WO2010129950A1 - Micro-arn régulant le remodelage cardiaque - Google Patents

Micro-arn régulant le remodelage cardiaque Download PDF

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Publication number
WO2010129950A1
WO2010129950A1 PCT/US2010/034227 US2010034227W WO2010129950A1 WO 2010129950 A1 WO2010129950 A1 WO 2010129950A1 US 2010034227 W US2010034227 W US 2010034227W WO 2010129950 A1 WO2010129950 A1 WO 2010129950A1
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mir
seq
inhibitor
antisense oligonucleotide
sequence
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PCT/US2010/034227
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English (en)
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Eric N. Olson
Eva Van Rooij
David M. Patrick
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Board Of Regents, The University Of Texas System
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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
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs

Definitions

  • the present invention relates to the treatment of cardiac disorders by administering agents that modulate the activity or expression of a microRNA (miRNA).
  • miRNA microRNA
  • the invention provides a method for treating or preventing cardiac disorders by inhibiting the expression or activity of miR-451 in the heart cells of a subject.
  • the invention provides a method for regulating cardiomyocyte survival by contacting the cardiomyocyte with an inhibitor of miR-451.
  • 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.
  • MicroRNAs 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). Following transport to the cytoplasm, the hairpin pre-miRNA is further processed by Dicer to produce a double-stranded miRNA.
  • RISC RNA- induced silencing complex
  • knockout mice lacking disease-inducing miRNAs are normal, but display aberrant responses to cardiac stress, suggesting the dedication of these miRNAs to disease-related processes rather than tissue homeostasis, and pointing to their potential as therapeutic targets.
  • these identified miRNAs represent potential novel therapeutic targets for the development of treatments for a variety of cardiovascular diesases.
  • the present invention is based, in part, on the discovery that the expression of miR- 451 is regulated in heart tissue following myocardial infarction and that overexpression of miR-451 in a cardiac-specific manner is sufficient to induce cardiac hypertrophy.
  • the inventors have surprisingly found that miR-451 regulates cardiomyocyte survival and cardiogenesis.
  • the present invention provides a method of treating or preventing pathologic cardiac hypertrophy, heart failure, cardiac remodeling, and myocardial infarction in a subject in need thereof.
  • the pathologic cardiac hypertrophy is associated with pulmonary arterial hypertension.
  • the method comprises administering an inhibitor of miR-451 to the subject, wherein the expression or activity of miR-451 is reduced in the heart cells of the subject following administration.
  • the inhibitor of miR-451 can be an antagomir or an antisense oligonucleotide.
  • the number of cardiomyocytes is increased in the subject following administration of the miR-451 inhibitor as compared to a subject not receiving the miR-451 inhibitor.
  • the present invention also includes a method of regulating cardiomyocyte survival.
  • the method comprises contacting a cardiomyocyte with an inhibitor of miR-451.
  • the cardiomyocyte can be in vitro or in vivo.
  • cardiomyocyte survival is enhanced following contact with the miR-451 inhibitor.
  • Akt signaling is increased in the cardiomyocyte following contact with the miR-451 inhibitor.
  • the expression of one or more genes regulated by miR-451 is increased in the cardiomyocyte following contact with the miR-451 inhibitor.
  • Genes targeted by miR-451 can include AKTIP, OSRl, DKKl, and DKK3.
  • FIG. 1 A. Northern blot analysis for miR-451 of RNA isolated from multiple mouse tissues.
  • FIG. 1 A. Rat vascular smooth muscle cells were differentiated for 6 days. Realtime RT-PCR analysis shows that miR-451 is induced during smooth muscle cell differentiation. B. C2C12 myoblasts were differentiated for 5 days. Real-time RT-PCR analysis shows that miR-451 levels are approximately 10-fold higher in differentiated versus undifferentiated myoblasts.
  • FIG. 3 Real-time RT-PCR analysis of RNA from tissue harvested from the border zone of an infarct at 6- and 48-hours post-myocardial infarction. MiR-451 is significantly downregulated as compared to sham operated heart tissue.
  • Figure 4 Schematic of targeting strategy for generation of miR-451 conditional knockout mice.
  • FIG. 1 Northern blot analysis (left panel) of cardiac tissue from a 6-week old transgenic mouse overexpressing miR-451 under the control of the ⁇ -MHC promoter. Densitometry analysis is shown in the right panel.
  • FIG. 1 Absolute (left panel) and normalized (right panel) heart weight/body weight ratios from ⁇ -MHC-miR-451 transgenic animals and wild-type litter mates at 6 weeks of age.
