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WO2010120969A1 - Ciblage de la famille mir-30 et de la famille let-7 pour le traitement de la maladie cardiaque - Google Patents

Ciblage de la famille mir-30 et de la famille let-7 pour le traitement de la maladie cardiaque Download PDF

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WO2010120969A1
WO2010120969A1 PCT/US2010/031147 US2010031147W WO2010120969A1 WO 2010120969 A1 WO2010120969 A1 WO 2010120969A1 US 2010031147 W US2010031147 W US 2010031147W WO 2010120969 A1 WO2010120969 A1 WO 2010120969A1
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mir
inhibitor
family
seq
expression
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Eric Olson
Eva Van Rooij
Robert Frost
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University of Texas System
University of Texas at Austin
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University of Texas System
University of Texas at Austin
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/712—Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose

Definitions

  • the present invention is related to the fields of cardiology and molecular biology.
  • the invention provides a method of designing an inhibitor to reduce or eliminate the expression of multiple members of a microRNA family. Such inhibitors are useful in the treatment or prevention of cardiovascular diseases.
  • Heart disease and its manifestations including coronary artery disease, myocardial infarction, congestive heart failure and cardiac hypertrophy, clearly present a major health risk in the United States today.
  • the cost to diagnose, treat and support patients suffering from these diseases is well into the billions of dollars.
  • Two particularly severe manifestations of heart disease are myocardial infarction and cardiac hypertrophy.
  • Myocardial infarction commonly known as a heart attack, is caused by a sudden and sustained lack of blood flow to the heart tissue, which is usually the result of a narrowing or occlusion of a coronary artery. Without adequate blood supply, the tissue becomes ischemic, leading to the death of cardiomyocytes (e.g. heart muscle cells) and vascular structures.
  • cardiomyocytes e.g. heart muscle cells
  • the necrotic tissue resulting from the death of the cardiomyocytes is generally replaced by scar tissue, which is not contractile, fails to contribute to cardiac function, and often plays a detrimental role in heart function by expanding during cardiac contraction, or by increasing the size and effective radius of the ventricle, for example, becoming hypertrophic.
  • Cardiac hypertrophy is an adaptive response of the heart to virtually all forms of cardiac disease, including those arising from hypertension, mechanical load, myocardial infarction, cardiac arrhythmias, endocrine disorders, and genetic mutations in cardiac contractile protein genes. While the hypertrophic response is initially a compensatory mechanism that augments cardiac output, sustained hypertrophy can lead to dilated cardiomyopathy (DCM), heart failure, and sudden death. In the United States, approximately half a million individuals are diagnosed with heart failure each year, with a mortality rate approaching 50%.
  • DCM dilated cardiomyopathy
  • MicroRNAs have recently been implicated in a number of biological processes including regulation of developmental timing, apoptosis, fat metabolism, and hematopoietic cell differentiation among others.
  • MiRNAs are small, non-protein coding RNAs of about 18 to about 25 nucleotides in length that are derived from individual miRNA genes, from introns of protein coding genes, or from poly-cistronic transcripts that often encode multiple, closely related miRNAs. See review by Carrington et al. ⁇ Science, Vol. 301(5631):336-338, 2003).
  • MiRNAs act as repressors of target mRNAs by promoting their degradation, when their sequences are perfectly complementary, or by inhibiting translation, when their sequences contain mismatches.
  • MiRNAs are transcribed by RNA polymerase II (pol II) or RNA polymerase III (pol III; see Qi et al. (2006) Cellular & Molecular Immunology, Vol. 3:411-419) and arise from initial transcripts, termed primary miRNA transcripts (pri-miRNAs), that are generally several thousand bases long.
  • Pri-miRNAs are processed in the nucleus by the RNase Drosha into about 70- to about 100-nucleotide hairpin-shaped precursors (pre-miRNAs).
  • the hairpin pre-miRNA is further processed by Dicer to produce a double-stranded miRNA.
  • the mature miRNA strand is then incorporated into the RNA-induced silencing complex (RISC), where it associates with its target mRNAs by base-pair complementarity.
  • RISC RNA-induced silencing complex
  • RISC RNA-induced silencing complex
  • miRNAs have been identified that are up-regulated in response to stresses, such as hypoxia, nutrient deprivation, and DNA damage (Leung and Sharp (2007) Cell, Vol. 130(4):581-585).
  • MiRNAs were first suggested to play a role in heart disease in a study of distinctive patterns of miRNA expression in the hearts of normal mice and mice that suffered from heart disease (van Rooij et al. (2006) Proc. Natl. Acad. ScL, Vol. 103(48):18255- 18260).
  • the present invention is based, in part, on the discovery that a single antisense oligonucleotide can be designed to target multiple members of a miRNA family.
  • the present invention provides a method of reducing expression of two or more members of a miRNA family, such as the miR-30 or let-7 family, in a cell by administering a single inhibitor capable of targeting two or more members of the miRNA family.
  • miRNA family inhibitors are useful in the treatment of cardiac diseases.
  • the present invention provides a method of reducing expression of two or more members of a miRNA family in a cell comprising contacting the cell with an inhibitor, wherein the inhibitor targets two or more members of the miRNA family, and wherein the expression or activity of the two or more miRNA family members is reduced in the cell following contact with the inhibitor.
  • the inhibitor can be an antisense oligonucleotide or an antagomir.
  • the inhibitor is an antisense oligonucleotide having at least one chemical modification (e.g., sugar or backbone modification).
  • the inhibitor is a nucleic acid comprising one or more binding sites for the miRNA family members.
  • the inhibitor is a nucleic acid comprising one or more linked antisense oligonucleotides for each of the two or more miRNA family members.
  • the miRNA family can include, for example, miR-30, let-7, miR-29, and miR-15.
  • the present invention also includes a method of designing a nucleic acid for inhibiting the expression or activity of some or all members of a miRNA family.
  • the method comprises aligning nucleotide sequences of some or all the members of a miRNA family; determining a consensus sequence from said aligned nucleotide sequences; and synthesizing a nucleic acid having a sequence that is complementary to said consensus sequence, wherein the synthesized nucleic acid is capable of inhibiting the expression or activity of some or all members of the miRNA family.
  • a nucleic acid having complementarity to a consensus sequence can be designed to allow for bulges and loops at regions or individual nucleotides lacking complementarity.
  • the miRNA family can include, for example, miR-30, let-7, miR-29, and miR-15.
  • the miRNA family is the miR-30 family.
  • the miRNA family is the let-7 family.
  • the present invention encompasses a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of at least one miR-30 family member, wherein the expression or activity of the at least one miR-30 family member is reduced in heart cells of the subject following administration of the inhibitor.
  • the at least one miR-30 family member can be miR-30a, miR-30b, miR-30c, miR-30d, and miR- 3Oe.
  • the expression or activity of two or more miR-30 family members is reduced following administration of the inhibitor.
  • the subject is human.
  • the present invention provides a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of at least one let-7 family member, wherein the expression or activity of the at least one let-7 family member is reduced in heart cells of the subject following administration of the inhibitor.
  • the at least one member of the let-7 family can be let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i.
  • the expression or activity of two or more let-7 family members is reduced following administration of the inhibitor.
  • the subject is human.
  • the present invention also includes a method of modulating calcium signaling in a cell comprising delivering to the cell a modulator of one or more miR-30 family members.
  • the cell may be in vitro or in vivo.
  • the modulator can be an agonist of miR-30 function or an inhibitor of miR-30 function.
  • calcium signaling is reduced in the cell following delivery of a miR-30 agonist.
  • calcium signaling is increased in the cell following delivery of a miR-30 inhibitor.
  • the expression or activity of one or more genes regulated by a miR-30 family member is modulated in the cell after delivery of a miR-30 modulator.
  • Genes regulated by miR-30 can include adrenergic receptor, endothelin receptor type A, regulator of G-protein signaling 2 (RGS2), calcium/calmodulin-dependent protein kinase II delta (CaMKIId), G protein-coupled receptor kinase 5, and Calpain 7.
