miRNA e.g., mature or precursor hsa-miR-130a or hsa-miR-130b
  • the target mRNA e.g., IL-6
  • 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.
  • hybridization means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases.
  • adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
  • Complementary refers to the capacity for precise pairing between two nucleotides.
  • 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
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • SDS sodium dodecyl sulfate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • antagomirs have various modifications for RNase protection and pharmacologic properties such as enhanced tissue and cellular uptake.
  • 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.
  • 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.
  • 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.
  • 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.
  • the inhibitory nucleic acid can be locked and includes a cholesterol moiety (e.g., a locked antagomir).
  • 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.
  • 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).
  • compositions of the invention can be an ophthalmic formulation.
  • Such ophthalmic formulations can be homogeneous or heterogeneous formulations.
  • 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.
  • exemplary absorption/permeation enhancers include methylsulfonylmethane, alone or in combination with dimethylsulfoxide, carboxylic acids and surfactants.
  • 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 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.
  • a vehicle such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like
  • 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.
  • 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.
  • the vehicle comprises water.
  • 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.
  • 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.
  • the fatty acid ester is castor oil.
  • 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.
  • the ophthalmic formulation comprises a surfactant.
  • 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.
  • the formulation of the invention comprises a nonionic surfactant.
  • 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.
  • the surfactant is Polysorbate 80 (ICI Americas, Inc., Wilmington, Del.).
  • useful buffers include, but are not limited to, acetate buffers, citrate buffers, phosphate buffers and borate buffers.
  • 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.
  • 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.
  • the buffering agent comprises boric acid.
  • the buffering agent comprises sodium citrate.
  • 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.
  • the tonicity agent is glycerin.
  • 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.
  • chelating agents include, but are not limited to, edetate salts like edetate disodium, edetate calcium disodium, edetate sodium, edetate trisodium, and edetate dipotassium.
  • 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.
  • 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.
  • 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.
  • 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.
  • each tray can be dispensed intact, and one vial or pack is used each time and immediately discarded after each use.
  • plastic ampules or vials or containers can be manufactured using blow-fill-seal (BFS) technology.
  • BFS blow-fill-seal
  • 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.
  • 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.
  • the formulation is packed in conventional vials/containers as sterile product.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • 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.
  • the pharmaceutical composition can be delivered as microspheres for slow release in the body.
  • 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.
  • 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.
  • IV intravenous
  • a pharmaceutically acceptable carrier e.g., water and Ringer's solution, an isotonic sodium chloride.
  • sterile fixed oils can be employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid can likewise be used in the preparation of injectables.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions and formulations can be delivered by the use of liposomes.
  • 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.
  • 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.
  • 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).
  • 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.
  • an anti-inflammatory compound e.g., non-steroidal anti-inflammatory drug
  • an alpha 2 adrenergic receptor agonist e.g., a beta- adrenergic receptor agonist
  • an immunosuppressant e.g., a calcineurin inhibitor (e.g., cyclosporine)
  • a lymphocyte associated antigen antagonist e.g., a beta-blocker
  • a prostaglandin analog e.g., a
  • 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 ⁇
  • 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, dac
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an RNA molecule may be encoded by a nucleic acid molecule comprised in a vector.
  • 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).
  • viruses bacteriophage, animal viruses, lentivirus, and plant viruses
  • artificial chromosomes e.g., YACs.
  • a vector may encode non-modified polypeptide sequences such as a tag or targeting molecule.
  • 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.
  • control sequences refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism.
  • vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described.
  • a transfection agent can be used.
  • a transfection agent e.g., a transfection reagent and/or delivery vehicle
  • a transfection agent can be a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and enhances their entry into cells.
  • 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.
  • miRNAs can be transfected at different concentrations.
  • Non- limiting examples of useful reagents for delivery of a nucleic acid 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).
  • 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.
  • 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.
  • the expression vector comprises a virus or engineered vector derived from a viral genome.
  • 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).
  • viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal
  • a nucleic acid e.g., DNA, including viral and nonviral vectors
  • 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.
  • 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.
  • 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.
  • 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.
  • RNA samples can be isolated from biological samples using any of a number of well-known procedures.
  • biological samples can be lysed in a guanidinium-based lysis buffer, optionally containing additional components to stabilize the RNA.
  • 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.
  • 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.).
  • 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.
  • crude samples e.g., blood, serum, plasma, or cell lysates
  • RNAse inhibitors are optionally added to the crude samples.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the diagnostic methods described above can identify subjects having or at risk of developing TED.
  • 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.
  • 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.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate
  • 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.
  • a relative decrease in the expression level of one or more of miR-130a and miR-130b is indicative of recovery from the disorder.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • kits examples 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.
  • reaction components used in an amplification and/or detection process may be provided in a variety of forms.
  • the components e.g., enzymes, nucleotide triphosphates, probes and/or primers
  • the components can be suspended in an aqueous solution or as a freeze- dried or lyophilized powder, pellet, or bead.
  • 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.
  • 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.
  • 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.
  • 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).
  • solid matrices e.g., glass, plastic, paper, foil, micro-particles and the like
  • 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.
  • 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.
  • 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.
  • expression of a target is reduced by 10%, 25%, 50%, 75%, or even 90-100%.
  • 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.
  • 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.
  • the percent homology between two amino acid or nucleotide s sequences is equivalent to the percent identity between 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.
  • the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • 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.
  • hybridization 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.
  • 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.
  • 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.
  • flanking sequence operably linked refers to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled to perform their usual function.
  • a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence.
  • 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.
  • 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.
  • 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.
  • the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment.
  • 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).
  • the subject is a human.
  • the subject is an experimental, non-human animal or animal suitable as a disease model.
  • “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.
  • prevent 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.
  • a 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.
  • 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.
  • 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.
  • 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.
  • “about” generally refers to plus or minus 10% of the indicated number.
  • “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.
  • 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.
  • the term "reference value” refers to a value that statistically correlates to a particular outcome when compared to an assay result.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • determining means 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.
  • prognosis 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.
  • 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.