  • B Overexpression of miR-451 induces cardiac hypertrophy which progresses into dilated cardiomyopathy.
  • Figure 7 H & E stained (top panels) and Masson trichome stained (bottom panels) sections from wild-type littermates and ⁇ -MHC-miR-451 transgenic animals at 6 weeks of age and post mortem.
  • Figure 8 H & E stained (top panels) and Masson trichome stained (bottom panels) sections from wild-type littermates and ⁇ -MHC-miR-451 transgenic animals at 6 weeks of age and post mortem. Magnif ⁇ cation-20x.
  • Figure 9 H & E stained section from an ⁇ -MHC-miR-451 transgenic animal post mortem showing loss of cadiomyoctyes.
  • FIG. 10 H & E stained sections at 5x (top panels) and 2Ox (bottom panels) from wild-type littermates and ⁇ -MHC-miR-451 transgenic animals at 6 weeks of age and post mortem illustrating abnormalities in the atria of the hearts of the ⁇ -MHC-miR-451 transgenics.
  • FIG. 11 A. Western blot analysis of cardiac tissue isolated from either transgenic mice overexpressing miR-451 or wild-type litter mates. Expression of GAPDH was used as a loading control. B. Schematic illustrating putative mechanism of miR-451 in cardiac remodeling.
  • GAT A4 binds to both conserved GATA binding sequences (451 -site 1 and
  • FIG. 14 Relative luciferase activities of COS cells co-transfected with GATA4 and a miR-451 luciferase reporter construct having a wild-type (WT) GATA binding site or a mutation in site- 1 , site-2, or both sites (double mutant). GAT A4 activation of the miR-451 luciferase reporter is dependent on both of the conserved GATA binding sites.
  • WT wild-type
  • MiRNAs act as negative regulators of gene expression by inhibiting the translation or promoting the degradation of target mRNAs. Because individual miRNAs often regulate the expression of multiple target genes with related functions, modulating the expression of a single miRNA can, in principle, influence an entire gene network and thereby modify complex disease phenotypes.
  • the present invention is based, in part, on the discovery of a miRNA that is highly expressed in the heart and is regulated in various cardiac disease states.
  • the inventors have surprisingly discovered that overexpression of miR-451 under the control of the ⁇ -MHC promoter induces cardiac hypertrophy and leads to significant cardiac remodeling.
  • overexpression of miR-451 causes abnormalities in the atria and cardiomyocyte loss.
  • the present invention provides a method of treating various cardiac disorders in a subject by inhibiting the expression or activity of miR-451 in heart cells, particularly cardiomyocytes.
  • 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).
  • the subject is a mammal. In other embodiments, the subject is a human.
  • miR-451 is clustered with miR-144 in an intergenic region of chromosome 17.
  • the mature miR-451 is processed from a bicistronic transcript also encoding miR-144.
  • the mature human sequence for miR-451 is 5'-
  • AAACCGUU ACCAUUACUGAGUU-S' (SEQ ID NO: 1), while the human precursor sequence for miR-451 (pre-miR-451 ) is 5'-
  • the present invention provides a method of treating or preventing pathologic cardiac hypertrophy, cardiac remodeling, myocardial infarction, or heart failure in a subject in need thereof comprising administering an inhibitor of miR-451 to the subject, wherein the expression or activity of miR-451 is reduced in the heart cells of the subject following administration.
  • “Heart cells” as used herein include cardiomyocytes, cardiac fibroblasts, and cardiac endothelial cells.
  • the expression or activity of miR-451 is reduced in cardiomyocytes of the subject following administration of a miR- 451 inhibitor.
  • the cardiac hypertrophy may be right ventricular hypertrophy associated with pulmonary arterial hypertension (PAH).
  • PAH pulmonary arterial hypertension
  • the present invention includes a method of preventing or treating PAH in a subject by administering to the subject an inhibitor of miR-451.
  • the invention provides a method of preventing or delaying cardiac hypertrophy associated with PAH from progressing into heart failure by administering to the subject an inhibitor of miR-451.
  • the subject in need thereof may be at risk for developing pathologic cardiac hypertrophy, cardiac remodeling, heart failure, or myocardial infarction.
  • Such a subject may exhibit one or more risk factors including, but not limited to, long standing uncontrolled hypertension, pulmonary arterial hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease or pathological hypertrophy.
  • the subject at risk may be diagnosed as having a genetic predisposition to cardiac hypertrophy or may have a familial history of cardiac hypertrophy.