  • the present invention also provides a method of improving endothelial function or promoting endothelial regeneration after vascular injury in a subject.
  • the method comprises administering to the subject an inhibitor of one or more miR-30 family members, wherein the expression or activity of the one or more miR-30 family members is reduced in endothelial cells of the subject following administration of the inhibitor.
  • the activity or expression of dimethylarginine dimethylaminohydrolase 1 is increased in the endothelial cells of the subject following administration of the inhibitor.
  • the activity or expression of nitric oxide synthase is increased in the endothelial cells of the subject following administration of the inhibitor.
  • the subject is human.
  • the present invention includes pharmaceutical compositions comprising miRNA family inhibitors as described herein.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and an inhibitor of two or more members of a miRNA family.
  • the miRNA family is the miR-30 family.
  • the miRNA family is the let-7 family.
  • the composition can be formulated for injection, for example, for parenteral administration or catheter administration.
  • FIG. 1 Downregulation of miR-30 in cardiac disease.
  • A Determination of the expression of the miR-30 family members in total cardiac RNA from human heart failure patients compared to healthy individuals by microRNA micro-array.
  • B Expression of the individual miR-30 family members after myocardial infarction (MI).
  • MI myocardial infarction
  • Expression of miRNAs was determined in pooled RNA by microRNA micro-array in comparison to sham- operated mice.
  • FIG. 1 Gene structure of miR-30 family members.
  • A Murine sequences of the miR- 30 family members (SEQ ID NOs: 50-54). While miR-30a, miR-30d and miR-30e differ each only in one nucleotide, miR-30b and miR-30c have a different 3 '-end.
  • B Four of the five miR- 30 genes are located as two paired clusters on chromosomes 4 and 15.
  • MiR-30a and a second copy of the miR-30c gene (miR-30c-2) are expressed independently from intergenic regions on chromosome 1.
  • the miR-30c-l and miR-30e cluster is located within intron 5 of the transcription factor Nfyc.
  • E exon.
  • FIG. 3 Expression pattern of miR-30.
  • A Detection of individual family members of miR-30 in diverse tissues of wild-type C57BL/6 mice by Northern blotting (5 ⁇ g total RNA per lane pooled from 3 animals). Due to cross-reactivity, the probes directed specifically against miR-30b and miR-30c detect two bands of which the lower one represents miR-30b and the upper one miR-30c, which is one nucleotide longer than the other miR-30 family members.
  • B Detection of miR-30 in primary neonatal rat fibroblasts (FB) and cardiomyocytes (NRCM) by Northern blotting. Ethidium bromide-stained acrylamide gels were used as loading controls.
  • miR-30 probes COS cells were transfected with plasmids expressing miR-30a, the miR-30b/d-cluster, miR-30c, or miR-30e. Northern blots of lysates of cells expressing the different miR-30 plasmids were probed with oligonucleotides directed to specific miR-30 family members.
  • FIG. 1 MiRNAs in training-induced growth of the adult heart.
  • A Representative record of the voluntary run activity of mice during a 10-day period.
  • B Ratio of ventricular weight to body weight in mice after 10 days of voluntary running (Run) compared to inactive mice (Ctr).
  • C MiRNA expression in hearts of trained mice relative to inactive mice.
  • D Expression of the miR-30 family members in the mouse heart after 10 days of voluntary running.
  • Overexpression of miR-30 increases protein synthesis in cardiomyocytes.
  • NRCM neonatal rat cardiomyocytes
  • NRCMs expression of miR-30a, mir- 30b and miR-30d, respectively, in NRCMs was determined by Northern blotting following adenovirus infection.
  • Cells were transfected with an adenovirus expressing miR-30a (left panel) or the miR-30b/d-cluster (right panel) and stimulated with isoproterenol and phenylephrine (Iso/PE, 2.5/25 ⁇ mol/L) or insulin-like growth factor-1 (IGF-I) or left untreated.
  • MOI multiplicity of infection
  • Cpm counts per minute.
  • FIG. 7 Knock-down of miR-30 by anti-miRs.
  • A Design of a specific anti-miR targeted to miR-30a (SEQ ID NO: 18). The first five nucleotides on the 5' and 3' ends are methylated at the 2' position of the sugar to prevent degradation.
  • B Northern blot analysis of lysates from isolated neonatal rat cardiomyocytes forty eight hours after transfection with the anti-miR-30a.
  • C Northern blot analysis of lysates from isolated neonatal rat cardiomyocytes forty eight hours after transfection with anti-miRs specifically directed against miR-30b or miR- 30c.
  • FIG. 8 Knock-down of miR-30 reduces protein synthesis in cardiomyocytes.
  • Neonatal rat cardiomyocytes were transfected with various combinations of anti-miRs targeted to miR-30a, miR-30b, and miR-30c and stimulated with isoproterenol and phenylephrine (Iso/PE, 2.5/25 ⁇ mol/L) or left untreated. Protein synthesis was measured by [ 3 H]-isoleucine incorporation.
  • FIG. 1 Maps of miR-30 transgenic mice develop severe heart failure.
  • MiR-30 targets molecules involved in Ca2+-signaling in the heart. Schematic of calcium signaling pathways and predicted rm ' R-30 targets (red outlines and red background). MiR-30 is predicted to target the adrenergic receptors (AR), the endothelin receptor type A (EtR-A), the regulator of G-protein signaling 2 (RGS2), calcium/calmodulin- dependent protein kinase II delta (CaMKIId), and Calpain 7. Several of these targets (red background) exhibited reduced mRNA expression in 3 -week old transgenic mice overexpressing miR-30a in cardiac tissue.
  • AR adrenergic receptors
  • EtR-A endothelin receptor type A
  • RGS2 regulator of G-protein signaling 2
  • CaMKIId calcium/calmodulin- dependent protein kinase II delta
  • Calpain 7 Several of these targets (red background) exhibited reduced mRNA expression in 3 -week old transgenic mice overexpressing miR-30a in cardiac
  • FIG. 11 Generation of miR-30 eraser mice.
  • A Schematic illustrating two different eraser constructs for downregulating expression of miR-30 family members in cardiac tissue. Nucleic acids containing sequences complementary to the mature miR-30 family members are placed under the control of the alpha myosin heavy chain promoter ( ⁇ MHC).
  • B Western blot analysis of cardiac tissue isolated from miR-30 eraser mice expressing antisense sequences to miR-30a, miR-30d, and miR-30e (30ade eraser). Expression of total CaMKII (left) and activated CaMKII (right) is increased in the eraser mice.
  • MiR-30 targets GRK-5 directly.
  • FIG. 13 Expression of let-7 family members in cardiac disease.
  • Transgenic mice overexpressing the let-7 a,d,f-cluster under the control of the alpha-MHC promoter exhibit a high variability in cardiomyocyte size (right two panels) relative to wild-type litter mates (left panel).
  • the present invention is based, in part, on the discovery that several miRNA families are regulated in various forms of cardiac disease, and all members of such miRNA families are regulated in the same direction.
  • the inventors have surprisingly found that a single inhibitor can be designed to target multiple members of a miRNA family.
  • the present invention provides a method of designing a nucleic acid for inhibiting the expression or activity of some or all members of a miRNA family. Such inhibitors are capable of reducing the expression or activity of some or all members of the miRNA family in a cell. These inhibitors can be used to treat various forms of heart disease in a subject.
  • the present invention also provides a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction in a subject in need thereof by administering to the subject an inhibitor of two or more members of a miRNA family.
  • the present invention provides a method of designing a nucleic acid for inhibiting the expression or activity of some or all members of a miRNA family comprising aligning nucleotide sequences of some or all the members of a miRNA family; determining a consensus sequence from said aligned nucleotide sequences; and synthesizing a nucleic acid having a sequence that is complementary to said consensus sequence, wherein the synthesized nucleic acid is capable of inhibiting the expression or activity of some or all members of the miRNA family.