  • determining the prognosis 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.
  • 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.
  • a“favorable prognosis” is an outcome that is relatively better than many other possible prognoses that could be associated with a particular condition
  • an unfavorable prognosis predicts an outcome that is relatively worse than many other possible prognoses that could be associated with a particular condition.
  • 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.
  • 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 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.
  • 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.
  • miR-130 miRNAs could function as 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).
  • AMPK activated protein kinase
  • AMPKB1 and AMPKA1 were downregulated by both miR-130a expression and 15d-PGJ2.
  • AMPKB1 subunit was dramatically reduced when exogenous miR-130a was introduced into TED fibroblasts.
  • AMPK is regulated by miR- 130a.
  • 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.
  • miR-130a/b blocks AMPK to increase lipid accumulation and adipogenesis.
  • assays were carried out to examine serum and plasma levels of miR-130a/b.
  • 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 ⁇
  • 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.
  • miR-130a 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.
  • miR-130a/b inhibitor(s) and AMPK activators could be novel TED therapeutics.
  • Activation or increased expression of AMPK can result in decreased inflammatory signaling and reduced lipid accumulation.
  • 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 display distinct phenotypes where Thyl OFs were more prone to form lipid rich adipocytes and more prone to produce inflammatory mediators.
  • 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.
  • FIG. 7 A Western blot assays here demonstrated a novel link between Thyl, miR-130a and TSHR.
  • 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).
  • GOFs GEFs
  • 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.
  • assays were carried out to examine roles of miR-130a in controlling inflammatory signaling in TED orbital fibroblasts.
  • 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.
  • 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.
  • orbital fibroblasts were treated with the control or the antagomir-miR-130a and treated with 5ng/mL interleukin- 1 beta to induce inflammatory signaling.
  • 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.
  • miR-130a controls inflammatory signaling in TED orbital fibroblasts.
  • miR-130a mimic expression increased both IL-6 and IL-8 while inhibition of miR-130a reduced expression of these inflammatory cytokines (FIG. 8A).
  • antagomir-miR-130a attenuated expression of the inflammatory mediators IF6 and IF8 (FIG. 8B).
  • antagomir-miR-130a attenuated expression of the inflammatory miRNAs, miR-146a and miR-155 (FIG. 8C).

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Abstract

La présente invention concerne des compositions et des méthodes pour bloquer l'accumulation des lipides ou l'inflammation dans des cellules ou des sujets, tels que ceux atteints de la maladie oculaire thyroïdienne
PCT/US2019/068936 2019-02-19 2019-12-30 Blocage de l'accumulation des lipides ou de l'inflammation dans la maladie oculaire thyroïdienne Ceased WO2020171889A1 (fr)

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Citations (156)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4667025A (en) 1982-08-09 1987-05-19 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US4835263A (en) 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use
US4876335A (en) 1986-06-30 1989-10-24 Wakunaga Seiyaku Kabushiki Kaisha Poly-labelled oligonucleotide derivative
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5013830A (en) 1986-09-08 1991-05-07 Ajinomoto Co., Inc. Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5112963A (en) 1987-11-12 1992-05-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Modified oligonucleotides
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5149797A (en) 1990-02-15 1992-09-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of rna and production of encoded polypeptides
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5220007A (en) 1990-02-15 1993-06-15 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
WO1994009699A1 (fr) 1992-10-30 1994-05-11 British Technology Group Limited Methode d'examen corporel
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5384253A (en) 1990-12-28 1995-01-24 Dekalb Genetics Corporation Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
WO1995006128A2 (fr) 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5407926A (en) 1987-12-29 1995-04-18 Alcon Laboratories, Inc. Ophthalmic composition
US5414077A (en) 1990-02-20 1995-05-09 Gilead Sciences Non-nucleoside linkers for convenient attachment of labels to oligonucleotides using standard synthetic methods
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5491133A (en) 1987-11-30 1996-02-13 University Of Iowa Research Foundation Methods for blocking the expression of specifically targeted genes
US5510475A (en) 1990-11-08 1996-04-23 Hybridon, Inc. Oligonucleotide multiple reporter precursors
