HK1156360B - Microrna (mirna) and downstream targets for diagnostic and therapeutic purposes - Google Patents

Description

micro-RNAs (MIRNAs) and downstream targets for diagnostic and therapeutic purposes

Technical Field

The present invention relates to the field of micro rna (mirna), in particular miR-21 and its downstream targets for the diagnosis, prevention and/or treatment of fibrosis and other diseases. The invention also relates to compositions, methods and uses for treating fibrosis. Such methods include modulating and inhibiting miRNA activity in a subject having fibrosis.

Background

Micrornas are a large class of small, non-coding RNAs that control a variety of biological processes, including the major signal transduction pathways resulting from the regulation of expression of complementary target mrnas (Ambros, 2004). Deregulation of micrornas in various disease entities is caused by genomic alterations (Mi et al, 2007), differential expression, or in some cases viral infection that alters microrna function as a tumor suppressor or oncogene. Recently, microRNAs have been implicated in the regulation of different cardiac functions in a series of elaborate genetic studies (Care et al, 2007; Yang et al, 2007). While these studies help to delineate the role of micrornas in cardiac physiology, growth, and morphogenesis, little is known about the detailed molecular mechanisms of micrornas in disease pathways in vivo. Studies have shown that single-stranded oligonucleotide microRNA antagonists silence endogenous microRNAs in vitro and in vivo, and thus have an effect on target mRNA and protein levels and metabolism (Kruetzfeldt et al, 2005; Esu et al, 2006). These findings indicate that the use of microrna antagonists confirms microrna function in vivo and perhaps more importantly, as new therapeutic modalities.

Summary of The Invention

The invention relates to a promoter region (promoter region) of microRNA, application of microRNA, particularly miR-21, and related components for diagnosing and preparing medicaments for treating and/or preventing fibrosis and/or fibrosis-related diseases. In addition, the invention relates to various antisense oligonucleotides directed against targets of miR-21. Target deficient cells of miR-21, promoter regions and miR-21 and knock-out organisms thereof are also included. Finally, the present invention relates to methods for diagnosing fibrosis and/or a fibrosis-related disease and methods of screening pharmaceutically active compounds for the treatment of fibrosis and/or a fibrosis-related disease.

Provided herein are methods for treating fibrosis, the methods comprising administering to a subject having or suspected of having fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase (nucleobase) sequence complementary to a miRNA.

The methods provided herein include identifying a subject having or suspected of having fibrosis; administering to the subject a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to the miRNA or a precursor thereof.

The methods provided herein include administering to a subject at risk of developing fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence complementary to a miRNA or a precursor thereof, thereby preventing fibrosis. In certain embodiments, the fibrosis is hepatic fibrosis. In certain embodiments, the fibrosis is pulmonary fibrosis. In certain embodiments, the fibrosis is skin fibrosis. In certain embodiments, the fibrosis is age-related fibrosis. In certain embodiments, the fibrosis is cardiac fibrosis. In certain embodiments, the fibrosis is renal fibrosis. In certain embodiments, the fibrosis is spleen fibrosis.

The methods provided herein comprise administering to a subject having or suspected of having fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence complementary to a miRNA or a precursor thereof, wherein the subject has at least one cardiac disease or disorder.

The methods provided herein include identifying a subject having or suspected of having fibrosis, wherein the subject has at least one heart disease or disorder; and administering to the subject a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to the miRNA or a precursor thereof.

In certain embodiments, the cardiac disease or disorder is selected from cardiac hypertrophy, hypertensive heart failure, diastolic heart failure, systolic heart failure, heart-related storage disease, cardiomyopathy, constrictive pericarditis, coronary artery disease, acute myocardial infarction, chronic myocardial infarction, right heart failure, arrhythmia, myocarditis-related fibrosis, and heart valve disease.

In certain embodiments, the cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, obstructive hypertrophic cardiomyopathy, ileus hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and diabetic cardiomyopathy.

In certain embodiments, the heart valve disease is selected from the group consisting of mitral valve stenosis, aortic valve stenosis, tricuspid valve stenosis, and pulmonary valve stenosis.

In certain embodiments, the heart valve disease is selected from mitral insufficiency, aortic insufficiency, tricuspid insufficiency, and pulmonary insufficiency.

In certain embodiments, the methods provided herein further comprise administering one or more additional agents.

In certain embodiments, administration improves cardiac weight gain, left ventricular dilation, or reduction of fractional shortening (attenuation of fractional shortening).

In certain embodiments, administration prevents cardiac weight gain, left ventricular dilation, or a decrease in fractional shortening.

In certain embodiments, the administration improves cardiac function.

In certain embodiments, administering comprises intravenous administration, subcutaneous administration, intraarterial administration, or intracardiac administration.

Provided herein are methods for treating fibrosis, the methods comprising administering to a subject having or suspected of having fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence complementary to a miRNA or a precursor thereof, wherein the subject has at least one liver disease or disorder.

The methods provided herein include identifying a subject having or suspected of having fibrosis, wherein the subject has at least one liver disease or disorder; and administering to the subject a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to the miRNA or a precursor thereof. In certain embodiments, the at least one liver disease or disorder is chronic liver injury. In certain embodiments, the at least one liver disease or condition is a hepatitis virus infection. In certain embodiments, the hepatitis infection is a hepatitis c infection. In certain embodiments, the at least one liver disease or disorder is non-alcoholic steatohepatitis. In certain embodiments, administering comprises intravenous administration or subcutaneous administration. In certain embodiments, the at least one liver disease or disorder is cirrhosis. In certain embodiments, the administration improves liver function.

Provided herein are methods for treating fibrosis, the methods comprising administering to a subject having or suspected of having fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence complementary to a miRNA or a precursor thereof, wherein the subject has at least one pulmonary disease or disorder.

The methods provided herein include identifying a subject having or suspected of having fibrosis, wherein the subject has at least one pulmonary disease or disorder; and administering to the subject a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to the miRNA or a precursor thereof. In certain embodiments, the at least one other pulmonary disease or condition is chronic obstructive pulmonary disease. In certain embodiments, administering comprises pulmonary administration.

Provided herein are methods for treating fibrosis, the methods comprising administering to a subject having or suspected of having fibrosis a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides and having a nucleobase sequence complementary to a miRNA or a precursor thereof, wherein the subject has at least another disease or disorder.

The methods provided herein include identifying a subject having or suspected of having fibrosis, wherein the subject has at least one other disease or disorder; and administering to the subject a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to the miRNA or a precursor thereof. In certain embodiments, the at least one other disease or disorder is pulmonary hypertension. In certain embodiments, at least one other disease or disorder is a vascular-related disease. In certain embodiments, the vascular-related disorder is selected from the group consisting of arterial stiffness, mediaclerosis, and arteriosclerosis. In certain embodiments, the at least one other disease or disorder is intestinal sclerosis. In certain embodiments, at least one other disease or disorder is systemic scleroderma. In certain embodiments, the at least one further disease or condition is selected from retroperitoneal fibrosis, proliferative fibrosis (proliferative fibrosis), proliferative fibrosis (neoplastic fibrosis), nephrogenic systemic fibrosis, congestive fibrosis (Objective fibrosis), mediastinal fibrosis, myelofibrosis, post vasectomy pain syndrome, rheumatoid arthritis.