  • a "consensus sequence” is a sequence containing one or more variable nucleotides, and is determined by aligning and comparing the nucleotide sequences of two or more miRNA family members. In a preferred embodiment, the mature sequences of the miRNA family members are compared. In another embodiment, the minor or star sequences of the miRNA family members are compared. Alternatively, in some embodiments, the pre-miRNA or pri-miRNA sequences of each of the miRNA family members can be aligned and compared to determine a consensus sequence.
  • the consensus sequence can be determined from nucleotide sequences of two or more members, three or more members, four or more members, five or more members, six or more members, seven or more members, eight or more members, or all members of a rm ' RNA family.
  • Any miRNA family can be used in the method as long as it would be desirable to inhibit expression of multiple members or all members of the miRNA family.
  • Non-limiting examples of miRNA families that are suitable for use in the methods of the invention include the miR-29 family (e.g.
  • miR-29a, miR-29b, and miR-29c the miR-15 family (miR-15, miR-16, miR-195, miR-424, and miR-497), the miR-30 family (miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e), and the let-7 family (let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i).
  • the miRNA family is the miR-30 family.
  • the miRNA family is the let-7 family.
  • the present invention also includes a method of reducing expression of two or more members of a miRNA family in a cell comprising contacting the cell with an inhibitor, wherein the inhibitor targets two or more members of the miRNA family, and wherein the expression or activity of the two or more miRNA family members is reduced in the cell following contact with the inhibitor.
  • the cell is in vitro, hi other embodiments, the cell is in vivo. In another embodiment, the cell is a cardiomyocyte.
  • target refers to the ability of the inhibitor to bind to and/or interfere with the endogenous activity of the miRNA molecule.
  • an inhibitor that targets two members of a miRNA family is capable of binding to and/or interfering with the endogenous activity of those two miRNA family members.
  • the inhibitor is a nucleic acid designed according to the methods of the invention as described herein.
  • the inhibitor of two or more members of a miRNA family is an antisense oligonucleotide.
  • the antisense oligonucleotides can include ribonucleotides, deoxyribonucleotides, or a combination thereof.
  • RNA sequences disclosed herein are used in embodiments that require deoxyribonucleotides, a thymidine residue is substituted for a uridine residue.
  • DNA sequences disclosed herein are used in embodiments that require ribonucleotides, a uridine residue is substituted for a thymidine residue.
  • the antisense oligonucleotides have at least one chemical modification (e.g., sugar or backbone modification).
  • suitable antisense oligonucleotides may be comprised of one or more "conformationally constrained” or bi cyclic sugar nucleoside modifications (BSN) that confer enhanced thermal stability to complexes formed between the oligonucleotide containing BSN and their complementary microRNA target strand.
  • BSN bi cyclic sugar nucleoside modifications
  • the antisense oligonucleotides contain at least one "locked nucleic acid.”
  • Locked nucleic acids contain the 2'-O, 4'-C-methylene ribonucleoside (structure A) wherein the ribose sugar moiety is in a "locked” conformation.
  • the antisense oligonucleotides contain at least one 2', 4'-C-bridged 2' deoxyribonucleoside (CDNA, structure B). See, e.g., U.S. Patent No. 6,403,566 and Wang et al. (1999) Bioorganic and Medicinal Chemistry Letters, Vol.
  • the antisense oligonucleotides contain at least one modified nucleoside having the structure shown in structure C.
  • the antisense oligonucleotides targeting two or more members of a miRNA family can contain combinations of BSN (LNA, CDNA and the like) or other modified nucleotides, and ribonucleotides or deoxyribonucleotides.
  • the antisense oligonucleotides can comprise peptide nucleic acids (PNAs), which contain a peptide-based backbone rather than a sugar-phosphate backbone.
  • PNAs peptide nucleic acids
  • Other modified sugar or phosphodiester modifications to the antisense oligonucleotide are also contemplated.
  • antisense oligonucleotides may contain include, but are not limited to, sugar modifications, such as 2'-O-alkyl ⁇ e.g., 2'-O-methyl, 2'-O- methoxyethyl), 2'-fluoro, and 4' thio modifications, and backbone modifications, such as one or more phosphorothioate, morpholino, or phosphonocarboxylate linkages (see, for example, U.S. Patent Nos. 6,693,187 and 7,067,641, which are herein incorporated by reference in their entireties).
  • antisense oligonucleotides targeting two or more miRNA family members contain 2'O-methyl sugar modifications on each base and are linked by phosphorothioate linkages.
  • Antisense oligonucleotides, particularly those of shorter lengths ⁇ e.g., less than 15 nucleotides) can comprise one or more affinity enhancing modifications, such as, but not limited to, LNAs, bicyclic nucleosides, phosphonoformates, T O-alkyl modifications and the like.
  • suitable antisense oligonucleotides are 2'-O-methoxyethyl "gapmers" which contain 2'-O-methoxyethyl-modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center. These "gapmers” are capable of triggering RNase H-dependent degradation mechanisms of RNA targets.
  • Other modifications of antisense oligonucleotides to enhance stability and improve efficacy such as those described in U.S. Patent No. 6,838,283, which is herein incorporated by reference in its entirety, are known in the art and are suitable for use in the methods of the invention.
  • the antisense oligonucleotide may be linked to a steroid, such as cholesterol moiety, a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand at its 3' end.
  • a steroid such as cholesterol moiety, a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand at its 3' end.
  • Antisense oligonucleotides useful for inhibiting the activity of two or more miRNA family members are about 5 to about 25 nucleotides in length, about 10 to about 30 nucleotides in length, or about 20 to about 25 nucleotides in length. In certain embodiments, antisense oligonucleotides targeting two or more miRNA family members are about 8 to about 18 nucleotides in length, and in other embodiments about 12 to about 16 nucleotides in length. Antisense oligonucleotides can comprise a sequence that is at least partially complementary to a mature miRNA sequence from one or more of the family members.
  • Partially complementary refers to a sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antisense oligonucleotide can be substantially complementary to a mature miRNA family member sequence, that is at least about 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antisense oligonucleotide comprises a sequence that is at least partially complementary to a consensus sequence derived from some or all of the members of the miRNA family.
  • the antisense oligonucleotide is 100% complementary to a consensus sequence derived from some or all of the members of the miRNA family.
  • the antisense oligonucleotides may be designed to allow for bulges or loops at regions or at individual sites lacking identity between family members. Such bulges or loops may range in length from two to about five or about 10 or about 15 or more nucleotides.
  • the antisense oligonucleotides are antagomirs.
  • “Antagomirs” are single-stranded, chemically-modified ribonucleotides that are at least partially complementary to at least one of the miRNA family members or a consensus sequence derived from the members of the miRNA family.
  • Antagomirs may comprise one or more modified nucleotides, such as bicyclic sugar nucleoside modifications or 2'-O-methyl-sugar modifications.
  • antagomirs comprise only modified nucleotides.
  • Antagomirs can also comprise one or more phosphorothioate linkages resulting in a partial or full phosphorothioate backbone.
  • antagomir can be linked to a steroid (e.g., cholesterol) or other moiety (e.g., a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand) at its 3' end.
  • a steroid e.g., cholesterol
  • other moiety e.g., a vitamin, a fatty acid, a carbohydrate or glycoside, a peptide, or other small molecule ligand
  • Antagomirs suitable for inhibiting two or more miRNA family members can be about 15 to about 50 nucleotides in length, more preferably about 18 to about 30 nucleotides in length, and most preferably about 20 to about 25 nucleotides in length.
  • the antagomirs can be at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a mature miRNA family member sequence or a consensus sequence derived from some or all miRNA family members.
  • the antagomir may be substantially complementary to a mature miRNA family member sequence or miRNA family consensus sequence, that is at least about 90%, 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antagomirs are 100% complementary to a consensus sequence derived from some or all of the members of the miRNA family.
  • Antagomirs may also be designed to form bulges or loops at regions lacking identity between miR family members.
  • the inhibitor of two or more members of a miRNA family is a nucleic acid comprising one or more linked antisense oligonucleotides for each of the two or more miRNA family members.
  • an inhibitor for three particular members of a miRNA family can contain at least one antisense oligonucleotide for each of those three particular miRNA family members.