  • administration of an inhibitor of miR-451 to the subject results in the improvement of one or more symptoms of cardiac hypertrophy, heart failure, or myocardial infarction in the subject, or in the delay in the transition from cardiac hypertrophy to heart failure.
  • the one or more improved symptoms may 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 cardiac fibrosis, decreased collagen deposition in cardiac muscle, decreased left and right ventricular wall stress, decreased wall tension, increased quality of life, and decreased disease related morbidity or mortality.
  • an inhibitor of miR-451 is an antisense oligonucleotide.
  • the antisense oligonucleotides can include ribonucleotides or deoxyribonucleotides or a combination thereof.
  • 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 bicyclic sugar nucleoside modifications (BSN) that confer enhanced thermal stability to complexes formed between the oligonucleotide containing BSN and their complementary microRNA target strand.
  • BSN bicyclic sugar nucleoside modifications
  • the antisense oligonucleotides contain at least one "locked nucleic acid.”
  • Locked nucleic acids (LNAs) 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. 9: 1147- 1150, both of which are herein incorporated by reference in their entireties.
  • the antisense oligonucleotides contain at least one modified nucleoside having the structure shown in structure C.
  • the antisense oligonucleotides targeting miR-451 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.
  • other chemical modifications that the antisense oligonucleotides can contain include, but are not limited to, sugar modifications, such as T- O-alkyl (e.g.
  • antisense oligonucleotides targeting miR-451 contain 2'0-methyl sugar modifications on each base and are linked by phosphorothioate linkages.
  • Antisense oligonucleotides can comprise one or more affinity enhancing modifications, such as, but not limited to, LNAs, bi cyclic nucleosides, phosphonoformates, 2' O-alkyl modifications and the like.
  • suitable antisense oligonucleotides are 2'-O-methoxyethyl "gapmers" which contain 2 '-O-methoxy ethyl -modified ribonucleotides on both 5' and 3' ends with at least ten deoxyribonucleotides in the center.
  • antisense oligonucleotide is 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.
  • antisense oligonucleotides useful for inhibiting the activity of miRNAs 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.
  • antisense oligonucleotides targeting miR-451 are about 8 to about 18 nucleotides in length, and in other embodiments about 12 to about 16 nucleotides in length. Any 8-mer or longer complementary to miR-451 may be used, i.e., any antimiR complementary to the 5' end of the miRNA and progressing across the full complementary sequence of the miRNA.
  • the antisense oligonucleotide has a sequence of 5'-AACUCAGUAAUGGUAACGGUUU-S ' (SEQ ID NO: 3). In another embodiment, the antisense oligonucleotide has a sequence of 5'- AACGGUUU-3' (SEQ ID NO: 4). In another embodiment, the antisense oligonucleotide has a sequence of 5'-GUAACGGUUU-3' (SEQ ID NO: 5). In another embodiment, the antisense oligonucleotide has a sequence of 5'-UGGUAACGGUUU-3' (SEQ ID NO: 6).
  • the antisense oligonucleotide has a sequence of 5'- AAUGGU AACGGUUU-3' (SEQ ID NO: 7). In still another embodiment, the antisense oligonucleotide has a sequence of 5'-GUAAUGGUAACGGUUU-S ' (SEQ ID NO: 8).
  • Antisense oligonucleotides can comprise a sequence that is at least partially complementary to a mature miR-451 sequence, e.g. at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% complementary to a mature miR-451 sequence.
  • the antisense oligonucleotide can be substantially complementary to a mature miR-451 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 100% complementary to a mature miR-451 sequence.
  • the antisense oligonucleotide is at least partially complementary to SEQ ID NO: 1.
  • the antisense oligonucleotides are antagomirs.
  • “Antagomirs” are single-stranded, chemically-modified ribonucleotides that are at least partially complementary to a miR-451 sequence.
  • Antagomirs may comprise one or more modified nucleotides, such as 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. To facilitate in vivo delivery and stability, the antagomir can be linked to a cholesterol or other moiety at its 3' end.
  • Antagomirs suitable for inhibiting miR-451 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 miR-451 sequence.
  • the antagomir may be substantially complementary to a mature miR-451 sequence, that is at least about 95%, 96%, 97%, 98%, or 99% complementary to a target polynucleotide sequence.
  • the antagomirs are 100% complementary to a mature miR-451 sequence.
  • inhibitors of miR-451 are antagomirs comprising a sequence that is perfectly complementary to the mature miR-451 sequence.