In certain embodiments, the administration ameliorates fibrosis. In certain embodiments, the administration slows further progression of fibrosis. In certain embodiments, the administration terminates further progression of fibrosis. In certain embodiments, the administration reduces fibrosis. In certain embodiments, the administration reduces the collagen content.

Provided herein are methods for treating a fibroproliferative disease, comprising administering to a subject having or suspected of having a fibroproliferative disease a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to a miRNA or a precursor thereof, thereby treating the fibroproliferative disease.

In certain embodiments, administration includes intravenous administration, subcutaneous administration, pulmonary administration, intraarterial administration, or intracardiac administration.

Provided herein are methods for inhibiting fibroblast proliferation, the methods comprising contacting a fibroblast with a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to a miRNA or a precursor thereof, thereby inhibiting fibroblast proliferation.

Provided herein are methods for stimulating apoptosis in fibroblasts, the methods comprising contacting fibroblasts with a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21 or a precursor thereof, thereby stimulating apoptosis in fibroblasts.

Provided herein are methods of increasing Sprouty1 protein in a fibroblast, the method comprising contacting the fibroblast with a compound comprising a modified oligonucleotide consisting of 12-30 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to a miRNA or a precursor thereof, thereby stimulating Sprouty1 protein expression.

Provided herein are compositions for inhibiting micrornas. In certain embodiments, the miRNA is miR-21. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to a nucleobase sequence having at least 80% identity to miR-21 or a precursor thereof. In certain embodiments, miR-21 has the amino acid sequence as set forth in SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof. In certain embodiments, the miR-21 precursor has the amino acid sequence as set forth in SEQ ID NO: 11, or a nucleotide sequence represented by formula (I).

In certain embodiments, the modified oligonucleotide consists of 12-30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 12 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 13 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 15-24 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 15 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 21 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 22 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 23 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 24 linked nucleosides.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is not mismatched by more than 2 with the nucleobase sequence of miR-21 or a precursor thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is not mismatched by more than 1 with the nucleobase sequence of miR-21 or a precursor thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is free of mismatches to the nucleobase sequence of miR-21 or a precursor thereof.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of SEQ id no: 12 is at least 15 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of seq id NO: 12 is at least 16 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of SEQ ID NO: 12 is at least 17 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of seq id NO: 12 is at least 18 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of SEQ ID NO: 12 is at least 19 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of seq id NO: 12 is at least 20 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of SEQ ID NO: 12 is at least 21 consecutive nucleobases of the nucleobase sequence of 12. In certain embodiments, the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence of SEQ ID NO: 12 is at least 22 consecutive nucleobases of the nucleobase sequence of 12.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide consists of SEQ ID NO: 12 in a sequence listing.

In certain embodiments, the compound comprises a modified oligonucleotide conjugated to a ligand. In certain embodiments having the following structure (III):

(5’)QxQz¹(Qy)_nQz²Qz³Qz⁴Q-L(3’)

(III)

wherein

Each Q is independently a 2' -O-methyl modified nucleoside;

one of A and B is S and the other is O;

y is

z¹、z²、z³And z⁴Each is independently x or y;

n＝6-17

l is

Wherein:

x is N (CO) R⁷Or NR⁷；

R¹、R³And R⁹Each of which is independently H, OH or-CH₂OR^bWith the proviso that R is¹、R³And R⁹At least one of which is OH, and R¹、R³And R⁹At least one of which is-CH₂OR^b；

R⁷Is R^dOr by NR^cR^dOr NHC (O) R^dSubstituted C₁-C₂₀An alkyl group;

R^cis H or C₁-C₆An alkyl group;

R^dis a carbohydrate radical; or a steroid-like alcohol group optionally linked to at least one sugar group; and

R^bis composed of

One of A and B is S and the other is O.

In certain embodiments, R^dIs cholesterol. In certain embodiments, z¹、z²、z³And z⁴Each of which isOne of A and B is S and the other is O.

In certain embodiments, R¹is-CH₂OR^b. In certain embodiments, R⁹Is OH. In certain embodiments, R¹And R⁹Is in the trans form. In certain embodiments, R¹And R³Is in the trans form. In certain embodiments, R³is-CH₂OR^b. In certain embodiments, R¹Is OH. In certain embodiments, R¹And R³Is in the trans form. In certain embodiments, R³And R⁹Is in the trans form. In certain embodiments, R⁹Is CH₂OR^b. In certain embodiments, X is NC (O) R⁷. In certain embodiments, R⁷is-CH₂(CH₂)₃CH₂NHC(O)R^d。

In certain embodiments, at least one internucleoside linkage is a modified internucleoside linkage. In certain embodiments, each internucleoside linkage is a modified internucleoside linkage. In certain embodiments, at least one internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, at least one nucleoside comprises a modified sugar. In certain embodiments, the plurality of nucleosides comprises a modified sugar. In certain embodiments, each nucleoside comprises a modified sugar. In certain embodiments, each nucleoside comprises a 2' -O-methoxyethyl sugar. In certain embodiments, each of the plurality of nucleosides comprises a 2 '-O-methoxyethyl sugar and each of the plurality of nucleosides comprises a 2' -fluoro sugar. In certain embodiments, each modified sugar is independently selected from a 2 ' -O-methoxyethyl sugar, a 2 ' -fluoro sugar, a 2 ' -O-methyl sugar, or a bicyclic sugar moiety. In certain embodiments, at least one nucleoside comprises a modified nucleobase. In certain embodiments, the modified nucleobase is a 5-methylcytosine. In certain embodiments, at least one nucleoside comprises a cytosine, wherein the cytosine is a 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 1is at least 90% complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 1is at least 95% complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ id no: 1is 100% complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 1is complementary over the full length. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 11 is at least 90% complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 11 is at least 95% complementary. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 11 are 100% complementary. In certain embodiments, the miR-21 nucleobase sequence consists of the nucleobase sequence of seq id NO: 1 in the sequence listing. In certain embodiments, the precursor nucleobase sequence consists of the nucleobase sequence of SEQ ID NO: 11 in the sequence listing.

In certain embodiments, the compounds comprising modified oligonucleotides are formulated into pharmaceutical compositions. In certain embodiments, the modified oligonucleotide is formulated as a pharmaceutical composition.

In certain embodiments, the compound consists of a modified oligonucleotide. In certain embodiments, the modified oligonucleotide is a single-stranded modified oligonucleotide. In certain embodiments, the modified oligonucleotide is an antisense oligonucleotide.

Provided herein are compositions for treating, preventing and/or ameliorating fibrosis. Also provided herein are compounds comprising modified oligonucleotides having nucleobase sequences complementary to mirnas for use in treating, preventing and/or ameliorating fibrosis.

Provided herein are antisense oligonucleotides complementary to miR-21 for use in the preparation of a medicament for treating and/or preventing fibrosis.

Provided herein are antisense oligonucleotides complementary to miR-21 for use in the treatment and/or prevention of fibrosis.

Provided herein are uses of miR-21 and/or antisense oligonucleotides directed against miR-21 for the treatment of fibrosis.