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5538880A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5545730A (en) 1984-10-16 1996-08-13 Chiron Corporation Multifunctional nucleic acid monomer
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5587371A (en) 1992-01-21 1996-12-24 Pharmacyclics, Inc. Texaphyrin-oligonucleotide conjugates
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5599928A (en) 1994-02-15 1997-02-04 Pharmacyclics, Inc. Texaphyrin compounds having improved functionalization
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5610042A (en) 1991-10-07 1997-03-11 Ciba-Geigy Corporation Methods for stable transformation of wheat
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5623065A (en) 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5652356A (en) 1995-08-17 1997-07-29 Hybridon, Inc. Inverted chimeric and hybrid oligonucleotides
US5652355A (en) 1992-07-23 1997-07-29 Worcester Foundation For Experimental Biology Hybrid oligonucleotide phosphorothioates
US5656610A (en) 1994-06-21 1997-08-12 University Of Southern California Producing a protein in a mammal by injection of a DNA-sequence into the tongue
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5700922A (en) 1991-12-24 1997-12-23 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US5702932A (en) 1992-07-20 1997-12-30 University Of Florida Microinjection methods to transform arthropods with exogenous DNA
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5736524A (en) 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
US5789215A (en) 1991-08-20 1998-08-04 Genpharm International Gene targeting in animal cells using isogenic DNA constructs
US5945100A (en) 1996-07-31 1999-08-31 Fbp Corporation Tumor delivery vehicles
US5981274A (en) 1996-09-18 1999-11-09 Tyrrell; D. Lorne J. Recombinant hepatitis virus vectors
US5994624A (en) 1997-10-20 1999-11-30 Cotton Incorporated In planta method for the production of transgenic plants
US6007839A (en) 1996-02-16 1999-12-28 The Liposome Company, Inc. Preparation of pharmaceutical compositions containing etherlipid-containing multiple lipid liposomes
US6060463A (en) 1994-04-04 2000-05-09 William Freeman Treatment of conditions of abnormally increased intraocular pressure by administration of phosphonylmethoxyalkyl nucleoside analogs and related nucleoside analogs
US6063400A (en) 1997-07-02 2000-05-16 Sdg, Inc. Targeted liposomal constructs for diagnostic and therapeutic uses
WO2001030337A2 (fr) 1999-10-22 2001-05-03 Orbon Corporation Traitement topique de l'hypertension oculaire, du glaucome, de la retinopathie ischemique et de la degenerescence maculaire liee a l'age par une formulation ophtalmique d'antagonistes de la dopamine
US6287860B1 (en) 2000-01-20 2001-09-11 Isis Pharmaceuticals, Inc. Antisense inhibition of MEKK2 expression
WO2001068053A2 (fr) 2000-03-10 2001-09-20 Insite Vision Incorporated Procedes et compositions pour traiter et prevenir des affections ophtalmiques de la partie posterieure de l'oeil
WO2004035779A1 (fr) 2002-08-27 2004-04-29 Anges Mg, Inc. Procede de transfert d'une biomolecule utilisant une enveloppe virale et composition et systeme associes
WO2004043480A2 (fr) 2002-11-07 2004-05-27 Peyman Gholam A Traitement d'une maladie oculaire
WO2004054512A2 (fr) 2002-12-13 2004-07-01 Genetix Pharmaceuticals, Inc. Vecteurs retroviraux therapeutiques destines a la therapie genique
WO2004058289A1 (fr) 2002-12-20 2004-07-15 Chakshu Research, Inc. Formulation ophtalmique pour la prevention et le traitement de pathologies oculaires
US20050101582A1 (en) 2003-11-12 2005-05-12 Allergan, Inc. Compositions and methods for treating a posterior segment of an eye
WO2006048662A1 (fr) 2004-11-03 2006-05-11 Ark Therapeutics Ltd. Baculovirus pseudotypé et emploi dudit baculovirus
US20070191294A1 (en) 2003-03-21 2007-08-16 Santaris Pharma A/S Short interfering rna (sirna) analogues
WO2008007073A2 (fr) 2006-07-11 2008-01-17 Pci Biotech As Procédé
WO2008071959A1 (fr) 2006-12-12 2008-06-19 Oxford Biomedica (Uk) Limited Vecteurs lentiviraux comprenant des micro-arn
US20080249039A1 (en) 2004-01-30 2008-10-09 Santaris Pharma A/S Modified Short Interfering Rna (Modified Sirna)
US20090181914A1 (en) 2004-11-09 2009-07-16 Santaris Pharma A/S Lna oligonucleotides and the treatment of cancer
WO2009149182A1 (fr) * 2008-06-04 2009-12-10 The Board Of Regents Of The University Of Texas System Modulation de l'expression génique par ciblage d'un petit arn endogène de promoteurs de gènes
WO2010040112A2 (fr) 2008-10-03 2010-04-08 Curna, Inc. Traitement de maladies liées à l'apolipoprotéine a1 par l'inhibition d'un produit de la transcription antisens naturel de l'apolipoprotéine a1
US20100234451A1 (en) 2007-11-26 2010-09-16 Enzon Pharmaceuticals, Inc. Lna antagonists targeting the androgen receptor
US20100249052A1 (en) 2007-03-26 2010-09-30 Alnylam Pharmaceuticals, Inc. Dsrna compositions and methods for treating hpv infections
WO2010129746A2 (fr) 2009-05-06 2010-11-11 Curna, Inc. Traitement de maladies liées à la tristétraproline (ttp) par l'inhibition d'un transcrit antisens naturel de ttp
US20100317718A1 (en) 2004-08-25 2010-12-16 Isis Pharmaceuticals, Inc. Modulation of hif1 beta expression
WO2011017697A1 (fr) * 2009-08-07 2011-02-10 New York University Compositions et méthodes pour traiter des troubles inflammatoires