  • an inhibitor of miR-451 is an antagomir having a sequence that is partially or perfectly complementary to 5'-AAACCGUUACCAUUACUGAGUU-S ' (SEQ ID NO: 1).
  • inhibitors of miR-451 are chemically-modified antisense oligonucleotides.
  • an inhibitor of miR-451 is a chemically- modified antisense oligonucleotide comprising a sequence substantially complementary to 5'- AAACCGUU ACCAUU ACUGAGUU-3' (SEQ ID NO: 1).
  • substantially complementary refers to a sequence that is at least about 95%, 96%, 97%, 98%, 99%, or 100% complementary to a target polynucleotide sequence (e.g. mature or precursor miRNA sequence).
  • Antisense oligonucleotides may comprise a sequence that is substantially complementary to a precursor miRNA sequence (pre-miRNA) for miR-451.
  • the antisense oligonucleotide comprises a sequence that is substantially complementary to a sequence located outside the stem-loop region of the pre-miR-451 sequence.
  • an inhibitor of miR-451 function is an antisense oligonucleotide having a sequence that is substantially complementary to a pre-miR-451 sequence (SEQ ID NO: 2).
  • any of the inhibitors of miR-451 described herein can be delivered to the target cell (e.g. heart cell) by delivering to the cell an expression vector encoding the miR-451 inhibitors.
  • 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 an inhibitor of miR-451 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 miR-451.
  • the phrase "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.
  • 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).
  • CMV human cytomegalovirus
  • the promoter is a tissue specific promoter.
  • muscle specific promoters Of particular interest are muscle specific promoters, and more particularly, cardiac specific promoters. These include the myosin light chain-2 promoter (Franz et al. (1994) Cardioscience, Vol.
  • alpha7 integrin promoter Ziober and Kramer (1996) J. Bio. Chem., Vol. 271(37):22915-22), 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 operably linked to a polynucleotide encoding a miR-451 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.
  • the present invention also includes methods for scavenging or clearing miR-451 inhibitors following treatment.
  • the method may comprise overexpressing binding sites for the miR-451 inhibitors in cardiac tissue.
  • the binding site regions preferably contain a sequence of the seed region for miR-451.
  • the seed region is the 5' portion of a miRNA spanning bases 2-8, which is important for target recognition.
  • the binding site may contain a sequence from the 3'UTR of one or more targets of miR-451, such as AKTIP, DKKl, DKK3, or OSRl .
  • the present invention provides a method of regulating cardiomyocyte survival comprising contacting a cardiomyocyte with an inhibitor of miR-451.
  • cardiomyocyte survival is enhanced following contact with the miR- 451 inhibitor.
  • Akt signaling is increased in the cardiomyocyte following contact with the miR-451 inhibitor.
  • One or more genes regulated by miR-451 may be increased in the cardiomyocyte following contact with the inhibitor.
  • Target genes of miR- 451 include, but are not limited to, AKTIP, DKKl , DKK3, or OSRl .
  • the expression of AKTIP is increased in the cardiomyocyte following contact with the miR- 451 inhibitor.
  • 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 cardiomyocyte can be in vitro or in vivo.
  • the present invention also includes pharmaceutical compositions comprising an inhibitor of miR-451. Where clinical applications are contemplated, pharmaceutical 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 a miR-451 inhibitor.
  • the pharmaceutical composition comprises and effective dose of a modified antisense oligonucleotide targeting miR-451 as described herein.
  • the pharmaceutical composition comprises a modified antisense oligonucleotide having a sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.
  • 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 20 mg/kg to about 200 mg/kg, about 40 mg/kg to about 160 mg/kg, or about 80 mg/kg to about 100 mg/kg.
  • the inhibitor of miR-451 is administered at a dosage of about 20 mg/kg to about 200 mg/kg.
  • the inhibitor of miR-451 is administered at a dosage of about 80 mg/kg.
  • the precise determination of what would be considered an effective dose may be based on factors individual to each patient, including their size, age, type of disorder (e.g. myocardial infarction, heart failure, or cardiac hypertrophy), and nature of inhibitor or agonist (e.g. antagomir, expression construct, antisense oligonucleotide, etc). Therefore, dosages can be readily ascertained by those of ordinary skill in the art from this disclosure and the knowledge in the art.
  • 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 oligonucleotide inhibitors of miR-451 function or constructs expressing particular miR-451 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 or expression constructs comprising miRNA inhibitors 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. Some non-limiting examples of catheter-based delivery methods or coronary isolation methods suitable for use in the present invention are disclosed in U.S.