Provided herein are uses of miR-21 and/or antisense oligonucleotides directed against miR-21 for diagnosing fibrosis.

Brief Description of Drawings

FIG. 1 miR-21 dysregulation and significant expression in cardiac fibroblasts in heart diseases

(a) Analysis of microRNA expression using microarrays. RNA was isolated from the left ventricular myocardium of a murine model of heart failure (β 1AR-TG mouse) at early (3 months of age), mid (6 months of age) and late (12 months) of heart failure. Expression is expressed as fold-regulation relative to wild-type control. miR-21 is labeled with red. Data were obtained from 3-4 independent hybridizations per group.

(b) Left panel: northern blot analysis of miR-21 expression in different stages of heart failure in β 1AR-TG mice. Right panel: quantitative analysis of the data of the upper graph.

(c) Left panel: northern blot analysis of miR-21 expression in non-failing and failing human left ventricular myocardium. Right panel: quantitative analysis of miR-21 mature form in non-failing and failing human left ventricular myocardium.

(d) The upper diagram: neonatal cardiomyocytes (neonatal cardiac yocyte) stained with 4', 6-diamidino-2-phenylindole (DAPI) and antibodies against alpha-actinin following transfection with synthetic miR (scarmbled-miR) (control-pre-miR, 50nM, 72 hours), synthetic miR-21(pre-miR-21, 50nM, 72 hours) and miR-21 inhibitors (anti-miR-21, 50nM, 72 hours). Cardiomyocytes were cultured under controlled conditions (see supplement methods) or stimulated with FCS (5%) for 48 hours. The following figures: quantitative analysis of the size of each cardiomyocyte (histogram analysis). Analysis was performed on n > 200 cardiomyocytes per group.

(e) The upper diagram: production of transgenic mice expressing miR-21 under the control of an alpha-MHC promoter. The middle graph is as follows: northern blots of mature miR-21 in wild-type (WT) and Transgenic (TG) animals. The following figures: stained heart cross sections of wild-type and miR-21-transgenic mice to determine basic morphology (HE staining) and collagen deposition (Sirius red).

(f) miR-21 expression in cardiac fibroblasts and cardiomyocytes. Northern blot (upper panel) and quantitative real-time PCR analysis (lower panel). All error bars represent SEM. N of a-f is 3-6.

FIG. 2 inhibition of Sprouty1 derepression of ERK-signaling and increased fibroblast survival by miR-21

(a) Spry1LacZ +/-mouse hearts were stained with X-gal. Visual (upper panel) and microscopic (lower panel) analysis showed LacZ was detected in cardiac fibroblasts. The short black bars represent 100 μm. The long black bars represent 10 μm.

(b, c) upper diagram: human failing left ventricle (b) and synthetic microRNA (scarmbled-microRNA) (Pre-miR) for co-cultured cardiomyocytes and fibroblasts^TMNegative control #2, 50nM, 72 hr), synthetic miR-21(pre-miR-21, 50nM, 72 hr) or miR-21 inhibitor (anti-miR-21, 50nM, 72 hr) were tested for SPRY1, ERK1/2 and ERK phosphate 1/2 after transfection. The following figures: quantitative analysis of western blot results.

(d) The upper diagram: SPRY1, ERK1/2, and ERK phosphate 1/2 were tested after siRNA-mediated knockdown (150nM, 72 hours) of SPRY1 or treatment with synthetic siRNA co-cultured heart cells (150nM, 72 hours). The following figures: quantitative analysis of western blot results.

(e) The upper diagram: FACS analysis of annexin V positive cardiac fibroblasts following treatment with synthetic microRNAs (control-pre-miR, 100nM, 72 h), synthetic miR-21(pre-miR-21, 100nM, 72 h), miR-21 antagonists (anti-miR-21, 100nM, 72 h) or corresponding controls (100nM, 72 h). The following figures: quantitative analysis of annexin V positive cells after the corresponding treatment.

(f) The method comprises the following steps FGF2 concentration in supernatants of cultured cardiac fibroblasts following treatment with synthetic micrornas (control pre-miR, 100nM, 72 hours), synthetic miR-21(pre-miR-21, 100nM, 72 hours), or miR-21 antagonists (anti-miR-21, 100nM, 72 hours) (upper panel), or siRNA-mediated SPRY1 knockdown (150nM, 72 hours) or treatment with synthetic siRNA (150nM, 72 hours) (lower panel). All error bars represent SEM. N of a-f is 3-6.

FIG. 3 therapeutic silencing of miR-21 prevents cardiac fibrosis and heart failure in vivo

(a) The upper diagram: fluorescence microscopy of Cy3 and DAPI staining in left ventricular heart tissue following injection of Cy3 labeled modified oligonucleotide via jugular vein. Controls received PBS injections. The following figures: mice subjected To Aortic Constriction (TAC) or sham surgery (sham operation) were injected 24 hours after surgery with either control (PBS, 200. mu.l) or antagomir-21 (200. mu.l; 80 mg/kg/day) for 3 consecutive days.

(b) The upper diagram: northern blot analysis of miR-21 expression in untreated (control) and antagomir-21 treated mice after TAC. The following figures: quantitative analysis of cardiac miR-21 expression.

(c) The upper diagram: western blot analysis of SPRY1, ERK1/2 and ERK phosphate 1/2 in mice treated with control or antagomir-21 after sham surgery or after TAC. The expression of G.beta.is shown as a housekeeping gene control (housekeepingcontrol). The following figures: quantitative analysis of western blot results.

(d) Cardiac sections were used to detect myocardial fibrosis in control and antagomir-21 treated mice after Sirius red staining.

(e) Quantitative analysis of myocardial fibrosis in control-treated mice and antagomir-21-treated mice (left panel) and heart weight/body weight (right panel).

(f) The upper diagram: complete transcriptome analysis of mouse genome in mouse heart tissue treated with control or antagomir-21 after sham surgery or after TAC. The red (green) region represents a significant (p < 0.05) induced (repressed) gene. The following figures: normalization of the up-regulated genes encoding proteins involved in myocardial fibrosis by antagomir-21 treatment after TAC.

(g) Echocardiography analysis. LVD, left ventricular diameter; FS, fractional shortening (fractional shortening).

(h) The mechanism proposed on the basis of derepression of cardiac ERK signaling through miR-21-mediated inhibition of SPRY 1. All error bars represent SEM, n of a-g is 3-6.

FIG. 4 transcriptional regulation of miR-21

(a) Sequence conservation within the miR-21-promoter region of a different species, as compared to the human miR-21 promoter. Bars indicate the degree of conservation.

(b) Luciferase activity of the human miR-21-promoter construct. The stepwise shortening of the native promoter enables the identification of the 117bp region responsible for miR-21 expression. n is 3.

(c) Luciferase data of human miR-21-promoter constructs following deletion/mutation of the respective transcription factor binding site. n is 3.

FIG. 5 knockdown of ubiquitously expressed miR-21 induces a cardiac phenotype in zebrafish

(a) Left panel: profiles of miR-21 morphans in MO control embryos and 80 hpf. A, an atrium; v, the ventricle; pericardial edema is emphasized by black arrows.