WO2014063155A1 (fr) * 2012-10-21 2014-04-24 University Of Rochester Thy1 (cd90) en tant que nouvelle thérapie pour réguler l'accumulation de tissu adipeux
US20140235697A1 (en) 2011-10-11 2014-08-21 The Brigham And Women's Hospital, Inc. Micrornas in neurodegenerative disorders
WO2014130945A1 (fr) * 2013-02-25 2014-08-28 Integrated Dna Technologies, Inc. Modifications de composés antisens
WO2015042435A1 (fr) * 2013-09-20 2015-03-26 Academia Sinica Traitement de maladies associées à pgc1-alpha par modulation des microarn mir-130a et mir-130b
US9209196B2 (en) 2011-11-30 2015-12-08 Sharp Kabushiki Kaisha Memory circuit, method of driving the same, nonvolatile storage device using the same, and liquid crystal display device
WO2016058061A1 (fr) * 2014-10-13 2016-04-21 Katholieke Universiteit Leuven Signatures de microarn pour troubles cardiaques
CN105903036A (zh) * 2015-07-15 2016-08-31 浙江大学 miR-130a反义核酸及其衍生物在Hippo-YAP信号通路抑制剂中的应用
CN106636368A (zh) * 2016-11-28 2017-05-10 山东大学 miR‑130a 在卵巢癌的诊断、治疗及预后中的应用

Patent Citations (174)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3687808A (en) 1969-08-14 1972-08-29 Univ Leland Stanford Junior Synthetic polynucleotides
US4469863A (en) 1980-11-12 1984-09-04 Ts O Paul O P Nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof
US5023243A (en) 1981-10-23 1991-06-11 Molecular Biosystems, Inc. Oligonucleotide therapeutic agent and method of making same
US4476301A (en) 1982-04-29 1984-10-09 Centre National De La Recherche Scientifique Oligonucleotides, a process for preparing the same and their application as mediators of the action of interferon
US4789737A (en) 1982-08-09 1988-12-06 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives and production thereof
US4667025A (en) 1982-08-09 1987-05-19 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US4835263A (en) 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use
US4605735A (en) 1983-02-14 1986-08-12 Wakunaga Seiyaku Kabushiki Kaisha Oligonucleotide derivatives
US5541313A (en) 1983-02-22 1996-07-30 Molecular Biosystems, Inc. Single-stranded labelled oligonucleotides of preselected sequence
US4948882A (en) 1983-02-22 1990-08-14 Syngene, Inc. Single-stranded labelled oligonucleotides, reactive monomers and methods of synthesis
US4824941A (en) 1983-03-10 1989-04-25 Julian Gordon Specific antibody to the native form of 2'5'-oligonucleotides, the method of preparation and the use as reagents in immunoassays or for binding 2'5'-oligonucleotides in biological systems
US4587044A (en) 1983-09-01 1986-05-06 The Johns Hopkins University Linkage of proteins to nucleic acids
US5118802A (en) 1983-12-20 1992-06-02 California Institute Of Technology DNA-reporter conjugates linked via the 2' or 5'-primary amino group of the 5'-terminal nucleoside
US5550111A (en) 1984-07-11 1996-08-27 Temple University-Of The Commonwealth System Of Higher Education Dual action 2',5'-oligoadenylate antiviral derivatives and uses thereof
US5545730A (en) 1984-10-16 1996-08-13 Chiron Corporation Multifunctional nucleic acid monomer
US5258506A (en) 1984-10-16 1993-11-02 Chiron Corporation Photolabile reagents for incorporation into oligonucleotide chains
US5552538A (en) 1984-10-16 1996-09-03 Chiron Corporation Oligonucleotides with cleavable sites
US5578717A (en) 1984-10-16 1996-11-26 Chiron Corporation Nucleotides for introducing selectably cleavable and/or abasic sites into oligonucleotides
US4828979A (en) 1984-11-08 1989-05-09 Life Technologies, Inc. Nucleotide analogs for nucleic acid labeling and detection
US5235033A (en) 1985-03-15 1993-08-10 Anti-Gene Development Group Alpha-morpholino ribonucleoside derivatives and polymers thereof
US5185444A (en) 1985-03-15 1993-02-09 Anti-Gene Deveopment Group Uncharged morpolino-based polymers having phosphorous containing chiral intersubunit linkages
US5034506A (en) 1985-03-15 1991-07-23 Anti-Gene Development Group Uncharged morpholino-based polymers having achiral intersubunit linkages
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683202B1 (fr) 1985-03-28 1990-11-27 Cetus Corp
US4762779A (en) 1985-06-13 1988-08-09 Amgen Inc. Compositions and methods for functionalizing nucleic acids
US5317098A (en) 1986-03-17 1994-05-31 Hiroaki Shizuya Non-radioisotope tagging of fragments
US4876335A (en) 1986-06-30 1989-10-24 Wakunaga Seiyaku Kabushiki Kaisha Poly-labelled oligonucleotide derivative
US5013830A (en) 1986-09-08 1991-05-07 Ajinomoto Co., Inc. Compounds for the cleavage at a specific position of RNA, oligomers employed for the formation of said compounds, and starting materials for the synthesis of said oligomers
US5276019A (en) 1987-03-25 1994-01-04 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5286717A (en) 1987-03-25 1994-02-15 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US5264423A (en) 1987-03-25 1993-11-23 The United States Of America As Represented By The Department Of Health And Human Services Inhibitors for replication of retroviruses and for the expression of oncogene products
US4904582A (en) 1987-06-11 1990-02-27 Synthetic Genetics Novel amphiphilic nucleic acid conjugates
US5585481A (en) 1987-09-21 1996-12-17 Gen-Probe Incorporated Linking reagents for nucleotide probes
US5405939A (en) 1987-10-22 1995-04-11 Temple University Of The Commonwealth System Of Higher Education 2',5'-phosphorothioate oligoadenylates and their covalent conjugates with polylysine