  • the antisense oligonucleotides for each of the family members can be adjacent to one another in the nucleic acid ⁇ e.g. no linker sequences) or can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more linker nucleotides.
  • the linker nucleotides can, in some embodiments, contain cleavage sequences for endonucl eases, such that each of the antisense oligonucleotides are liberated from the larger nucleic acid sequence following delivery of the nucleic acid sequence to the cell or expression of the nucleic acid in the cell, or alternatively may be separated by cleavage in vitro.
  • the inhibitor nucleic acid can contain one or more, two or more, three or more, four or more, or five or more antisense oligonucleotides for each family member.
  • the inhibitor targets two or more family members of the miR-30 family.
  • the miR-30 family consists of five family members: miR-30a, miR-30b, miR-30c, miR- 30d, and miR-30e.
  • miR-30b and miR-30d are expressed as a cluster from an intergenic region of chromosome 8.
  • miR-30e and one copy of miR-30c are expressed as a cluster from an intron of the gene encoding the NYFC transcription factor on chromosome 1.
  • MiR-30a and a second copy of miR-30c (miR-30c-2) are expressed independently from introns of protein coding genes on chromosome 6.
  • the seed region ⁇ e.g.
  • each of the pre- miRNA sequences for each miR-30 family member is processed into a mature sequence and a star sequence.
  • the star sequence is processed from the other arm of the stem loop structure.
  • the pre-miRNA ⁇ e.g. stem-loop sequences), mature, and star sequences for each of the miR-30 family members is given below:
  • the expression or activity of two or more, three or more, or four or more miR-30 family members can be reduced in the cell following contact with the inhibitor.
  • the inhibitor reduces the activity or expression of miR-30a, miR-30b, and miR-30c in the cell.
  • the inhibitor reduces the activity or expression of miR-30a, miR-30d, and miR-30e in the cell.
  • inhibitors that reduce the expression of miR-30a, miR-30d, and miR-30e include, but are not limited to, nucleic acids comprising the sequence of 5'- CUUCCAGUCGAGGAUGUUU ACA-3' (SEQ ID NO: 18); 5'CUUCCAGUCUAGGAUGUUUACA-S' (SEQ ID NO: 60); 5'- CUUCCAGUCGGGGAUGUUUACA-3' (SEQ ID NO: 63); or 5'-CUUCCAGUCAAGGAUGUUUACA-S' (SEQ ID NO: 64).
  • the expression or activity of all five miR-30 family members e.g.
  • the inhibitor for reducing the expression or activity of all five miR-30 family members in a cell is a nucleic acid comprising the sequence of 5'- CUUCGAGUCGAGGAUGUUUACA-3' (SEQ ID NO: 61). In other embodiments, the inhibitor for reducing the expression or activity of all five miR-30 family members in a cell is a nucleic acid comprising one or more miR-30 binding sites.
  • miR-30 binding site refers to a nucleotide sequence that is capable of binding a mature sequence of miR- 30a, miR-30b, miR-30c, miR-30d, and miR-30e.
  • a miR-30 binding site comprises a sequence that is at least partially complementary (e.g. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to the miR-30 seed sequence (SEQ ID NO: 19).
  • the miR-30 binding site has a sequence that is complementary to SEQ ID NO: 19.
  • the nucleic acid comprising one or more miR-30 binding sites may be from about 20 to about 500 nucleotides in length, about 25 to about 400 nucleotides in length, about 30 to about 300 nucleotides in length, about 40 to about 200 nucleotides in length, or about 50 to about 100 nucleotides in length.
  • the nucleic acid maybe 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 500 nucleotides in length.
  • the nucleic acid may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 miR-30 binding sites.
  • the multiple miR-30 binding sites may be adjacent or may be separated by spacers of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.
  • the inhibitor is a nucleic acid comprising one or more linked antisense oligonucleotides for each of the two or more miR-30 family members.
  • the nucleic acid comprises one or more linked antisense oligonucleotides for each of miR-30a, miR-30d, and miR-30e.
  • the nucleic acid can have a sequence comprising SEQ ID NO: 18, SEQ ID NO: 63, and SEQ ID NO: 64 separated by one or more linker nucleotides.
  • the nucleic acid comprises two or more linked antisense oligonucleotides for each of miR-30a, miR-30d, and miR-30e (see Example 4 and Figure 1 IA).
  • the nucleic acid comprises one or more linked antisense oligonucleotides for each of miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e.
  • the nucleic acid can have a sequence comprising SEQ ID NO: 18, SEQ ID NO: 63, SEQ ID NO: 64, 5'- AGCUGAGUGUAGGAUGUUUACA-3' (SEQ ID NO: 65), and 5'- GCUGAGAGUGUAGGAUGUUUACA-S' (SEQ ID NO: 66) separated by one or more linker nucleotides.
  • the nucleic acid comprises two or more linked antisense oligonucleotides for each of miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e (see Example 4 and Figure 1 IA).
  • the inhibitor targets two or more family members of the let-7 family.
  • the let-7 family consists of eight family members: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i.
  • a first copy of let-7a is expressed as a cluster with let-7d and a first copy of let-7f (let-7f-l) from an intergenic region of chromosome 9.
  • a second copy of let-7a (let-7a-2) is expressed as a cluster with miR-100 from an intergenic region on chromosome 11.
  • let-7a A third copy of let-7a (let-7a-3) is expressed as a cluster with let-7b from a coding region of the RP4 gene on chromosome 22.
  • Let-7c is expressed as a cluster with miR-99a from within an open reading frame on chromosome 21, while let-7e is expressed as a cluster with miR-99b and miR-125a from an intergenic region on chromosome 19.
  • a second copy of let-7f (let-7f-2) is expressed in a cluster with miR-98 from within an intron of the HUWE-I gene on the X-chromosome.
  • Let-7g is expressed from an intron of the WDR82 gene on chromosome 3
  • let-7i is expressed from an intergenic region of chromosome 12.
  • the seed region for all let-7 family members is highly conserved (GAGGU AGU AG (SEQ ID NO: 49)).
  • Each of the pre- miRNA sequences for each let-7 family member is processed into a mature sequence and a star sequence.
  • the star sequence is processed from the other arm of the stem loop structure.
  • the pre- miRNA (e.g. stem-loop sequences), mature, and star sequences for each of the let-7 family members is given below:
  • the expression or activity of two or more, three or more, four or more, five or more, six or more, or seven or more let-7 family members can be reduced in the cell following contact with the inhibitor.
  • the inhibitor reduces the activity or expression of let-7a, let-7d, and let-7f in the cell.
  • the inhibitor reduces the activity or expression of let-7a, let-7b, let-7c, let-7d, and let-7f in the cell.
  • the expression or activity of all eight let-7 family members e.g. let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i
  • the expression or activity of all eight let-7 family members e.g. let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i
  • the inhibitor for reducing the expression or activity of all eight let-7 family members in a cell is a nucleic acid comprising the sequence of 5'-AACUAUACAACCUACUACCUCA-S' (SEQ ID NO: 62).
  • the inhibitor for reducing the expression or activity of all eight let-7 family members in a cell is a nucleic acid comprising one or more let-7 binding sites.
  • let-7 binding site refers to a nucleotide sequence that is capable of binding a mature sequence of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i.
  • a let-7 binding site comprises a sequence that is at least partially complementary (e.g. 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) to the let-7 seed sequence (SEQ ID NO: 49).
  • the let-7 binding site has a sequence that is complementary to SEQ ID NO: 49.
  • the nucleic acid comprising one or more let-7 binding sites may be from about 20 to about 500 nucleotides in length, about 25 to about 400 nucleotides in length, about 30 to about 300 nucleotides in length, about 40 to about 200 nucleotides in length, or about 50 to about 100 nucleotides in length.
  • the nucleic acid may be 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 500 nucleotides in length.
  • the nucleic acid may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 let-7 binding sites.
  • the multiple let-7 binding sites may be adjacent or may be separated by spacers of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides.
