Micro-rna family that modulates fibrosis and uses thereof

Abstract

The present invention relates to the identification of a microRNA family, designated miR-29a-c, that is a key regulator of fibrosis in cardiac tissue. The inventors show that members of the miR-29 family are down-regulated in the heart tissue in response to stress, and are up-regulated in heart tissue of mice that are resistant to both stress and fibrosis. Also provided are methods of modulating expression and activity of the miR-29 family of miRNAs as a treatment for fibrotic disease, including cardiac hypertrophy, skeletal muscle fibrosis other fibrosis related diseases and collagen loss-related disease.

1 . A method of treating cardiac fibrosis, cardiac hypertrophy, or heart failure in a subject in need thereof comprising:
 (a) identifying a subject having cardiac fibrosis, cardiac hypertrophy or heart failure; and   (b) administering to said subject an agonist of miR-29a-c expression or function.   
     
     
         2 . The method of  claim 1 , wherein the agonist of miR-29a-c is a polynucleotide comprising the mature sequence of miR-29a, miR-29b, miR-29c, or combinations thereof. 
     
     
         3 . The method of  claim 2 , wherein the polynucleotide comprises a sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         4 . The method of  claim 2 , wherein the agonist of miR-29a-c is administered by parenteral administration or direct injection into cardiac tissue. 
     
     
         5 . The method of  claim 4 , wherein the parenteral administration is intravenous or subcutaneous. 
     
     
         6 . The method of  claim 2 , wherein the agonist of miR-29a-c is administered by oral, transdermal, sustained release, controlled release, delayed release, suppository, catheter or sublingual administration. 
     
     
         7 . The method of  claim 1 , further comprising administering to said subject a second therapy 
     
     
         8 . The method of  claim 7 , wherein said second therapy is selected from the group consisting of a beta blocker, an ionotrope, a diuretic, ACE-I, AII antagonist, BNP, a Ca ++ -blocker, an endothelin receptor antagonist, and an HDAC inhibitor. 
     
     
         9 . The method of  claim 7 , wherein said second therapy is administered at the same time as the agonist of miR-29a-c. 
     
     
         10 . The method of  claim 7 , wherein said second therapy is administered either before or after the agonist of miR-29a-c. 
     
     
         11 . The method of  claim 1 , wherein one or more symptoms of cardiac fibrosis, cardiac hypertrophy or heart failure is improved in the subject following administration of the agonist of miR-29a-c. 
     
     
         12 . The method of  claim 11 , wherein said one or more improved symptoms is 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, decreased disease related morbidity or mortality, or combinations thereof. 
     
     
         13 . The method of  claim 1 , wherein administration of the agonist of miR-29a-c delays the transition from cardiac hypertrophy to heart failure in the subject. 
     
     
         14 . A method of preventing pathologic hypertrophy or heart failure in a subject in need thereof comprising:
 (a) identifying a subject at risk of developing pathologic cardiac hypertrophy or heart failure; and   (b) promoting the expression or activity of miR-29a-c in cardiac cells of said subject.   
     
     
         15 . The method of  claim 14 , wherein promoting expression or activity of miR-29a-c comprises delivering to the cardiac cells an agonist of miR-29a-c or an expression vector encoding miR-29a-c. 
     
     
         16 . The method of  claim 15 , wherein the agonist of miR-29a-c is a polynucleotide comprising the mature sequence of miR-29a, miR-29b, miR-29c, or combinations thereof. 
     
     
         17 . The method of  claim 15 , wherein the expression vector comprises a sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. 
     
     
         18 . The method of  claim 14 , wherein the subject at risk exhibits one or more risk factors selected from the group consisting of long standing uncontrolled hypertension, uncorrected valvular disease, chronic angina, recent myocardial infarction, congenital predisposition to heart disease, and pathological hypertrophy. 
     
     
         19 . The method of  claim 14 , wherein the subject at risk has been diagnosed as having a genetic predisposition to cardiac hypertrophy. 
     
     
         20 . The method of  claim 14 , wherein the subject at risk has a familial history of cardiac hypertrophy. 
     
     
         21 . A transgenic, non-human mammal, the cells of which fail to express a functional miR-29a, miR29b, and/or miR29c. 
     
     
         22 . The transgenic mammal of  claim 21 , wherein said mammal is a mouse. 
     
     
         23 . A transgenic, non-human mammal, the cells of which comprise a miR-29a-c coding region under the control of a heterologous promoter active in the cells of said non-human mammal. 
     
     
         24 . The transgenic mammal of  claim 23 , wherein said mammal is a mouse. 
     