US5188897A (en) 1987-10-22 1993-02-23 Temple University Of The Commonwealth System Of Higher Education Encapsulated 2',5'-phosphorothioate oligoadenylates
US5525465A (en) 1987-10-28 1996-06-11 Howard Florey Institute Of Experimental Physiology And Medicine Oligonucleotide-polyamide conjugates and methods of production and applications of the same
US5112963A (en) 1987-11-12 1992-05-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Modified oligonucleotides
US5491133A (en) 1987-11-30 1996-02-13 University Of Iowa Research Foundation Methods for blocking the expression of specifically targeted genes
US5403711A (en) 1987-11-30 1995-04-04 University Of Iowa Research Foundation Nucleic acid hybridization and amplification method for detection of specific sequences in which a complementary labeled nucleic acid probe is cleaved
US5407926A (en) 1987-12-29 1995-04-18 Alcon Laboratories, Inc. Ophthalmic composition
US5082830A (en) 1988-02-26 1992-01-21 Enzo Biochem, Inc. End labeled nucleotide probe
US5519126A (en) 1988-03-25 1996-05-21 University Of Virginia Alumni Patents Foundation Oligonucleotide N-alkylphosphoramidates
US5453496A (en) 1988-05-26 1995-09-26 University Patents, Inc. Polynucleotide phosphorodithioate
US5278302A (en) 1988-05-26 1994-01-11 University Patents, Inc. Polynucleotide phosphorodithioates
US5109124A (en) 1988-06-01 1992-04-28 Biogen, Inc. Nucleic acid probe linked to a label having a terminal cysteine
US5216141A (en) 1988-06-06 1993-06-01 Benner Steven A Oligonucleotide analogs containing sulfur linkages
US5262536A (en) 1988-09-15 1993-11-16 E. I. Du Pont De Nemours And Company Reagents for the preparation of 5'-tagged oligonucleotides
US5512439A (en) 1988-11-21 1996-04-30 Dynal As Oligonucleotide-linked magnetic particles and uses thereof
US5599923A (en) 1989-03-06 1997-02-04 Board Of Regents, University Of Tx Texaphyrin metal complexes having improved functionalization
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5391723A (en) 1989-05-31 1995-02-21 Neorx Corporation Oligonucleotide conjugates
US5256775A (en) 1989-06-05 1993-10-26 Gilead Sciences, Inc. Exonuclease-resistant oligonucleotides
US5416203A (en) 1989-06-06 1995-05-16 Northwestern University Steroid modified oligonucleotides
US4958013A (en) 1989-06-06 1990-09-18 Northwestern University Cholesteryl modified oligonucleotides
US5464765A (en) 1989-06-21 1995-11-07 Zeneca Limited Transformation of plant cells
US5302523A (en) 1989-06-21 1994-04-12 Zeneca Limited Transformation of plant cells
US5451463A (en) 1989-08-28 1995-09-19 Clontech Laboratories, Inc. Non-nucleoside 1,3-diol reagents for labeling synthetic oligonucleotides
US5254469A (en) 1989-09-12 1993-10-19 Eastman Kodak Company Oligonucleotide-enzyme conjugate that can be used as a probe in hybridization assays and polymerase chain reaction procedures
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5399676A (en) 1989-10-23 1995-03-21 Gilead Sciences Oligonucleotides with inverted polarity
US5264564A (en) 1989-10-24 1993-11-23 Gilead Sciences Oligonucleotide analogs with novel linkages
US5264562A (en) 1989-10-24 1993-11-23 Gilead Sciences, Inc. Oligonucleotide analogs with novel linkages
US5292873A (en) 1989-11-29 1994-03-08 The Research Foundation Of State University Of New York Nucleic acids labeled with naphthoquinone probe
US5455233A (en) 1989-11-30 1995-10-03 University Of North Carolina Oligoribonucleoside and oligodeoxyribonucleoside boranophosphates
US5166315A (en) 1989-12-20 1992-11-24 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5405938A (en) 1989-12-20 1995-04-11 Anti-Gene Development Group Sequence-specific binding polymers for duplex nucleic acids
US5486603A (en) 1990-01-08 1996-01-23 Gilead Sciences, Inc. Oligonucleotide having enhanced binding affinity
US5578718A (en) 1990-01-11 1996-11-26 Isis Pharmaceuticals, Inc. Thiol-derivatized nucleosides
US5538877A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5538880A (en) 1990-01-22 1996-07-23 Dekalb Genetics Corporation Method for preparing fertile transgenic corn plants
US5366878A (en) 1990-02-15 1994-11-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5220007A (en) 1990-02-15 1993-06-15 The Worcester Foundation For Experimental Biology Method of site-specific alteration of RNA and production of encoded polypeptides
US5149797A (en) 1990-02-15 1992-09-22 The Worcester Foundation For Experimental Biology Method of site-specific alteration of rna and production of encoded polypeptides
US5214136A (en) 1990-02-20 1993-05-25 Gilead Sciences, Inc. Anthraquinone-derivatives oligonucleotides
US5414077A (en) 1990-02-20 1995-05-09 Gilead Sciences Non-nucleoside linkers for convenient attachment of labels to oligonucleotides using standard synthetic methods
US5563253A (en) 1990-03-08 1996-10-08 Worcester Foundation For Biomedical Research Linear aminoalkylphosphoramidate oligonucleotide derivatives
US5541306A (en) 1990-03-08 1996-07-30 Worcester Foundation For Biomedical Research Aminoalkylphosphotriester oligonucleotide derivatives
US5321131A (en) 1990-03-08 1994-06-14 Hybridon, Inc. Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling
US5536821A (en) 1990-03-08 1996-07-16 Worcester Foundation For Biomedical Research Aminoalkylphosphorothioamidate oligonucleotide deratives
US5470967A (en) 1990-04-10 1995-11-28 The Dupont Merck Pharmaceutical Company Oligonucleotide analogs with sulfamate linkages