Upper right panel: at 80 hours post-fertilization (80hpf), the control-morpholino (MO-control) injected hearts showed normal heart morphology. Cardiac cyclization (loop), well developed endocardium and myocardium, with ventricles (V) and atria (a) separated by Atrioventricular (AV) rings. The following figures: due to the loss of ventricular contractility, miR-21 morphine (injected MO-1) developed pericardial edema and showed tail shortening, but other organ systems developed normally.

(b) Inhibition of miR-21 function by two different morpholino-modified antisense oligonucleotides (MO-1, n-496 and MO-2, n-460) resulted in the same phenotype in > 90% of injected embryos. Data were from 3 independent injections per group.

(c) The Fractional Shortening (FS) at 48, 72, 96 and 120hpf of ventricles of MO-control (n-6) and miR-21 morphine injected with two different morpholinos (MO-1, n-8 and MO-2, n-8) indicates myocardial function. The ventricular FS of the miR-21morphant ventricles decreased sharply with time.

FIG. 6 transfection efficiency in cultured cardiomyocytes

(a) Cultured neonatal cardiomyocytes were transfected with Cy-3-labeled miR-21(50nM, 72 h, Ambion, USA) and stained with DAPI (right panel). For comparison, cultured cardiomyocytes were stained with DAPI only (left panel).

(b) Northern blot analysis of miR-21 expression in cultured cardiomyocytes following transfection with synthetic microRNAs (control-pre-miR, 50nM, 72 hours), miR-21 inhibitors (anti-miR-21, 50nM, 72 hours) or synthetic miR-21(pre-miR-21, 50nM, 72 hours).

All error bars represent SEM; a) and n of b) is 3-6.

FIG. 7 microRNA binding sites within the 3' UTR of the Spry1 gene

The upper diagram: alteration of expression of various micrornas with binding sites within the 3' UTR of the Spry1 gene.

The following figures: the grey color indicates microRNAs with binding sites within the 3' UTR as analyzed by the microRNA microarray. Cds ═ coding sequence.

FIG. 8 miR-21 real-time PCR expression data

In addition to the determination by northern blot, miR-21 expression in mouse left ventricular tissue was analyzed by real-time PCR after sham surgery, sham surgery + antagomir-21 treatment, TAC and TAC + antagomir-21 treatment (left panel), and in hearts of wild-type mice and miR-21 transgenic mice (right panel). n is 4-6 per group.

Data not shown:

screening of the putative miR-21 targets revealed 22 known potential target genes, of which 8 were shown to be expressed in cardiac tissue. The combination of 3 different target prediction tools identified Spry1(sprouty1) as a very likely candidate target.

Transcriptional regulation of miR-21

Compared with the human miR-21 promoter, sequence conservation exists in miR-21 promoter regions of different species. Following deletion/mutation of each transcription factor binding site, the human miR-21 promoter construct showed luciferase activity. The stepwise shortening of the native promoter enables the identification of the 117bp region responsible for miR-21 expression. n is 3.

Transfection efficiency in cultured cardiomyocytes

Neonatal cardiomyocytes in culture were transfected with Cy-3-labeled miR-21(50nM, 72 h, Ambion, USA) and stained with DAPI. For comparison, cultured cardiomyocytes were stained with DAPI only. Northern blots of miR-21 expression in cultured cardiomyocytes were analyzed after transfection with synthetic microRNAs (control-pre-miR, 50nM, 72 hours), miR-21 inhibitors (anti-miR-21, 50nM, 72 hours), or synthetic miR-21(pre-miR-21, 50nM, 72 hours).

microRNA binding sites within the 3' UTR of the Spry1 gene

There was a change in the expression of various micrornas with binding sites within the 3' UTR of the Spry1 gene. microRNAs with binding sites within the 3' UTR were analyzed by microRNA microarrays.

miR-21 real-time PCR expression data

In addition to being determined by northern blot, miR-21 expression was analyzed by real-time PCR in left ventricular tissue of mice after sham surgery, sham surgery + miR-21 antagonist treatment, TAC and TAC + miR-21 antagonist treatment, and in wild-type and miR-21 transgenic mouse hearts.

Detailed Description

Definition of

"fibrosis" refers to the formation or development of excess fibrous connective tissue in an organ or tissue. In certain embodiments, fibrosis occurs as a repair or reactive process. In certain embodiments, fibrosis occurs in response to injury or damage. The term "fibrosis" is understood to mean the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reactive process, as opposed to the formation of fibrous tissue as a normal component of an organ or tissue.

Antisense oligonucleotides are understood to be oligonucleotides having a certain sequence which is complementary to another sequence, in particular a sequence which is complementary to miR-21. Targets for miR-21 are also understood to include downstream targets for miR-21. It is important to note that inhibition of miR-21, e.g., by an oligonucleotide having a sequence complementary to at least miR-21, will derepress or even over-express the target of miR-21 (e.g., Sprouty, etc.).

"subject" refers to a human or non-human animal selected for treatment or therapy.

A subject "suspected of having" is a subject who exhibits one or more clinical signs of a disease or disorder.

A "subject suspected of having fibrosis" refers to a subject exhibiting one or more clinical signs of fibrosis.

"preventing" refers to delaying or preventing the onset, development or progression of a condition or disease for a period of time, including weeks, months or years.

"treating" or "treatment" refers to the application of one or more specific methods to cure or ameliorate a disease. In certain embodiments, the specific method is administration of one or more drugs.

"ameliorating" refers to reducing the severity of at least one marker of a condition or disease. In certain embodiments, ameliorating comprises delaying or slowing the progression of one or more markers of a condition or disease. The severity of the marker can be determined by subjective or objective measurements known to those skilled in the art.

By "subject in need" is meant a subject identified as in need of a certain therapy or treatment.

"administering" refers to providing a drug or composition to a subject, including but not limited to administration by a medical professional and self-administration.

By "parenteral administration" is meant administration by injection or infusion.

Parenteral administration includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarterial, and intracranial administration.

By "subcutaneous administration" is meant administration just under the skin.

By "intravenous administration" is meant administration into a vein.

By "intraarterial administration" is meant administration into an artery.

By "intracardiac administration" is meant administration into the heart. In certain embodiments, the intracardiac administration is via a catheter. In certain embodiments, the intracardiac administration is performed by open heart surgery.

By "pulmonary administration" is meant administration to the lung.

"improving liver function" refers to a change in liver function towards normal parameters. In certain embodiments, liver function is assessed by measuring molecules present in the blood of the subject. For example, in certain embodiments, improvement in liver function is measured by a decrease in liver transaminase levels in the blood.

"pharmaceutical composition" refers to a mixture of substances, including drugs, suitable for administration to an individual. For example, the pharmaceutical composition may comprise the modified oligonucleotide and a sterile aqueous solution.

"drug" refers to a substance that provides a therapeutic effect when administered to a subject.

By "active pharmaceutical ingredient" is meant a substance in a pharmaceutical composition that provides the desired effect.

"target nucleic acid," "target RNA transcript," and "nucleic acid target" all refer to any nucleic acid capable of being targeted by an antisense compound.

"Targeting" refers to the process of designing and selecting a sequence of nucleobases that can hybridize to a target nucleic acid and induce a desired effect.

"Targeted to" refers to a nucleic acid molecule having a nucleobase sequence that can hybridize to a target nucleic acid to induce a desired effect. In certain embodiments, the desired effect is to reduce a certain target nucleic acid.