     
         25 . The transgenic mammal of  claim 23 , wherein said promoter is a tissue specific promoter. 
     
     
         26 . The transgenic mammal of  claim 25 , wherein the tissue specific promoter is a muscle specific promoter or a fibroblast-specific promoter. 
     
     
         27 . The transgenic mammal of  claim 25 , wherein the tissue specific promoter is a heart muscle-specific promoter. 
     
     
         28 . A transgenic, non-human mammalian cell lacking one or both native alleles of miR-29a, miR-29b, and/or miR-29c. 
     
     
         29 . The cell of  claim 28 , wherein said cell lacks all of said native miR-29a-c alleles. 
     
     
         30 . A method of treating myocardial infarction in a subject in need thereof comprising promoting expression or activity of miR-29a-c in cardiac cells of said subject. 
     
     
         31 . A method of preventing cardiac hypertrophy and dilated cardiomyopathy in a subject in need thereof comprising promoting expression or activity of miR-29a-c in cardiac cells of said subject. 
     
     
         32 . A method of inhibiting progression of cardiac hypertrophy in a subject in need thereof comprising promoting expression or activity of miR-29a-c in cardiac cells of said subject. 
     
     
         33 . A method of treating or preventing a tissue fibrosis in a subject comprising:
 (a) identifying a subject having or at risk of tissue fibrosis; and   (b) increasing the expression and/or activity of miR-29a-c in skeletal muscle or fibroblast cells of the subject.   
     
     
         34 . The method of  claim 33 , wherein said tissue fibrosis is cardiac fibrosis, scleroderma, skeletal muscle fibrosis, hepatic fibrosis, kidney fibrosis, pulmonary fibrosis, or diabetic fibrosis. 
     
     
         35 . The method of  claim 33 , wherein increasing the expression and/or activity of miR-29a-c comprises administering an agonist of miR-29a-c to the subject. 
     
     
         36 . The method of  claim 35 , wherein the agonist of miR-29a-c is a polynucleotide comprising a sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         37 . The method of  claim 33 , wherein increasing the expression and/or activity of miR-29a-c comprises administering to the subject an expression vector that encodes miR-29a-c. 
     
     
         38 . The method of  claim 37 , wherein the expression vector is a viral expression vector. 
     
     
         39 . The method of  claim 38 , wherein the viral expression vector is an adenoviral expression vector. 
     
     
         40 . The method of  claim 37 , wherein the expression vector is a non-viral expression vector. 
     
     
         41 . The method of  claim 40 , wherein the non-viral expression vector is comprised within a lipid vehicle. 
     
     
         42 . The method of  claim 35 , wherein the agonist of miR-29a-c is comprised within a lipid vehicle. 
     
     
         43 . The method of  claim 33 , further comprising administering a non-miR-29a-c anti-fibrotic therapy to the subject. 
     
     
         44 . A method for identifying a modulator of miR-29a-c comprising:
 (a) contacting a cell with a candidate compound;   (b) assessing miR-29a-c activity or expression; and   (c) comparing the activity or expression in step (b) with the activity or expression of miR-29a-c in the absence of the candidate compound,   wherein a difference between the measured activities or expression of miR-29a-c indicates that the candidate compound is a modulator of miR-29.   
     
     
         45 . The method of  claim 44 , wherein the cell is contacted with the candidate compound in vitro. 
     
     
         46 . The method of  claim 44 , wherein the cell is contacted with the candidate compound in vivo. 
     
     
         47 . The method of  claim 44 , wherein the modulator of miR-29a-c is an agonist of miR-29a-c. 
     
     
         48 . The method of  claim 44 , wherein the modulator of miR-29a-c is an antagonist of miR-29a-c. 
     
     
         49 . The method of  claim 44 , wherein the candidate compound is a protein, a peptide, a polypeptide, a polynucleotide, an oligonucleotide or a small molecule. 
     
     
         50 . The method of  claim 44 , wherein assessing the miR-29a-c activity or expression comprises assessing the expression of miR-29a-c. 
     
     
         51 . The method of  claim 50 , wherein assessing the expression of miR-29a-c comprises northern blotting or RT-PCR. 
     
     
         52 . The method of  claim 44 , wherein assessing the miR-29a-c activity or expression comprises assessing the activity of miR-29a-c. 
     
     
         53 . The method of  claim 52 , wherein assessing the activity of miR-29a-c comprises assessing expression or activity of a gene regulated by miR-29a-c. 
     
     
         54 . The method of  claim 53 , wherein the gene regulated by miR-29a-c is COL1A1, COL1A2, COL1A3 and/or FBN1. 
     