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5514785A (en) 1990-05-11 1996-05-07 Becton Dickinson And Company Solid supports for nucleic acid hybridization assays
US5618704A (en) 1990-07-27 1997-04-08 Isis Pharmacueticals, Inc. Backbone-modified oligonucleotide analogs and preparation thereof through radical coupling
US5610289A (en) 1990-07-27 1997-03-11 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogues
US5218105A (en) 1990-07-27 1993-06-08 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5138045A (en) 1990-07-27 1992-08-11 Isis Pharmaceuticals Polyamine conjugated oligonucleotides
US5688941A (en) 1990-07-27 1997-11-18 Isis Pharmaceuticals, Inc. Methods of making conjugated 4' desmethyl nucleoside analog compounds
US5602240A (en) 1990-07-27 1997-02-11 Ciba Geigy Ag. Backbone modified oligonucleotide analogs
US5541307A (en) 1990-07-27 1996-07-30 Isis Pharmaceuticals, Inc. Backbone modified oligonucleotide analogs and solid phase synthesis thereof
US5489677A (en) 1990-07-27 1996-02-06 Isis Pharmaceuticals, Inc. Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms
US5608046A (en) 1990-07-27 1997-03-04 Isis Pharmaceuticals, Inc. Conjugated 4'-desmethyl nucleoside analog compounds
US5623070A (en) 1990-07-27 1997-04-22 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5677437A (en) 1990-07-27 1997-10-14 Isis Pharmaceuticals, Inc. Heteroatomic oligonucleoside linkages
US5245022A (en) 1990-08-03 1993-09-14 Sterling Drug, Inc. Exonuclease resistant terminally substituted oligonucleotides
US5677439A (en) 1990-08-03 1997-10-14 Sanofi Oligonucleotide analogues containing phosphate diester linkage substitutes, compositions thereof, and precursor dinucleotide analogues
US5567810A (en) 1990-08-03 1996-10-22 Sterling Drug, Inc. Nuclease resistant compounds
US5623065A (en) 1990-08-13 1997-04-22 Isis Pharmaceuticals, Inc. Gapped 2' modified oligonucleotides
US5177196A (en) 1990-08-16 1993-01-05 Microprobe Corporation Oligo (α-arabinofuranosyl nucleotides) and α-arabinofuranosyl precursors thereof
US5512667A (en) 1990-08-28 1996-04-30 Reed; Michael W. Trifunctional intermediates for preparing 3'-tailed oligonucleotides
US5214134A (en) 1990-09-12 1993-05-25 Sterling Winthrop Inc. Process of linking nucleosides with a siloxane bridge
US5561225A (en) 1990-09-19 1996-10-01 Southern Research Institute Polynucleotide analogs containing sulfonate and sulfonamide internucleoside linkages
US5596086A (en) 1990-09-20 1997-01-21 Gilead Sciences, Inc. Modified internucleoside linkages having one nitrogen and two carbon atoms
US5510475A (en) 1990-11-08 1996-04-23 Hybridon, Inc. Oligonucleotide multiple reporter precursors
US5384253A (en) 1990-12-28 1995-01-24 Dekalb Genetics Corporation Genetic transformation of maize cells by electroporation of cells pretreated with pectin degrading enzymes
US5714331A (en) 1991-05-24 1998-02-03 Buchardt, Deceased; Ole Peptide nucleic acids having enhanced binding affinity, sequence specificity and solubility
US5371241A (en) 1991-07-19 1994-12-06 Pharmacia P-L Biochemicals Inc. Fluorescein labelled phosphoramidites
US5571799A (en) 1991-08-12 1996-11-05 Basco, Ltd. (2'-5') oligoadenylate analogues useful as inhibitors of host-v5.-graft response
US5789215A (en) 1991-08-20 1998-08-04 Genpharm International Gene targeting in animal cells using isogenic DNA constructs
US5610042A (en) 1991-10-07 1997-03-11 Ciba-Geigy Corporation Methods for stable transformation of wheat
US5587361A (en) 1991-10-15 1996-12-24 Isis Pharmaceuticals, Inc. Oligonucleotides having phosphorothioate linkages of high chiral purity
US5700922A (en) 1991-12-24 1997-12-23 Isis Pharmaceuticals, Inc. PNA-DNA-PNA chimeric macromolecules
US5565552A (en) 1992-01-21 1996-10-15 Pharmacyclics, Inc. Method of expanded porphyrin-oligonucleotide conjugate synthesis
US5587371A (en) 1992-01-21 1996-12-24 Pharmacyclics, Inc. Texaphyrin-oligonucleotide conjugates
US5595726A (en) 1992-01-21 1997-01-21 Pharmacyclics, Inc. Chromophore probe for detection of nucleic acid
US5633360A (en) 1992-04-14 1997-05-27 Gilead Sciences, Inc. Oligonucleotide analogs capable of passive cell membrane permeation
US5434257A (en) 1992-06-01 1995-07-18 Gilead Sciences, Inc. Binding compentent oligomers containing unsaturated 3',5' and 2',5' linkages
US5272250A (en) 1992-07-10 1993-12-21 Spielvogel Bernard F Boronated phosphoramidate compounds
US5702932A (en) 1992-07-20 1997-12-30 University Of Florida Microinjection methods to transform arthropods with exogenous DNA
US5652355A (en) 1992-07-23 1997-07-29 Worcester Foundation For Experimental Biology Hybrid oligonucleotide phosphorothioates
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
WO1994009699A1 (fr) 1992-10-30 1994-05-11 British Technology Group Limited Methode d'examen corporel
US5574142A (en) 1992-12-15 1996-11-12 Microprobe Corporation Peptide linkers for improved oligonucleotide delivery
US5476925A (en) 1993-02-01 1995-12-19 Northwestern University Oligodeoxyribonucleotides including 3'-aminonucleoside-phosphoramidate linkages and terminal 3'-amino groups
US5466677A (en) 1993-03-06 1995-11-14 Ciba-Geigy Corporation Dinucleoside phosphinates and their pharmaceutical compositions
US5663312A (en) 1993-03-31 1997-09-02 Sanofi Oligonucleotide dimers with amide linkages replacing phosphodiester linkages
US5539082A (en) 1993-04-26 1996-07-23 Nielsen; Peter E. Peptide nucleic acids