"modulation" refers to fluctuations in function or activity. In certain embodiments, modulation refers to an increase in gene expression. In certain embodiments, modulation refers to a reduction in gene expression.

"expression" refers to any function or step whereby the coding information of a gene is converted into a structure that exists and operates within the cell.

A "5 'target site" refers to a nucleobase of a target nucleic acid that is complementary to the most 5' nucleobase of a particular oligonucleotide.

A "3 'target site" refers to a nucleobase of a target nucleic acid that is complementary to the nucleobase at the 3' most end of a particular oligonucleotide.

"region" refers to the portion of a nucleic acid to which nucleotides are attached. In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to a region of the target nucleic acid. For example, in certain such embodiments, the modified oligonucleotide is complementary to a region of the miRNA stem-loop sequence. In certain such embodiments, the modified oligonucleotide is 100% complementary to a region of the miRNA stem-loop sequence.

"segment" refers to a smaller portion or sub-portion of a region.

"nucleobase sequence" refers to the order of consecutive nucleobases in the 5 'to 3' direction, regardless of any sugar, linkage and/or nucleobase modification.

"contiguous nucleobases" refers to nucleobases that are immediately adjacent to one another in a nucleic acid.

"nucleobase complementarity" refers to the ability of two nucleobases to pair non-covalently through hydrogen bonds.

"complementary" means that a first nucleobase sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% identical or at least 60%, 65%, 70%, 75%, 85%, 95%, 97%, 98%, or 99% identical to the complementary sequence of a second nucleobase sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 85, 90, 100 or more nucleobases, or that two sequences hybridize under stringent hybridization conditions. In certain embodiments, a modified oligonucleotide having a nucleobase sequence 100% complementary to a miRNA or a precursor thereof may not be 100% complementary to a miRNA or a precursor thereof over the full-length modified oligonucleotide.

"complementarity" refers to the ability of a first nucleic acid to base pair with a second nucleic acid.

By "full-length complementarity" is meant that each nucleobase of a first nucleic acid is capable of pairing with each nucleobase at a corresponding position on a second nucleic acid. For example, in certain embodiments, the modified oligonucleotide in which each nucleobase is complementary to a nucleobase in a miRNA has full-length complementarity to the miRNA.

"percent complementary" refers to the number of complementary nucleobases in a nucleic acid divided by the length of the nucleic acid. In certain embodiments, the percent complementarity of a modified oligonucleotide refers to the number of nucleobases complementary to a target nucleic acid divided by the number of nucleobases of the modified oligonucleotide. In certain embodiments, the percent complementarity of the modified oligonucleotide refers to the number of nucleobases complementary to the miRNA divided by the number of nucleobases of the modified oligonucleotide.

"percent binding region" refers to the percentage of region complementary to the oligonucleotide region. The percent binding region is calculated by dividing the number of nucleobases of the target region complementary to the oligonucleotide by the length of the target region. In certain embodiments, the percent binding region is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

"percent identity" refers to the number of nucleobases of a first nucleic acid that are identical to the nucleobases at the corresponding position of a second nucleic acid divided by the total number of nucleobases in the first nucleic acid.

As used herein, "substantial identity" can mean that a first nucleobase sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical or 100% identical to a second nucleobase sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases.

"hybridization" refers to the annealing of complementary nucleic acids by nucleobase complementarity.

A "mismatch" refers to a nucleobase of a first nucleic acid that is not capable of pairing with a nucleobase at a corresponding position in a second nucleic acid.

"non-complementary nucleobases" refers to two nucleobases that are not capable of pairing by hydrogen bonds.

"identical" means having the same nucleotide base sequence.

"miRNA" or "miR" refers to non-coding RNAs between 18 and 25 nucleobases in length that hybridize to and regulate expression of coding RNAs. In certain embodiments, the miRNA is the product of cleavage of a pre-miRNA by a dicer. Examples of miRNAs can be found in the miRNA database known as miRBase (http:// microrna. sanger. ac. uk /).

"Pre-miRNA" or "Pre-miR" refers to a non-coding RNA that has a hairpin structure and contains a miRNA. In certain embodiments, the pre-miRNA is the product of cleavage of pri-miR by a double-stranded RNA-specific ribonuclease known as Drosha.

"stem-loop sequence" refers to an RNA having a hairpin structure and containing a mature miRNA sequence. The Pre-miRNA sequence and the stem-loop sequence may overlap. Examples of stem-loop sequences can be found in the miRNA database known as miRBase (http:// microrna. sanger. ac. uk /).

"Pri-miRNA" or "Pri-miR" refers to non-coding RNAs that have a hairpin structure that serves as a substrate for the double-stranded RNA-specific ribonuclease Drosha.

"miRNA precursor" refers to a transcript derived from genomic DNA and comprising a non-coding structural RNA comprising one or more miRNA sequences. For example, in certain embodiments, the miRNA precursor is a pre-miRNA. In certain embodiments, the miRNA precursor is a pri-miRNA.

"monocistronic transcript" refers to a miRNA precursor containing a single miRNA sequence.

"polycistronic transcript" refers to a miRNA precursor containing two or more miRNA sequences.

"Seed region" refers to the 2-6 th or 2-7 th nucleotides from the 5' end of the mature miRNA sequence.

"oligomeric compound" refers to a compound that comprises a polymer of linked monomeric subunits.

"oligonucleotide" refers to a polymer of linked nucleosides, each of which can be modified or unmodified, independently of the other.

"naturally occurring internucleoside linkage" refers to the 3 'to 5' phosphodiester linkage between nucleosides.

"native sugar" refers to a sugar that is present in DNA (2 '-H) or RNA (2' -OH).

"Natural nucleobase" refers to a nucleobase that is unmodified with respect to its naturally occurring form.

"internucleoside linkage" refers to a covalent bond between adjacent nucleosides.

"linking nucleosides" refers to nucleosides joined by covalent bonds.

A "nucleobase" refers to a heterocyclic moiety that is capable of noncovalently pairing with another nucleobase.

"nucleoside" refers to a nucleobase linked to a sugar.

"nucleotide" refers to a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

"miR antagonist" refers to a modified oligonucleotide that is complementary to a miRNA or a precursor thereof. For example, a "miR-X antagonist" refers to a modified oligonucleotide having a nucleobase complementary to miR-X. In certain embodiments, the antagonist is a miR-21 antagonist.

"modified oligonucleotide" refers to an oligonucleotide having one or more modifications relative to a naturally occurring terminus, sugar, nucleobase, and/or internucleoside linkage.

"Single-stranded modified oligonucleotide" refers to a modified oligonucleotide that is not hybridized to a complementary strand.

"modified internucleoside linkage" refers to any change resulting from a naturally occurring internucleoside linkage.

"phosphorothioate internucleoside linkage" refers to a linkage between nucleosides in which one of the non-bridging atoms is a sulfur atom.

"modified sugar" refers to a substitution and/or any alteration from a native sugar.

"modified nucleobase" refers to any substitution and/or alteration from a natural nucleobase.

"5-methylcytosine" refers to cytosine modified by a methyl group attached at the 5' position.