  • the inhibitor is a nucleic acid comprising one or more linked antisense oligonucleotides for each of the two or more let-7 family members.
  • the nucleic acid comprises one or more linked antisense oligonucleotides for each of let-7a, let-7d, and let-7f.
  • the nucleic acid comprises one or more linked antisense oligonucleotides for each of let-7a, let-7b, let-7c, let-7d, and let-7f.
  • the nucleic acid comprises one or more linked antisense oligonucleotides for each of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i.
  • the antisense oligonucleotides for each let-7 family member can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more linker nucleotides.
  • the miRNA family inhibitors of the invention can be delivered to a cell by an expression vector encoding any of inhibitor nucleic acids as described herein.
  • a "vector” is a composition of matter which can be used to deliver a nucleic acid of interest to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.
  • An expression construct can be replicated in a living cell, or it can be made synthetically.
  • the terms "expression construct,” “expression vector,” and “vector,” are used interchangeably to demonstrate the application of the invention in a general, illustrative sense, and are not intended to limit the invention.
  • an expression vector for expressing a miRNA family inhibitor comprises a promoter operably linked to a polynucleotide encoding an antisense oligonucleotide, wherein the sequence of the expressed antisense oligonucleotide is partially or perfectly complementary to a mature sequence of one member of the miRNA family or a consensus sequence derived from some or all members of the miRNA family.
  • an expression vector for expressing a miRNA family inhibitor comprises a promoter operably linked to a polynucleotide encoding a nucleic acid comprising one or more miRNA family binding sites.
  • operably linked or "under transcriptional control” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.
  • the expression vector may encode a single nucleic acid that comprises one or more linked antisense oligonucleotides for each member of the miRNA family, wherein the single nucleic acid is operably linked to a promoter.
  • a "promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene.
  • Suitable promoters include, but are not limited to RNA pol I, pol II, pol III, and viral promoters (e.g. human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat).
  • the promoter is a tissue specific promoter.
  • muscle specific promoters and more particularly, cardiac specific promoters. These include the myosin light chain-2 promoter (Franz et al. (1994) Cardioscience, Vol. 5(4):235-43; Kelly et al. (1995) J. Cell Biol, Vol. 129(2):383-396), the alpha actin promoter (Moss et al.
  • the brain natriuretic peptide promoter (LaPointe et al. (1996) Hypertension, Vol. 27(3 Pt 2):715-22) and the alpha B-crystallin/small heat shock protein promoter (Gopal-Srivastava (1995) J. MoI. Cell. Biol, Vol. 15(12):7081-7090), alpha myosin heavy chain promoter (Yamauchi-Takihara et al. (1989) Proc. Natl. Acad. ScL USA, Vol. 86(10):3504-3508) and the ANF promoter (LaPointe et al. (1988) J. Biol. Chem., Vol. 263(19):9075-9078).
  • the promoter is the alpha myosin heavy chain promoter.
  • the promoter operably linked to a polynucleotide encoding a miRNA family inhibitor may be an inducible promoter.
  • Inducible promoters are known in the art and include, but are not limited to, tetracycline promoter, metallothionein HA promoter, heat shock promoter, steroid/thyroid hormone/retinoic acid response elements, the adenovirus late promoter, and the inducible mouse mammary tumor virus LTR.
  • Methods of delivering expression constructs and nucleic acids to cells are known in the art and can include, for example, calcium phosphate co-precipitation, electroporation, microinjection, DEAE-dextran, lipofection, transfection employing polyamine transfection reagents, cell sonication, gene bombardment using high velocity microprojectiles, and receptor- mediated transfection.
  • the miRNA family inhibitors as described herein can be employed to treat various cardiac diseases in which the miRNA family is dysregulated. For instance, the inventors surprisingly discovered that all five members of the miR-30 family are downregulated in cardiac tissue from failing hearts, and overexpression of miR-30 family members leads to heart failure and premature death (see Examples 1 and 3). The let-7 family is upregulated in cardiac disease states and appears to control cardiomyocyte growth and proliferation (see Example 5). Other miRNA families also appear to play a role in cardiac hypertrophy and cardiac remodeling. The miR-15 family members (miR-15, miR-16, miR-195, miR-424, and miR-497) are upregulated in response to stress, and inhibition of this family increases cell survival following infarction.
  • MiR- 29 family members are predicted to target IGF-I, AMP- activated Kinase and PPAR delta, all of which have been shown to be involved in the regulation of physiological cardiomyocyte hypertrophy. Therefore, downregulation of the miR-29 family in myocytes during physiological training promotes hypertrophy of cardiac and skeletal myocytes.
  • the present invention also provides a method of treating or preventing pathologic cardiac hypertrophy, heart failure, cardiac remodeling, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of two or more members of a miRNA family, wherein the expression or activity of the two or more miRNA family members is reduced in heart cells of the subject following administration of the inhibitor.
  • the miRNA family can include, but is not limited to, miR-30, let-7, miR-15, and miR-29.
  • the term "subject" or “patient” refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like), hi some embodiments, the subject is a mammal. In other embodiments, the subject is a human.
  • primates e.g., chimpanzees and other apes and monkey species
  • farm animals e.g., cattle, sheep, pigs, goats and horses
  • domestic mammals e.g., dogs and cats
  • laboratory animals e.g., rodents such as mice, rats,
  • the present invention includes a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of at least one miR- 30 family member, wherein the expression or activity of the at least one miR-30 family member is reduced in heart cells of the subject following administration of the inhibitor.
  • the at least one miR-30 family can be selected from the group consisting of miR-30a, miR-30b, miR-30c, miR- 30d, and miR-30e.
  • the expression or activity of two or more miR-30 family members is reduced following administration of the inhibitor.
  • the expression or activity of miR-30a, miR-30d, and miR-30e is reduced following administration of the inhibitor. In other embodiments, the expression or activity of miR-30a, miR-30b, and miR-30c is reduced following administration of the inhibitor. In certain embodiments, the expression or activity of miR-30a, miR-30b, miR-30c, miR-30d, and rm ' R-30e is reduced following administration of the inhibitor.
  • the inhibitor can be any of the inhibitory molecules described herein.
  • the inhibitor can be an antisense oligonucleotide, a modified antisense oligonucleotide, an antagomir, or a nucleic acid containing one or more miR-30 binding sites.
  • the inhibitor targets a mature sequence of one or more miR-30 family members (e.g. SEQ ID NOs: 2, 5, 9, 13, and 16) or a star sequence of one or more miR-30 family members (e.g. SEQ ID NOs: 3, 6, 10, 11, 14, and 17).
  • inhibitors of miR-30 family members are antisense oligonucleotides or antagomirs comprising a sequence that is partially or perfectly complementary to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17.
  • inhibitors of one or more miR-30 family members are chemically- modified antisense oligonucleotides.
  • an inhibitor of a miR-30 family member is a chemically-modified antisense oligonucleotide comprising a sequence substantially complementary to a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16, and SEQ ID NO: 17.
  • substantially complementary refers to a sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% complementary to a target polynucleotide sequence.
  • Antisense oligonucleotides may comprise a sequence that is substantially complementary to a precursor miRNA sequence (pre-miRNA) for one or more miR-30 family members (e.g. pre- miR-30a, pre-miR-30b, pre-miR-30c, pre-miR-30d, or pre-miR-30e).
  • pre-miRNA precursor miRNA sequence
  • the antisense oligonucleotide comprises a sequence that is substantially complementary to a sequence located outside the stem-loop region of the pre-miRNA sequence.
  • an inhibitor of a miR-30 family member is an antisense oligonucleotide having a sequence that is substantially complementary to a pre-miRNA sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 15.
  • the present invention includes a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction in a subject in need thereof comprising administering to the subject an inhibitor of at least one let-7 family member, wherein the expression or activity of the at least one let-7 family member is reduced in heart cells of the subject following administration of the inhibitor.
  • the at least one let-7 family can be selected from the group consisting of let-7 a, let-7b, let-7c, let-7d, let-7 e, let- 7f, let-7 g, and let-7i.