WO1995006128A2 (fr) 1993-08-25 1995-03-02 Dekalb Genetics Corporation Plantes de mais transgeniques fertiles et leurs procedes de production
US5719262A (en) 1993-11-22 1998-02-17 Buchardt, Deceased; Ole Peptide nucleic acids having amino acid side chains
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5599928A (en) 1994-02-15 1997-02-04 Pharmacyclics, Inc. Texaphyrin compounds having improved functionalization
US5625050A (en) 1994-03-31 1997-04-29 Amgen Inc. Modified oligonucleotides and intermediates useful in nucleic acid therapeutics
US6060463A (en) 1994-04-04 2000-05-09 William Freeman Treatment of conditions of abnormally increased intraocular pressure by administration of phosphonylmethoxyalkyl nucleoside analogs and related nucleoside analogs
US5656610A (en) 1994-06-21 1997-08-12 University Of Southern California Producing a protein in a mammal by injection of a DNA-sequence into the tongue
US5597696A (en) 1994-07-18 1997-01-28 Becton Dickinson And Company Covalent cyanine dye oligonucleotide conjugates
US5580731A (en) 1994-08-25 1996-12-03 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5591584A (en) 1994-08-25 1997-01-07 Chiron Corporation N-4 modified pyrimidine deoxynucleotides and oligonucleotide probes synthesized therewith
US5736524A (en) 1994-11-14 1998-04-07 Merck & Co.,. Inc. Polynucleotide tuberculosis vaccine
US5652356A (en) 1995-08-17 1997-07-29 Hybridon, Inc. Inverted chimeric and hybrid oligonucleotides
US5780448A (en) 1995-11-07 1998-07-14 Ottawa Civic Hospital Loeb Research DNA-based vaccination of fish
US6007839A (en) 1996-02-16 1999-12-28 The Liposome Company, Inc. Preparation of pharmaceutical compositions containing etherlipid-containing multiple lipid liposomes
US5945100A (en) 1996-07-31 1999-08-31 Fbp Corporation Tumor delivery vehicles
US5981274A (en) 1996-09-18 1999-11-09 Tyrrell; D. Lorne J. Recombinant hepatitis virus vectors
US6063400A (en) 1997-07-02 2000-05-16 Sdg, Inc. Targeted liposomal constructs for diagnostic and therapeutic uses
US5994624A (en) 1997-10-20 1999-11-30 Cotton Incorporated In planta method for the production of transgenic plants
WO2001030337A2 (fr) 1999-10-22 2001-05-03 Orbon Corporation Traitement topique de l'hypertension oculaire, du glaucome, de la retinopathie ischemique et de la degenerescence maculaire liee a l'age par une formulation ophtalmique d'antagonistes de la dopamine
US6287860B1 (en) 2000-01-20 2001-09-11 Isis Pharmaceuticals, Inc. Antisense inhibition of MEKK2 expression
WO2001068053A2 (fr) 2000-03-10 2001-09-20 Insite Vision Incorporated Procedes et compositions pour traiter et prevenir des affections ophtalmiques de la partie posterieure de l'oeil
WO2004035779A1 (fr) 2002-08-27 2004-04-29 Anges Mg, Inc. Procede de transfert d'une biomolecule utilisant une enveloppe virale et composition et systeme associes
WO2004043480A2 (fr) 2002-11-07 2004-05-27 Peyman Gholam A Traitement d'une maladie oculaire
WO2004054512A2 (fr) 2002-12-13 2004-07-01 Genetix Pharmaceuticals, Inc. Vecteurs retroviraux therapeutiques destines a la therapie genique
WO2004058289A1 (fr) 2002-12-20 2004-07-15 Chakshu Research, Inc. Formulation ophtalmique pour la prevention et le traitement de pathologies oculaires
US20070191294A1 (en) 2003-03-21 2007-08-16 Santaris Pharma A/S Short interfering rna (sirna) analogues
US20050101582A1 (en) 2003-11-12 2005-05-12 Allergan, Inc. Compositions and methods for treating a posterior segment of an eye
US20080249039A1 (en) 2004-01-30 2008-10-09 Santaris Pharma A/S Modified Short Interfering Rna (Modified Sirna)
US20100317718A1 (en) 2004-08-25 2010-12-16 Isis Pharmaceuticals, Inc. Modulation of hif1 beta expression
WO2006048662A1 (fr) 2004-11-03 2006-05-11 Ark Therapeutics Ltd. Baculovirus pseudotypé et emploi dudit baculovirus
US20090181914A1 (en) 2004-11-09 2009-07-16 Santaris Pharma A/S Lna oligonucleotides and the treatment of cancer
WO2008007073A2 (fr) 2006-07-11 2008-01-17 Pci Biotech As Procédé
WO2008071959A1 (fr) 2006-12-12 2008-06-19 Oxford Biomedica (Uk) Limited Vecteurs lentiviraux comprenant des micro-arn
US20100249052A1 (en) 2007-03-26 2010-09-30 Alnylam Pharmaceuticals, Inc. Dsrna compositions and methods for treating hpv infections
US20100234451A1 (en) 2007-11-26 2010-09-16 Enzon Pharmaceuticals, Inc. Lna antagonists targeting the androgen receptor
WO2009149182A1 (fr) * 2008-06-04 2009-12-10 The Board Of Regents Of The University Of Texas System Modulation de l'expression génique par ciblage d'un petit arn endogène de promoteurs de gènes
WO2010040112A2 (fr) 2008-10-03 2010-04-08 Curna, Inc. Traitement de maladies liées à l'apolipoprotéine a1 par l'inhibition d'un produit de la transcription antisens naturel de l'apolipoprotéine a1
WO2010129746A2 (fr) 2009-05-06 2010-11-11 Curna, Inc. Traitement de maladies liées à la tristétraproline (ttp) par l'inhibition d'un transcrit antisens naturel de ttp
WO2011017697A1 (fr) * 2009-08-07 2011-02-10 New York University Compositions et méthodes pour traiter des troubles inflammatoires
US20140235697A1 (en) 2011-10-11 2014-08-21 The Brigham And Women's Hospital, Inc. Micrornas in neurodegenerative disorders
US9209196B2 (en) 2011-11-30 2015-12-08 Sharp Kabushiki Kaisha Memory circuit, method of driving the same, nonvolatile storage device using the same, and liquid crystal display device
WO2014063155A1 (fr) * 2012-10-21 2014-04-24 University Of Rochester Thy1 (cd90) en tant que nouvelle thérapie pour réguler l'accumulation de tissu adipeux