"2 ' -O-methyl sugar" or "2 ' -OMe sugar" refers to a sugar having an O-methyl modification at the 2 ' position.

"2 ' -O-methoxyethyl sugar" or "2 ' -MOE sugar" refers to a sugar having an O-methoxyethyl modification at the 2 ' position.

"2 ' -O-fluoro sugar" or "2 ' -F sugar" refers to a sugar having a fluorine modification at the 2 ' position.

By "bicyclic sugar moiety" is meant a sugar modified by bridging two non-geminal ring atoms.

"2 ' -O-methoxyethyl nucleoside" refers to a 2 ' -modified nucleoside having a 2 ' -O-methoxyethyl sugar modification.

"2 ' -fluoro nucleoside" refers to a 2 ' -modified nucleoside having a 2 ' -fluoro sugar modification.

A "2 ' -O-methyl" nucleoside refers to a 2 ' -modified nucleoside having a 2 ' -O-methyl sugar modification.

"bicyclic nucleoside" refers to a 2' -modified nucleoside having a bicyclic sugar moiety.

"Motif (Motif)" refers to the pattern of modified and/or unmodified nucleobase, sugar and/or internucleoside linkages in an oligonucleotide.

By "fully modified oligonucleotide" is meant that each nucleobase, each sugar, and/or each internucleoside linkage is modified.

By "consistently modified oligonucleotide" is meant that each nucleobase, each sugar, and/or each internucleoside linkage has the same modification throughout the modified oligonucleotide.

By "stable modification" is meant modification of a nucleoside in the presence of a nuclease such that the stability of the modified oligonucleotide is increased relative to the stability provided by the phosphodiester internucleoside linkage of 2' -deoxynucleosides. For example, in certain embodiments, the stabilizing modification is a stabilizing nucleoside modification. In certain embodiments, the stabilizing modification is an internucleoside linkage modification.

By "stabilized nucleoside" is meant a modified nucleoside that increases the stability of an oligonucleotide to nucleases relative to the stability provided by a 2' -deoxynucleoside. In one embodiment, the stabilizing nucleoside is a 2' -modified nucleoside.

"stabilized internucleoside linkages" refers to internucleoside linkages that provide increased nuclease stability of an oligonucleotide relative to the stability provided by phosphodiester internucleoside linkages. In one embodiment, the stabilized internucleoside linkage is a phosphorothioate internucleoside linkage.

Overview

Modified oligonucleotides complementary to miR-21 are found herein to be drugs for inhibiting miR-21. In certain embodiments, the modified oligonucleotide is administered to a subject having a disease characterized by upregulation of miR-21. In certain embodiments, the disease is cancer. In certain embodiments, the disease is heart failure. In certain embodiments, the disease is fibrosis. Fibrosis is caused by the production of excess fibrous connective tissue by an organ or tissue in response to injury or damage. If left untreated, fibrosis can lead to a variety of diseases in tissues such as the heart, lungs, kidneys, liver and skin.

It was found herein that miR-21 derived from fibroblasts plays a decisive role in fibrosis. The increase of the level of MiR-21 promotes the survival of fibroblasts, while the inhibition of endogenous miR-21 induces the death of apoptotic cells. Thus, identified herein is a mechanism by which miR-21 regulates fibroblast survival. Abnormal fibroblast proliferation and survival can lead to fibrosis. Thus, modified oligonucleotides complementary to miR-21 are drugs for treating fibrosis. In certain embodiments, the modified oligonucleotide complementary to miR-21 is an antisense oligonucleotide complementary to miR-21.

The inventors of the present invention demonstrated a key role for miR-21 and SPRY1 derived from fibroblasts in the heart (fig. 3 h). The data indicate that stress-induced and miRNA-mediated activation of ERK-MAP kinase activity can significantly modulate cardiac fibrosis. This study represents the first example of the application of microrna therapy in disease models. In particular, miR-21 is antagonized in murine models, preventing structural and functional deterioration. These findings suggest a new therapeutic entry point for heart failure and show broad therapeutic potential for microrna antagonists.

One aspect of the invention relates to the use of miR-21, an antisense oligonucleotide directed against miR-21 and/or a target of miR-21 in the preparation of a medicament for treating and/or preventing fibrosis and/or a fibrosis-associated disease.

The present invention relates in particular to specific cardiac diseases involving fibrosis (fibrosis-related diseases), such as cardiac hypertrophy, hypertensive heart disease, diastolic and systolic heart failure; heart-related storage diseases such as fabry disease (m.fabry); cardiomyopathies, for example dilated cardiomyopathy, hypertrophic cardiomyopathy with or without obstruction, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and other forms of cardiomyopathy, such as diabetic cardiomyopathy, constrictive pericarditis, coronary artery disease, myocardial infarction, acute and chronic right heart failure, arrhythmias caused by fibrosis, fibrosis associated with myocarditis; heart valve diseases (e.g. sclerosis) leading to valve stenosis or insufficiency, such as mitral valve stenosis and/or insufficiency, aortic valve stenosis and/or insufficiency, tricuspid valve stenosis and/or insufficiency, pulmonary valve stenosis and/or insufficiency.

A further aspect of the invention relates to other diseases involving fibrosis (fibrosis-related diseases), although not related to the cardiac system. Non-limiting examples are pulmonary fibrosis; chronic obstructive pulmonary disease; pulmonary hypertension; liver fibrosis caused by toxic environment; hepatitis and/or secondary right heart failure; fibrosis of the skin, such as the development of keloid after injury; vascular-related diseases, such as age or hypertension-related arterial stiffness, mediaclerosis, arteriosclerosis; age-related fibrosis of different organs; intestinal sclerosis, such as during crohn's disease; systemic scleroderma, CREST syndrome, and the like; renal fibrosis; proliferative fibrosis and/or rheumatoid arthritis.

Further well-known fibrosis-associated diseases and disorders are, for example, endocardial myocardial fibrosis and idiopathic cardiomyopathy, cirrhosis of the liver caused by hepatic fibrosis, idiopathic pulmonary fibrosis of the lung, diffuse parenchymal lung disease, mediastinal fibrosis, myelofibrosis, postvasectomy pain syndrome, retroperitoneal fibrosis and nephrogenic systemic fibrosis.

All aspects of the invention, in particular those mentioned in the appended claims, can be used for the diagnosis, treatment and/or prevention of the diseases, disorders and conditions mentioned above, given as possible examples. The listed diseases are not limiting.

In one embodiment, the invention relates to strategies for modulating miR-21. In another embodiment, the invention relates to strategies for modulating, e.g., overexpressing and/or upregulating, miR-21 targets. Possible modulation are implantable devices such as viral vectors, liposome formulations, and the like; gene transfer systems, sponges, and the like.

Sprouty (SPRY1) was identified herein as a target for miR-21. Both miR-21 and SPRY1 are expressed within cardiac fibroblasts, among other cell types. miR-21 expression is increased in cardiac fibroblasts, induces a strong repression of SPRY protein expression, and further enhances ERK-MAP kinase activation. In a preferred embodiment, the target is selected from Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and Rtn 4.