  • the expression or activity of two or more let-7 family members is reduced following administration of the inhibitor.
  • the expression or activity of let-7a, let-7d, and let-7f is reduced following administration of the inhibitor. In other embodiments, the expression or activity of let-7a, let-7b, let-7c, let-7d, and let-7f is reduced following administration of the inhibitor. In certain embodiments, the expression or activity of let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i is reduced following administration of the inhibitor.
  • the inhibitor targets a mature sequence of one or more let-7 family members (e.g. SEQ ID NOs: 21, 24, 27, 33, 36, 39, 44, and 47) or a star sequence of one or more miR-30 family members (e.g. SEQ ID NOs: 22, 25, 28, 30, 34, 37, 40, 42, 45, and 48).
  • let-7 family members e.g. SEQ ID NOs: 21, 24, 27, 33, 36, 39, 44, and 47
  • a star sequence of one or more miR-30 family members e.g. SEQ ID NOs: 22, 25, 28, 30, 34, 37, 40, 42, 45, and 48.
  • inhibitors of let-7 family members are antisense oligonucleotides or antagomirs comprising a sequence that is partially or perfectly complementary to a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 48.
  • inhibitors of one or more let-7 family members are chemically- modified antisense oligonucleotides.
  • an inhibitor of a let-7 family member is a chemically-modified antisense oligonucleotide comprising a sequence substantially complementary to a sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 48.
  • Antisense oligonucleotides may comprise a sequence that is substantially complementary to a precursor rm ' RNA sequence (pre-miRNA) for one or more let-7 family members (e.g. pre- let-7a, pre-let-7b, pre-let-7c, pre-let-7d, pre-let-7e, pre-let-7f, pre-let-7g, and pre-let-7i).
  • the antisense oligonucleotide comprises a sequence that is substantially complementary to a sequence located outside the stem-loop region of the pre-miRNA sequence.
  • an inhibitor of a let-7 family member is an antisense oligonucleotide having a sequence that is substantially complementary to a pre-miRNA sequence selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 46.
  • a miRNA family inhibitor e.g. inhibitor of a miR-30 or let- 7 family member
  • administration of a miRNA family inhibitor results in the improvement of one or more symptoms of pathologic cardiac hypertrophy, heart failure, cardiac remodeling, or myocardial infarction in the subject, or delays the transition from cardiac hypertrophy to heart failure.
  • the one or more improved symptoms can be, for example, increased exercise capacity, increased cardiac ejection volume, decreased left ventricular end diastolic pressure, decreased pulmonary capillary wedge pressure, increased cardiac output, increased cardiac index, lowered pulmonary artery pressures, decreased left ventricular end systolic and diastolic dimensions, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease related morbidity or mortality.
  • inhibitors of the miR-30 or let-7 family members can prevent cardiac hypertrophy and its associated symptoms from arising.
  • administration of an inhibitor of one or more miR-30 or let-7 family members to a subject suffering from myocardial infarction can reduce infarct size by decreasing the loss of heart cells.
  • cardiac function is stabilized in a subject suffering from myocardial infarction following administration of an inhibitor of one or more miR-30 or let-7 family members.
  • the present invention also encompasses a method of modulating calcium signaling in a cell comprising delivering to the cell a modulator of one or more miR-30 family members.
  • the one or more miR-30 family members is selected from the group consisting of miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e.
  • the cell is a cardiomyoctye.
  • the cardiomyoctye can be in vitro or in vivo.
  • a "modulator" is a molecule that regulates the expression or activity of one or more miR-30 family members. Modulators can be agonists of miR-30 family member function or they can be inhibitors of miR-30 family member function. Modulators can include proteins, peptides, polypeptides, polynucleotides, oligonucleotides, or small molecules.
  • the modulator is an agonist of one or more miR-30 family members, and calcium signaling is reduced in the cell following delivery of the agonist.
  • the expression or activity of one or more genes regulated by a miR-30 family member can be reduced in the cell following delivery of the miR-30 agonist.
  • the expression or activity of one or more genes selected from the group consisting of adrenergic receptor, endothelin receptor type A, regulator of G-protein signaling 2 (RGS2), calcium/calmodulin-dependent protein kinase II delta (CaMKIId), G protein-coupled receptor kinase 5, and Calpain 7 is reduced in the cell following delivery of the agonist.
  • the modulator is an inhibitor of one or more miR-30 family members, and calcium signaling is increased in the cell following delivery of the inhibitor.
  • the expression or activity of one or more genes regulated by a miR-30 family member can be increased in the cell following delivery of the miR-30 inhibitor.
  • the expression or activity of one or more genes selected from the group consisting of adrenergic receptor, endothelin receptor type A, regulator of G-protein signaling 2 (RGS2), calcium/calmodulin- dependent protein kinase II delta (CaMKIId), G protein-coupled receptor kinase 5, and Calpain 7 is increased in the cell following delivery of the inhibitor.
  • the present invention also provides a method of improving endothelial function or promoting endothelial regeneration after vascular injury in a subject in need thereof.
  • the method comprises administering to the subject an inhibitor of one or more miR- 30 family members, wherein the expression or activity of the one or more miR-30 family members is reduced in endothelial cells of the subject following administration of the inhibitor.
  • the one or more miR-30 family members can be miR-30a, miR-30b, miR-30c, miR-30d, or miR-30e.
  • Endothelial function is the normal function of endothelial cells lining vessels, and can include, but is not limited to, mediation of coagulation, platelet adhesion, immune function, control of volume and electrolyte content of the intravascular and extravascular spaces, production of nitric oxide, and response to vasoactive substances.
  • the activity or expression of dimethylarginine dimethylaminohydrolase 1 is increased in the endothelial cells of the subject following administration of the inhibitor.
  • the activity or expression of nitric oxide synthase is increased in the endothelial cells of the subject following administration of the inhibitor.
  • vasodilation of vessels is increased in the subject following administration of the inhibitor.
  • the present invention also includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an effective dose of an inhibitor of two or more members of a miRNA family.
  • the miRNA family can be miR-30, let-7, miR-15, or miR-29.
  • the miRNA family is the miR-30 family.
  • the miRNA family is the let-7 family.
  • An "effective dose" is an amount sufficient to effect a beneficial or desired clinical result.
  • An effective dose of an miRNA inhibitor of the invention may be about 1 mg/kg to about 100 mg/kg, about 2.5 mg/kg to about 50 mg/kg, or about 5 mg/kg to about 25 mg/kg.
  • compositions will be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • the pharmaceutical composition comprises an effective dose of an inhibitor of two or more members of the miR-30 family.
  • the inhibitor reduces the activity or expression of rm ' R-30a, miR-30b, and miR-30c.
  • the inhibitor reduces the activity or expression of miR-30a, miR-30d, and miR-30e.
  • the inhibitor can be a nucleic acid comprising a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 60, SEQ ID NO: 63, and SEQ ID NO: 64.
  • the inhibitor reduces the activity or expression of miR-30a, miR-30b, miR-30c, miR-30d, and miR- 30e.
  • the inhibitor can be a nucleic acid comprising the sequence of SEQ ID NO: 61. In other such embodiments, the inhibitor can be a nucleic acid comprising one or more miR-30 binding sites. The one or more miR-30 binding sites can have a sequence that is at least partially complementary to a miR-30 seed sequence (SEQ ID NO: 19).
  • the pharmaceutical composition comprises an effective dose of an inhibitor of two or more members of the let-7 family. In some embodiments, the inhibitor reduces the activity or expression of let-7a, let-7d, and let-7f. In other embodiments, the inhibitor reduces the activity or expression of let-7a, let-7b, let-7c, let-7d, and let-7f.
  • the inhibitor reduces the activity or expression of let-7a, let-7b, let-7c, let-7d, let- 7e, let-7f, let-7g, and let-7i.
  • the inhibitor can be a nucleic acid comprising the sequence of SEQ ID NO: 62.
  • the inhibitor can be a nucleic acid comprising one or more let-7 binding sites.
  • the one or more let-7 binding sites can have a sequence that is at least partially complementary to a let-7 seed sequence (SEQ ID NO: 49).