     
         55 . A pharmaceutical composition comprising an agonist of miR-29a-c. 
     
     
         56 . The pharmaceutical composition of  claim 55 , wherein said agonist is an expression vector encoding miR-29a-c. 
     
     
         57 . The pharmaceutical composition of  claim 55 , wherein said agonist is a polynucleotide comprising the mature sequence of miR-29a, miR-29b, miR-29c. 
     
     
         58 . The pharmaceutical composition of  claim 57 , wherein said polynucleotide comprises a sequence of SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         59 . The pharmaceutical composition of  claim 55 , wherein said agonist is comprised in a lipid delivery vehicle. 
     
     
         60 . The pharmaceutical composition of  claim 55 , wherein the composition is formulated for injection. 
     
     
         61 . The pharmaceutical composition of  claim 55  in combination with a kit for parenteral administration. 
     
     
         62 . The pharmaceutical composition of  claim 61 , wherein parenteral administration is intravenous or subcutaneous. 
     
     
         63 . The pharmaceutical composition of  claim 55  in combination with a kit for catheter administration. 
     
     
         64 . A pharmaceutical composition comprising an antagonist of miR-29a-c. 
     
     
         65 . The pharmaceutical composition of  claim 64 , wherein the antagonist of miR-29a-c is an antagomir of miR-29a-c. 
     
     
         66 . The pharmaceutical composition of  claim 64 , wherein the antagonist of miR-29a-c comprises a sequence that is complementary to the mature sequence of miR-29a, miR-29b, miR-29c, or combinations thereof. 
     
     
         67 . The pharmaceutical composition of  claim 64 , wherein the composition is formulated for topical administration. 
     
     
         68 . The pharmaceutical composition of  claim 67 , wherein the composition is a gel, cream, lotion, or ointment. 
     
     
         69 . A method of inducing collagen deposition in a tissue comprising contacting said tissue with an antagonist of miR-29a-c. 
     
     
         70 . The method of  claim 69 , wherein said antagonist is an antagonist of miR-29a, miR-29b, or miR-29c. 
     
     
         71 . The method of  claim 69 , wherein said antagonist is an antagomir of miR-29a-c. 
     
     
         72 . The method of  claim 69 , wherein said antagonist comprises a sequence that is complementary to the mature sequence of miR-29a, miR-29b, miR-29c, or combinations thereof. 
     
     
         73 . The method of  claim 69 , wherein said antagonist comprises a sequence that is complementary to SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20. 
     
     
         74 . The method of  claim 69 , wherein said tissue is facial tissue. 
     
     
         75 . The method of  claim 74 , wherein said facial tissue is forehead tissue, a lip, a cheek, a chin, an eyebrow, an eyelid, under the eye, or near the mouth. 
     
     
         76 . The method of  claim 69 , wherein said tissue is hand tissue, neck tissue, arm tissue, leg tissue, stomach tissue, or breast tissue. 
     
     
         77 . The method of  claim 69 , wherein said tissue comprises a wound, a skin graft, scar tissue, wrinkles, lax skin, sun damage, chemical damage, heat damage, cold damage, and/or stretch marks. 
     
     
         78 . The method of  claim 69 , wherein contacting comprises injection into said tissue, injection into vasculature that feeds said tissue, or topical application. 
     
     
         79 . The method of  claim 78 , wherein topical application comprises use of an ointment, cream, gel, salve, or balm. 
     
     
         80 . The method of  claim 78 , further comprising use of a pressure bandage or dressing. 
     
     
         81 . The method of  claim 69 , wherein said antagonist is contacted with said tissue more than once. 
     
     
         82 . The method of  claim 81 , wherein said antagonist is contacted with said tissue 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 or 100 times. 
     
     
         83 . The method of  claim 81 , wherein said antagonist is contacted with said tissue over 2, 3, 4, 5, or 6 days, 1, 2, 3, or 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or 1, 2, 3, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 years. 
     
     
         84 . The method of  claim 69 , further comprising contacting said tissue with a second agent. 
     
     
         85 . The method of  claim 84 , wherein said second agent is topical vitamin A, topical vitamin C, or vitamin E. 
     
     
         86 . The method of  claim 69 , further comprising subjecting said tissue to a second treatment. 
     
     
         87 . The method of  claim 86 , wherein said second treatment comprises a chemical peel, laser treatment, dermaplaning, or dermabrasion. 
     
     
         88 . The method of  claim 69 , wherein said tissue is in a subject that suffers from Ehler's-Danlos syndrome or Vitamin C deficiency.