One aspect of the invention relates to a polypeptide directed against (or complementary to) SEQ ID NO: 2 to SEQ ID NO: 4. sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 antisense oligonucleotides for the diagnosis, treatment and/or prevention of fibrosis and/or fibrosis-related diseases. In certain aspects, the polypeptide has a sequence that is identical to a sequence selected from Sprouty1(SPRY1), particularly SEQ ID NO: 7 or SEQ ID NO: 8. tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 target complementary antisense oligonucleotides interfere with the ability of microRNAs (e.g., miR-21) to bind to a target site and inhibit expression of a selected target. In certain aspects, such antisense oligonucleotides comprise one or more nucleoside modifications.

One aspect of the invention relates to a nucleic acid molecule lacking miR-21, SEQ ID NO: 2 to SEQ ID NO: 4. sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn 4.

One aspect of the invention relates to a nucleic acid molecule lacking miR-21, SEQ ID NO: 2 to SEQ ID NO: 4. sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. a non-human mammalian knockout organism of Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 as a disease model for fibrosis and/or fibrosis related diseases. Also included are transgenic animals, particularly non-human mammals that overexpress miR-21.

Herein, it was found that miR-21 derived from fibroblasts exerts an effect on heart failure. Analysis of left ventricular heart tissue samples from subjects with end-stage heart failure due to idiopathic dilated cardiomyopathy revealed increased miR-21 expression and de-repression of SPRY1 protein expression. In addition, ERK-MAP kinase was activated in samples from these subjects, as evidenced by an increase in the phosphorus-ERK/ERK ratio. It is found herein that administration of a modified oligonucleotide complementary to miR-21 results in a significant reduction in cardiac fibrosis in an animal model of heart failure. Heart failure is characterized by cardiac fibrosis, as well as other parameters. Thus, modified oligonucleotides complementary to miR-21 are drugs for treating cardiac fibrosis.

It has also been found herein that in animal models of heart failure, a reduction in cardiac fibrosis is accompanied by a reduction in the increase in heart weight. Furthermore, assessment of cardiac function by echocardiography revealed that administration of a modified oligonucleotide complementary to miR-21 prevented left ventricular dilation and normalized fractional shortening parameters. Heart failure may be characterized by increased heart weight, decreased left ventricular dilation and fractional shortening, and other parameters. Thus, modified oligonucleotides complementary to miR-21 are therapeutic agents for the treatment, amelioration, and prevention of heart failure associated with cardiac fibrosis.

One aspect of the present invention relates to promoter regions of micrornas (mirnas) comprising modifications of calcium/cAMP response element protein (CREB) and/or Serum Response Factor (SRF) binding sites for the diagnosis, prevention and/or treatment of fibrosis and/or fibrosis related diseases.

In one embodiment, the promoter region, the gene of mirR-21 itself and/or the 3' UTR of the various messenger RNAs may contain polymorphisms, mutations, particularly point mutations, deletions, truncations and/or inversions. All these changes in the wild-type sequence lead to the reformation of a new miR-21 binding site, or to the deletion of a miR-21 binding site. In another embodiment, the modification of the promoter region is selected from the group consisting of a point mutation, truncation, deletion, and inversion. Furthermore, the promoter region may be selected from SEQ ID NO: 2 to SEQ ID NO: 4.

diagnostic applications

One aspect of the invention relates to the use of miR-21, antisense oligonucleotides directed against miR-21 and/or targets of miR-21 for diagnosing fibrosis and/or a fibrosis-associated disease. Antisense oligonucleotides are understood to be oligonucleotides having a certain sequence which is complementary to another sequence, in particular to miR-21. Targets for miR-21 are also understood to include downstream targets for miR-21. It is important to note that inhibition of miR-21, e.g., by an oligonucleotide having a sequence complementary to at least miR-21, will derepress or even over-express the target of miR-21 (e.g., Sprouty, etc.).

In one aspect, the present invention relates to a method for diagnosing fibrosis and/or a fibrosis-related disease, the method comprising the steps of:

(a) providing a sample of a patient believed to have fibrosis and/or a fibrosis-related disease;

(b) determination of miR-21, Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. expression of Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn 4;

wherein the miR-21 level is increased and/or the level of Sprouty1(SPRY1), in particular the protein of SEQ ID NO: 7 or SEQ ID NO: 8. a decreased level of Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 indicates fibrosis and/or a fibrosis-related disease or predisposition thereto (predisposition).

In one aspect, the present invention relates to a method for screening a pharmaceutically active compound for the treatment and/or prevention of fibrosis and/or a fibrosis-related disease or a predisposition therefor, said method comprising the steps of:

(a) providing a polypeptide comprising miR-21, Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. samples of Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn 4;

(b) contacting a candidate substance with a sample;

(c) determining the effect of the candidate substance on the sample;

wherein miR-21, Sprouty1(SPRY1), in particular SEQ ID NO: 7 or SEQ ID NO: 8. variations of Tgfbi, Krit1, Pitx2, Fasl, Nfib, Lnx1 and/or Rtn4 indicate pharmaceutically active compounds.

Certain diseases or disorders

Provided herein are methods for treating a subject having or suspected of having fibrosis. Also provided herein are methods for treating a subject who has been identified as having or suspected of having fibrosis. In certain embodiments, such methods comprise administering to the subject a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

Also provided herein are methods for preventing fibrosis in a subject at risk of developing fibrosis. In certain embodiments, such methods comprise administering to a subject at risk of developing fibrosis a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

In certain embodiments, the fibrosis is hepatic fibrosis. In certain embodiments, the fibrosis is pulmonary fibrosis. In certain embodiments, the fibrosis is skin fibrosis. In certain embodiments, the fibrosis is cardiac fibrosis. In certain embodiments, the fibrosis is renal fibrosis. In certain embodiments, the fibrosis is pulmonary fibrosis. In certain embodiments, the fibrosis is age-related fibrosis. In certain embodiments, the fibrosis is spleen fibrosis.

In certain embodiments, a subject having or suspected of having fibrosis has at least one heart disease or disorder. In certain embodiments, a subject identified as having or suspected of having fibrosis has at least one cardiac disease or disorder. In certain embodiments, such methods comprise administering to the subject a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

In certain embodiments, the cardiac disease or disorder is cardiac hypertrophy. In certain embodiments, the cardiac disease or disorder is cardiomyopathy. In certain embodiments, the cardiomyopathy is dilated cardiomyopathy, obstructive hypertrophic cardiomyopathy, non-obstructive hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, or diabetic cardiomyopathy.

In certain embodiments, the cardiac disease or disorder is coronary artery disease. In certain embodiments, the cardiac disease or disorder is a heart-related storage disease, constrictive pericarditis, acute myocardial infarction, chronic myocardial infarction, arrhythmia, or myocarditis-related fibrosis.

In certain embodiments, the cardiac disease or disorder is heart failure. In certain embodiments, the heart failure is hypertensive heart failure, diastolic heart failure, systolic heart failure, or right heart failure.

In certain embodiments, the heart disease or disorder is heart valve disease. In certain embodiments, the heart valve disease is mitral valve stenosis, aortic valve stenosis, tricuspid valve stenosis, or pulmonary valve stenosis. In certain embodiments, the heart valve disease is mitral insufficiency, aortic insufficiency, tricuspid insufficiency, or pulmonary insufficiency.