WO2014130945A1 (fr) * 2013-02-25 2014-08-28 Integrated Dna Technologies, Inc. Modifications de composés antisens
WO2015042435A1 (fr) * 2013-09-20 2015-03-26 Academia Sinica Traitement de maladies associées à pgc1-alpha par modulation des microarn mir-130a et mir-130b
WO2016058061A1 (fr) * 2014-10-13 2016-04-21 Katholieke Universiteit Leuven Signatures de microarn pour troubles cardiaques
CN105903036A (zh) * 2015-07-15 2016-08-31 浙江大学 miR-130a反义核酸及其衍生物在Hippo-YAP信号通路抑制剂中的应用
CN106636368A (zh) * 2016-11-28 2017-05-10 山东大学 miR‑130a 在卵巢癌的诊断、治疗及预后中的应用

Non-Patent Citations (61)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2005
AL-MUHAMMED, J., MICROENCAPSUL, vol. 13, 1996, pages 293 - 306
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
BALLANTYNE CM ET AL.: "Markers of inflammation and their clinical significance", ATHEROSCLER, vol. 6, no. 2, May 2005 (2005-05-01), pages 21 - 9, XP004897500, DOI: 10.1016/j.atherosclerosissup.2005.02.005
BENTONDAVIS, SCIENCE, vol. 196, 1977, pages 180
BERGERKIMMEL: "Guide to Molecular Cloning Techniques", 1987, ACADEMIC PRESS
BRAASCH ET AL., BIOCHEMISTRY, vol. 41, no. 14, 2002, pages 4503 - 4510
BROPHY, EUR. J. CLIN. PHARMACOL., vol. 24, 1983, pages 103 - 108
BUSTIN S., J MOL ENDOCRINOL., vol. 25, 2000, pages 169 - 93
CHONN, CURR. OPIN. BIOTECHNOL., vol. 6, 1995, pages 698 - 708
CROOKE ET AL., J. PHARMACOL. EXP. THER., vol. 277, 1996, pages 923 - 937
CZECH, N., ENGL. J. MED., vol. 354, 2006, pages 1194 - 1195
DE MESMAEKER ET AL., ACE. CHEM. RES., vol. 28, 1995, pages 366 - 374
E. MEYERSW. MILLER, COMPUT. APPL. BIOSCI., vol. 4, 1988, pages 11 - 17
EYLES, J., PHARM. PHARMACOL., vol. 49, 1997, pages 669 - 674
FOTHERBY, CONTRACEPTION, vol. 54, 1996, pages 59 - 69
FREDERICK M.: "Ausubel, Current Protocols in Molecular Biology", 2003, JOHN WILEY & SONS
GAO, PHARM. RES., vol. 12, 1995, pages 857 - 863
GEBEYEHU ET AL., NUCL. ACIDS RES., vol. 15, 1987, pages 4513
GENESIS, vol. 30, no. 3, 2001
GINZINGER D., EXP HEMATOL., vol. 30, 2002, pages 503 - 12
GRONING, PHARMAZIE, vol. 51, 1996, pages 337 - 341
GRUNSTEINHOGNESS, PROC. NATL. ACAD. SCI. U.S.A., vol. 72, 1975, pages 3961
HEASMAN, J., DEV. BIOL., vol. 243, 2002, pages 209 - 214
HIDALGO-ARAGONES, J., STEROID BIOCHEM. MOL. BIOL., vol. 58, 1996, pages 611 - 617
JOHNSON, J., PHARM. SCI., vol. 84, 1995, pages 1144 - 1146
JOSEPH SAMBROOKDAVID W. RUSSELL: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
KABANOV ET AL., FEBS LETT., vol. 259, 1990, pages 327 - 330
KIM A LENNOX ET AL: "Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide Modifier", MOLECULAR THERAPY-NUCLEIC ACIDS, vol. 2, 27 August 2013 (2013-08-27), US, pages 1 - 19, XP055473296, ISSN: 2162-2531, DOI: 10.1038/mtna.2013.46 *
KORNBERG, A.: "DNA Replication", 1980, W. H. FREEMAN & CO., pages: 75 - 77
KRUTZFELDT ET AL., NATURE, vol. 438, 2005, pages 685 - 689
LACERRA ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 97, 2000, pages 9591 - 9596
LENNOX ET AL., NUCLEIC ACIDS, vol. 2, 2013, pages e117
LETSINGER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 86, 1989, pages 6553 - 6556
LIU ET AL., INT. J. MOL. SCI., vol. 9, 2008, pages 978 - 999
MANCHARAN ET AL., NUCLEOSIDES & NUCLEOTIDES, vol. 14, 1995, pages 969 - 973
MANOHARAN ET AL., ANN. N.Y. ACAD. SCI., vol. 660, 1992, pages 306 - 309
MANOHARAN ET AL., BIOORG. MED. CHEM. LETT., vol. 3, 1993, pages 2765 - 2770
MANOHARAN ET AL., BIOORG. MED. CHEM. LETT., vol. 4, 1994, pages 1053 - 1060
MANOHARAN ET AL., TETRAHEDRON LETT., vol. 36, 1995, pages 3651 - 3654
MARQUEZMCCAFFREY, HUMAN GENE THER., vol. 19, no. 1, 2008, pages 27 - 38
MISHRA ET AL., BIOCHIM. BIOPHYS. ACTA, vol. 1264, 1995, pages 229 - 237
NASEVICIUS ET AL., NAT. GENET., vol. 26, 2000, pages 216 - 220
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
NIELSEN ET AL., SCIENCE, vol. 254, 1991, pages 1497 - 1500
NING WANG ET AL: "Mechanism of MicroRNA-146a/Notch2 Signaling Regulating IL-6 in Graves Ophthalmopathy", CELLULAR PHYSIOLOGY AND BIOCHEMISTRY., vol. 41, no. 4, 8 March 2017 (2017-03-08), CH, pages 1285 - 1297, XP055686331, ISSN: 1015-8987, DOI: 10.1159/000464430 *
OBERHAUSER ET AL., NUCL. ACIDS RES., vol. 20, 1992, pages 533 - 538
ONG Y. ET AL., HEMATOLOGY, vol. 7, 2002, pages 59 - 67
OSTRO, AM. J. HOSP. PHARM., vol. 46, 1989, pages 1576 - 1587
RAO, J., BIOMATER SCI. POLYM. ED., vol. 7, 1995, pages 623 - 645
ROBERTSON ET AL., SILENCE, vol. 1, 2010, pages 10
ROHATAGI, J., CLIN. PHARMACOL., vol. 35, 1995, pages 1187 - 1193
ROHATAGI, PHARMAZIE, vol. 50, 1995, pages 610 - 613
SHEA ET AL., NUCL. ACIDS RES., vol. 18, 1990, pages 3777 - 3783
SUN YOUNG JANG ET AL: "Role of miR-146a in the Regulation of Inflammation in an In Vitro Model of Graves' Orbitopathy", INVEST OPHTHALMOL VIS SCI, vol. 57, August 2016 (2016-08-01), pages 4027 - 4034, XP055686334, Retrieved from the Internet <URL:https://doi.org/10.1167/iovs.16-19213> *
SVINARCHUK ET AL., BIOCHIMIE, vol. 75, 1993, pages 49 - 54
THAKER V., METHODS MOL BIOL., vol. 115, 1999, pages 379 - 402
TJWA, ANN, ALLERGY ASTHMA IMMUNOL., vol. 75, 1995, pages 107 - 111
VAN ROOIJ ET AL., CIRC. RES., vol. 103, no. 9, 2008, pages 919 - 928
WANG ET AL., J. AM. CHEM. SOC., vol. 122, 2000, pages 8595 - 8602
ZHANGMADDEN, GENOME RES., vol. 7, 1997, pages 649 - 656

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