  • the pharmaceutical composition comprises an effective dose of a miRNA family inhibitor, wherein the inhibitor is a nucleic acid comprising one or more linked antisense oligonucleotides for each of the two or more miRNA family members.
  • Pharmaceutical compositions comprising effective doses of any of the inhibitors described herein are also contemplated.
  • Colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes, may be used as delivery vehicles for the inhibitors of miRNA family function, polynucleotides encoding miRNA family inhibitors, or constructs expressing particular miRNA family inhibitors.
  • Commercially available fat emulsions that are suitable for delivering the nucleic acids of the invention to cardiac and skeletal muscle tissues include Intralipid®, Liposyn®, Liposyn® II, Liposyn® III, Nutrilipid, and other similar lipid emulsions.
  • a preferred colloidal system for use as a delivery vehicle in vivo is a liposome (i.e., an artificial membrane vesicle).
  • a liposome i.e., an artificial membrane vesicle.
  • the preparation and use of such systems is well known in the art.
  • Exemplary formulations are also disclosed in US 5,981,505; US 6,217,900; US 6,383,512; US 5,783,565; US 7,202,227; US 6,379,965; US 6,127,170; US 5,837,533; US 6,747,014; and WO03/093449, which are herein incorporated by reference in their entireties.
  • Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising the inhibitor polynucleotides (e.g. liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically acceptable or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or polynucleotides of the compositions.
  • the active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cardiac tissue. Pharmaceutical compositions comprising miRNA inhibitors, polynucleotides encoding miRNA family inhibitors or expression constructs comprising inhibitor sequences may also be administered by catheter systems or systems that isolate coronary circulation for delivering therapeutic agents to the heart. Various catheter systems for delivering therapeutic agents to the heart and coronary vasculature are known in the art.
  • solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations generally contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use or catheter delivery include, for example, sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Generally, these preparations are sterile and fluid to the extent that easy injectability exists.
  • Preparations should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • Appropriate solvents or dispersion media may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions may be prepared by incorporating the active compounds in an appropriate amount into a solvent along with any other ingredients (for example as enumerated above) as desired, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the desired other ingredients, e.g., as enumerated above.
  • the preferred methods of preparation include vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient(s) plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • compositions of the present invention generally may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include, for example, acid addition salts (formed with the free amino groups of the protein) derived from inorganic acids (e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups of the protein can also be derived from inorganic bases (e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) or from organic bases (e.g., isopropylamine, trimethylamine, histidine, procaine and the like.
  • inorganic acids e.g., hydrochloric or phosphoric acids, or from organic acids (e.g., acetic, oxalic, tartaric, mandelic, and the like.
  • Salts formed with the free carboxyl groups of the protein can also be
  • solutions are preferably administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution generally is suitably buffered and the liquid diluent first rendered isotonic for example with sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is known to those of skill in the art, particularly in light of the present disclosure.
  • a single dose may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
  • the pharmaceutical compositions of the invention can be packaged in a kit for parenteral administration.
  • Example 1 MiR-30 family members are downregulated in cardiac tissue following heart failure and myocardial infarction
  • RNA isolated from cardiac tissue from human heart failure patients suffering from idiopathic dilated cardiomyopathy and healthy control individuals were down-regulated in the samples from failing hearts.
  • Figure IA To determine if down-regulation of the miR-30 family was characteristic of cardiac disease, expression of the individual miR-30 family members was examined in mice following induction of myocardial infarction by ligation of the coronary artery. Total RNA isolated from the area adjacent to the infarcted region (border zone) was collected three and fourteen days following induction of the infarction. As shown in Figure IB, all five miR-30 family members were down- regulated in comparison to sham controls.
  • the miR-30 family consists of five members: miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e.
  • the murine mature sequences for the miR-30 family members are shown in Figure 2A. While miR-30a, miR-30d and miR-30e differ each only in one nucleotide, miR-30b and miR-30c have a different 3 '-end. However, all five members share the same seed region having a sequence of UGU AAACAUCC (SEQ ID NO: 19). In mice, the majority of the miR-30 genes are located as two paired clusters.
  • MiR-30c-l is clustered with miR-30e on chromosome 4, and miR-30b is clustered with rm ' R-30d in an intergenic region on chromosome 15 ( Figure 2B).
  • MiR-30a and miR-30c-2 are expressed independently from intergenic regions on chromosome 1.
  • the miR-30c-l/miR-30e cluster is located within intron 5 of the transcription factor Nfyc ( Figure 2B).
  • pre-miR-30a is longer than the other pre-miRNAs of the miR-30 family, which is reflected by the slower migration of the upper band in lysates from cells expressing miR-30a ( Figure 4).
  • cardiac miRNA expression was determined in a setting of physiological cardiac remodeling (e.g. running- induced cardiac hypertrophy), which is thought to be beneficial and to increase cardiac output (Figure 5A).
  • Mice were allowed to run voluntarily on an exercise wheel in their home cage for ten days and their activity was recorded (Figure 5A).
  • the ratio of ventricular weight to body weight was increased in mice that were allowed to voluntarily run as compared to sedentary controls ( Figure 5B), reflecting physiological cardiac hypertrophy in response to the exercise.
  • Expression of miRNAs in cardiac tissue from animals that experienced running revealed that several miRNAs, including all members of the miR-30 family, were downregulated (Figure 5C and D).
  • Example 2 MiR-30 regulates protein synthesis in cardiomyocytes in response to hypertrophic stimuli
  • adenoviruses carrying a transgene encoding for miR-30a or the miR-30b/miR-30d cluster were generated.
  • Isolated neonatal rat cardiomyocytes (NRCM) were infected with adenoviruses expressing either lacZ, miR-30a or the miR-30b/d-cluster.
  • Expression of the specific miR-30 family member was increased in cells following infection with the adenovirus expressing the miR-30 family member ( Figure 6A).
  • NRCMs transfected with increasing titers of an adenovirus expressing miR-30a (Figure 6B, left) or the miR-30b/d-cluster ( Figure 6B, right) were stimulated with isoproterenol and phenylephrine (Iso/PE, 2.5/25 ⁇ mol/L), insulin-like growth factor-1 (IGF-I), or left untreated.
  • Iso/PE isoproterenol and phenylephrine
  • IGF-I insulin-like growth factor-1
  • Protein synthesis was determined by [ 3 H]-isoleucine incorporation in the isolated NRCMs following stimulation.
  • Overexpression of miR-30, in combination with hypertrophic stimuli like isoproterenol or IGF-I appeared to increase protein synthesis (Figure 6B), which is likely due to hypertrophy of the cardiomyocytes.
  • NRCMs were transfected with various combinations of anti-miRs directed to miR-30a, miR-30b, and miR-30c. Protein synthesis was measured by [ 3 H]-isoleucine incorporation following stimulation with isoproterenol and phenylephrine. Interestingly, knockdown of miR-30 in cardiomyocytes in the presence of the hypertrophic stimulus isoproterenol / PE reduced the level of protein synthesis, suggesting that a reduction in cardiomyocyte hypertrophy occurred (Figure 8).
  • Example 3 Overexpression of miR-30 causes heart failure and premature death [0096]
  • transgenic mice overexpressing one or more of the miR-30 family members in cardiac tissue were generated.
  • MiR-30a, the miR-30b/d cluster, miR-30c-2 and miR-30e were overexpressed specifically in the heart under the control of the ⁇ -myosin heavy chain (MHC) promoter.
  • MHC ⁇ -myosin heavy chain
  • Northern analysis confirmed that the miR-30 family member and its precursor were upregulated in cardiac tissue isolated from independent transgenic lines (data not shown).
  • Transgenic mice overexpressing miR-30a or miR-30e specifically in the heart exhibited significant cardiac hypertrophy as compared to wild-type litter mates ( Figure 9A).
  • MiR-30a and miR-30e transgenic animals developed severe dilative heart failure and died prematurely (Figure 9B). These data provide further evidence that the miR-30 family contributes to cardiac hypertrophy, and downregulation of the miR-30 family could be an effective therapeutic approach to treating heart disease.