  • compositions would normally be administered as pharmaceutically acceptable compositions as described herein.
  • the active compounds may also be administered parenterally or intraperitoneally.
  • 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. In many cases, it will be preferable to include 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. [0056] 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.
  • any other ingredients for example as enumerated above
  • 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.
  • 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
  • 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.
  • miR-451 expression appears to be developmentally regulated.
  • Real-time RT-PCR analysis for miR-451 was performed on RNA from either whole animal or whole heart collected at embryonic days 12.5 and 15.5, and post-natal days 1, 3, and 5.
  • MiR-451 expression was induced in the heart approximately 5-fold from E12.5 to E15.5 ( Figure ID). Expression is reduced during the early post-natal period and then increases again in the adult.
  • MiR-451 knockout mice were generated to further elucidate the role of miR-451 in cardiac disease ( Figure 4). Specifically, a miR-451 targeting vector was generated by digesting a fragment (5' arm) extending upstream of the miR-451 coding region, which included the miR-144 coding region, and ligating the fragment into the pGKneoF2L2dta targeting plasmid upstream of the loxP sites and the Fit-flanked neomycin cassette. A second fragment (3' arm) was digested and ligated into the vector between the neomycin resistance and Dta negative selection cassettes.
  • a miR-451 targeting vector was generated by digesting a fragment (5' arm) extending upstream of the miR-451 coding region, which included the miR-144 coding region, and ligating the fragment into the pGKneoF2L2dta targeting plasmid upstream of the loxP sites and the Fit-flanked neo
  • MiR-451 targeted ES clones were identified and used for blastocyst injection. The resulting chimeric mice will be bred to C57BL/6 to obtain germline transmission of the mutant allele. MiR-451 knockout animals are expected to exhibit less cardiac hypertrophy in response to stress stimuli and more cell survival.
  • MiR-451 targets genes involved in cell survival and cardiogenesis
  • miR-451 is predicted to target genes involved in cell survival and cardiogenesis, including DKKl , DKK3, Akt interacting protein (AKTIP), and odd-skipped related 1 (OSR) (Table 1).
  • Western blot analysis of heart tissue isolated from transgenic mice overexpressing miR-451 showed a downregulation of both DKKl and AKTIP ( Figure 1 IA) suggesting that these two proteins are in vivo targets of miR-451.
  • the current data suggest that miR-451 influences cardiac remodeling by reducing cell survival and cardiogenesis, including atrial development, by repressing its targets AKTIP, DKKl, DKK3, and OSRl .
  • MiR-451 is encoded on a polycistronic primary transcript with miR-144 and the enhancer region upstream of the common precursor sequence is conserved across several mammalian species (See Dore et al). The enhancer region upstream of the miR-451 primary sequence contains two independent GATA binding sequences (Dore et al.).
  • luciferase constructs consisting of the enhancer region upstream of the pri -miR-451 sequence were generated.
  • the responsiveness of the miR-451 enhancer region to GAT A4 in COS cells was examined ( Figure 14).
  • GAT A4 activation of the miR-451 luciferase reporter was dependent on both GATA binding sequences in the enhancer region as demonstrated by mutational analysis in transfected COS cells ( Figure 14).

Abstract

La présente invention a pour objet une méthode de traitement ou de prévention d'une hypertrophie cardiaque pathologique, d'un remodelage cardiaque, d'un infarctus du myocarde ou d'une insuffisance cardiaque chez un sujet par l'inhibition de l'expression ou de l'activité de miR-451 dans les cellules cardiaques du sujet. L'invention a également pour objet des méthodes de régulation de la survie des cardiomyocytes et de la signalisation Akt par la modulation de l'expression ou de l'activité de miR-451.
PCT/US2010/034227 2009-05-08 2010-05-10 Micro-arn régulant le remodelage cardiaque WO2010129950A1 (fr)

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Publication number Priority date Publication date Assignee Title
US20130150256A1 (en) * 2010-06-11 2013-06-13 Jane Synnergren Novel micrornas for the detection and isolation of human embryonic stem cell-derived cardiac cell types
US9416360B2 (en) 2010-11-05 2016-08-16 MiRagen Therapeutics, Inc. Base modified oligonucleotides
WO2013093870A1 (fr) 2011-12-23 2013-06-27 International Centre For Genetic Engineering And Biotechnology - Icgeb Microarn pour la régénération cardiaque par l'intermédiaire d'induction de la prolifération de cardiomyocytes

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