  • MiR-30 family members target calcium signaling molecules [0097] Based on sequence analysis, miR-30 is predicted to target several molecules involved in calcium signaling and handling within the cell. Such targets include the adrenergic receptors (AR), the endothelin receptor type A (EtR-A), the regulator of G-protein signaling 2 (RGS2), calcium/calmodulin-dependent protein kinase II delta (CaMKIId) and Calpain 7 ( Figure 10). Messenger RNA expression of several of these predicted targets, including EtR-A, RGS2, Calpain 7, and CaMKIId, was reduced in 3-week old transgenic mice overexpressing miR-30a under the control of the ⁇ MHC promoter (e.g.
  • MiR-30 eraser mice were generated to further explore in vivo targets of the miR-30 family. Specifically, a nucleic acid containing sequences complementary to the mature miR-30a, miR-30d, and miR-30e sequences (ade eraser) or a nucleic acid containing sequences complementary to the mature miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e sequences (abcde eraser) was placed under the control of the ⁇ MHC promoter (Figure 1 IA). Transgenic mice overexpressing these nucleic acids exhibited a downregulation of the miR-30 family members in cardiac tissue.
  • ade eraser a nucleic acid containing sequences complementary to the mature miR-30a, miR-30b, miR-30c, miR-30d, and miR-30e sequences
  • a luciferase gene fused to the 3'UTR from GRK5 was co-expressed in COS cells with either a miR-30b/miR-30d expression construct or a let-7a/let-7d/let-7f expression construct.
  • Expression of the miR-30b/d-cluster repressed the expression of luciferase, while expression of the let-7 cluster, which has no predicted target site in the 3'UTR of GRK5, had no effect (Figure 12B).
  • DDAHl dimethylarginine dimethylaminohydrolase 1
  • ADMA asymmetric dimethylarginine
  • MMA NG-monomethyl-L-arginine
  • Inhibition of DDAH leads to accumulation of methylarginines, which in turn blocks nitric oxide synthesis causing vasoconstriction.
  • Reduced DDAH function has been implicated in the impairment of vascular relaxation observed in patients afflicted with cardiovascular disease. Expression of DDAHl was reduced in miR-30a transgenic mice indicating that DDAHl is an in vivo target of miR-30.
  • a downregulation of miR-30 was observed following vascular injury (data not shown). Such a downregulation of miR-30 would result in increased DDAHl levels, followed by decreased ADMA and increased NOS activity, which would facilitate vascular relaxation. Therefore, downregulation of miR-30 after vascular injury would enhance endothelial function and restore endothelial regeneration.
  • Example 5 Let-7 is upregulated in a cardiac hypertrophy model
  • FIG. 13A Another miRNA family that has been implicated in cardiac disease is the let-7 family. All members of the let-7 family were found to be upregulated in cardiac tissue isolated from human heart failure patients (data not shown). Similarly, members of the let-7 family are also upregulated in calcineurin transgenic mice, a model of stress-induced cardiac hypertrophy ( Figure 13A). There are eight members of the let-7 family: let-7a, let-7b, let-7c, let-7d, let-7e, let-7f, let-7g, and let-7i. In mice, let-7a-l, let-7d, and let-7f-l are clustered in an intergenic region on chromosome 13, and let-7c-2 and let-7b are clustered in an intergenic region on chromosome 15.
  • Let-7e is clustered with miR-99b and miR-125a in an intergenic region on chromosome 17.
  • Let-7g is expressed from an intron of a protein coding gene on chromosome 9 and let-7i is expressed from an intergenic region on chromosome 10.
  • the expression pattern of let-7 shows that the let-7a-l/let-7d/let-7f-l cluster and the let-7b/let-7c-2 cluster are highly expressed in the heart (data not shown).
  • FIG. 14 The cardiac tissue shown in Figure 14 is from a transgenic line in which the transgene likely integrated in the X-chromosome.

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Abstract

La présente invention concerne un procédé de conception d'un inhibiteur capable de cibler de multiples membres d'une famille d'ARNmi. La présente invention concerne, en particulier, des inhibiteurs capables d'inhiber l'expression ou l'activité d'au moins deux membres de la famille miR-30 ou de la famille let-7. L'invention concerne également des procédés d'utilisation desdits inhibiteurs en vue du traitement de la maladie cardiovasculaire.
PCT/US2010/031147 2009-04-15 2010-04-15 Ciblage de la famille mir-30 et de la famille let-7 pour le traitement de la maladie cardiaque Ceased WO2010120969A1 (fr)

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US9580708B2 (en) 2011-09-14 2017-02-28 Rana Therapeutics, Inc. Multimeric oligonucleotides compounds
US20150315587A1 (en) * 2011-09-14 2015-11-05 Rana Therapeutics, Inc. Multimeric oligonucleotides compounds having cleavable linkers
US10704046B2 (en) 2011-09-14 2020-07-07 Translate Bio Ma, Inc. Multimeric oligonucleotide compounds
US10093924B2 (en) 2011-09-14 2018-10-09 Translate Bio Ma, Inc. Multimetric oligonucleotide compounds
US9732341B2 (en) 2011-09-14 2017-08-15 Translate Bio Ma, Inc. Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers
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US9457046B2 (en) 2011-10-17 2016-10-04 PharmalHungary 2000 Kft. Compounds for the treatment of ischemic injury
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EP3399039A1 (fr) 2011-10-17 2018-11-07 PharmaHungary 2000 Kft. Composé pour traiter une lésion ischémique
US12391943B2 (en) 2011-12-23 2025-08-19 King's College London MicroRNAs for cardiac regeneration through induction of cardiac myocyte proliferation
US11236332B2 (en) * 2011-12-23 2022-02-01 King's College London MicroRNAs for cardiac regeneration through induction of cardiac myocyte proliferation
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US20150315585A1 (en) * 2012-09-14 2015-11-05 Rana Therapeutics, Inc. Multimeric oligonucleotide compounds having non-nucleotide based cleavable linkers
US10844375B2 (en) 2012-09-14 2020-11-24 Translate Bio Ma, Inc. Multimeric oligonucleotide compounds having non-nucleotide based cleavable linkers
US9624471B2 (en) 2013-06-12 2017-04-18 University Of Washington Through Its Center For Commercialization Methods for maturing cardiomyocytes and uses thereof
WO2014202973A1 (fr) * 2013-06-17 2014-12-24 Ucl Business Plc Agonistes de ddah1 utilisés pour le traitement d'un dysfonctionnement endothélial
WO2015066611A3 (fr) * 2013-11-01 2015-07-02 The Trustees Of Columbia University In The City Of New York Profils des microarn présents en cas d'insuffisance cardiaque : procédés et systèmes de détection et d'utilisation
US10968451B2 (en) * 2014-10-14 2021-04-06 Texas Tech University System Multiplexed shRNAs and uses thereof
US20180028557A1 (en) * 2015-02-13 2018-02-01 Translate Bio MA, Inc Hybrid oligonucleotides and uses thereof
US10758558B2 (en) * 2015-02-13 2020-09-01 Translate Bio Ma, Inc. Hybrid oligonucleotides and uses thereof
CN107683341A (zh) * 2015-05-08 2018-02-09 新加坡科技研究局 用于慢性心力衰竭的诊断和预后的方法
JP2018515097A (ja) * 2015-05-08 2018-06-14 エージェンシー フォー サイエンス，テクノロジー アンド リサーチ 慢性心不全の診断と予後予測の方法
WO2017062659A1 (fr) * 2015-10-06 2017-04-13 University Of Virginia Patent Foundation Compositions et procédés pour traiter la rétinopathie diabétique
US10870852B2 (en) 2015-10-06 2020-12-22 University Of Virginia Patent Foundation Compositions and methods for treating diabetic retinopathy
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US20190093106A1 (en) * 2015-10-06 2019-03-28 University Of Virginia Patent Foundation Compositions and methods for treating diabetic retinopathy
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