Provided herein are methods for treating a subject having or suspected of having fibrosis, wherein the subject has a liver disease or disorder. In certain embodiments, a subject identified as having or suspected of having fibrosis has at least one liver disease or disorder. In certain embodiments, such methods comprise administering to the subject a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21. In certain embodiments, the liver disease or disorder is chronic liver injury. In certain embodiments, the liver disease or disorder is a hepatitis virus infection. In certain embodiments, the hepatitis infection is a hepatitis c infection. In certain embodiments, the liver disease or disorder is non-alcoholic steatohepatitis. In certain embodiments, the liver disease or disorder is cirrhosis.

Provided herein are methods for treating a subject having or suspected of having fibrosis, wherein the subject has at least one pulmonary disease or disorder. Also provided herein are methods for treating a subject identified as having or suspected of having fibrosis, wherein the subject has at least one pulmonary disease or disorder. In certain embodiments, such methods comprise administering to the subject a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21. In certain embodiments, the pulmonary disease or disorder is chronic obstructive pulmonary disease.

Provided herein are methods for treating a subject having or suspected of having fibrosis, wherein the subject has at least one other disease or disorder. Also provided herein are methods for treating a subject identified as having or suspected of having fibrosis, wherein the subject has at least one other disease or disorder. In certain embodiments, such methods comprise administering to the subject a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21. In certain embodiments, one other disease or disorder is pulmonary hypertension. In certain embodiments, one other disease or disorder is a vascular-related disease. In certain such embodiments, the vascular-related disease is arterial stiffness, mediaclerosis, or arteriosclerosis. In certain embodiments, one other disease or disorder is intestinal sclerosis. In certain embodiments, another disease or disorder is systemic scleroderma. In certain embodiments, one other disease or condition is retroperitoneal fibrosis, proliferative fibrosis, nephrogenic systemic fibrosis, congestive fibrosis, mediastinal fibrosis, myelofibrosis, post vasectomy pain syndrome, or rheumatoid arthritis.

Provided herein are methods for treating a subject having a fibroproliferative disease. In certain embodiments, such methods comprise administering to a subject having or suspected of having a fibroproliferative disorder a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

The invention also relates to miRNA-21 based cancer therapies comprising targeting specific cancers by modulating SPRY-1 mediated ERK signaling in cancer cells. miR-21 is known to be overexpressed in many different cancer types, including esophageal cancer, colon adenocarcinoma, breast cancer, glioma, glioblastoma, ovarian cancer, hepatocellular carcinoma, head and neck cancer, chronic lymphocytic leukemia, pancreatic cancer, and the like, to name just a few of these cancers.

The invention therefore also relates to the diagnosis, prevention and/or treatment of the aforementioned cancer types. All the features mentioned in the claims can be combined with the disclosure of these cancer types.

Certain routes of administration

In certain embodiments, administering to the subject comprises parenteral administration. In certain embodiments, administering to the subject comprises intravenous administration. In certain embodiments, administering to the subject comprises subcutaneous administration.

In certain embodiments, administering to the subject comprises intraarterial administration. In certain embodiments, administering to the subject comprises intracardiac administration. Suitable methods for intracardiac administration include the use of catheters or administration during open heart surgery.

In certain embodiments, administering comprises pulmonary administration. In certain embodiments, pulmonary administration comprises delivering the nebulized oligonucleotide to the lungs of the subject by inhalation. After inhalation of the aerosolized oligonucleotide by the subject, the oligonucleotide spreads within cells of both normal and inflamed lung tissue, including alveolar macrophages, eosinophils, epithelial cells, vascular endothelial cells, and bronchial epithelial cells. Suitable devices for delivering the pharmaceutical composition comprising the modified oligonucleotide include, but are not limited to, standard nebulizer devices. Formulations and methods for adjusting droplet size using nebulizer devices to target specific portions of the respiratory tract and lungs are well known to those skilled in the art. Other suitable devices include dry powder inhalers or metered dose inhalers.

In certain embodiments, the pharmaceutical composition is administered to achieve local rather than systemic exposure. For example, pulmonary administration delivers pharmaceutical compositions to the lungs, minimizing systemic exposure.

Other suitable routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, intrathecal, intraventricular, intraperitoneal, intranasal, intraocular, intramuscular, intramedullary, and intratumoral.

Certain clinical results

In certain embodiments, the methods herein provide a clinically desirable result to a subject having or suspected of having fibrosis.

In certain embodiments, the clinically desirable result is an improvement in cardiac weight gain. In certain such embodiments, the clinically desirable result is improved left ventricular dilation. In certain embodiments, the clinically desirable result is a reduction in the improvement in foreshortening score. In certain embodiments, the clinically desirable result is prevention of cardiac weight gain. In certain such embodiments, the clinically desirable result is prevention of left ventricular dilation. In certain embodiments, the clinically desirable result is prevention of a decrease in the fractional shortening.

In certain embodiments, the clinically desirable result is improved cardiac function.

In certain embodiments, the clinically desirable result is improved fibrosis. In certain embodiments, the clinically desirable result is a slowing of further progression of fibrosis. In certain embodiments, the clinically desirable result is the termination of further progression of fibrosis. In certain embodiments, a clinically desirable result is reduced fibrosis. In certain embodiments, the clinically desirable result is a reduction in collagen content.

In certain embodiments, a therapeutically desirable result is improved liver function. Liver function can be assessed by liver function tests, which measure blood levels of hepatic transaminase, as well as other parameters. In certain embodiments, a subject with abnormal liver function has elevated blood liver transaminase. Blood liver transaminases include alanine transaminase (ALT) and aspartate transaminase (AST). In certain embodiments, the subject with abnormal liver function has elevated blood bilirubin. In certain embodiments, the subject has an abnormal blood albumin level. In certain embodiments, the methods provided herein alter ALT, AST, bilirubin, and/or albumin levels in the blood such that one or more of these levels are closer to normal limits.

In certain embodiments, liver function in a subject is assessed by the Child-Pugh classification system, which defines three classes of liver function. In this classification system, a score is given to a measurement value of one of the following five categories: bilirubin levels, albumin levels, prothrombin time, ascites and encephalopathy. There is a score of 1 for each of the following characteristics: blood bilirubin is less than 2.0 mg/dl; the blood albumin is more than 3.5 mg/dl; prothrombin time is less than 1.7 International Normalized Ratio (INR); absence of ascites; or the absence of encephalopathy. There are 2 points for each of the following features: 2-3mg/dl of blood bilirubin; 3.5-2.8mg/dl of blood bilirubin; prothrombin time 1.7-2.3 INR; mild to moderate ascites; or mild encephalopathy. There are 3 points for each of the following features: bilirubin is greater than 3.0 mg/dl; blood albumin is less than 2.8 mg/dl; prothrombin time greater than 2.3 INR; ascites severe to refractory; or severe encephalopathy. The scores are added, the score of A is 5-6, the score of B is 7-9 and the score of C is 10-15. In certain embodiments, the methods provided herein result in improved liver function, as determined by the Child-Pugh classification system.

Certain cell phenotypes

Provided herein are methods for inhibiting fibroblast proliferation. Also provided herein are methods for stimulating apoptosis in fibroblasts. Also provided herein are methods for increasing Sprouty1 protein in fibroblasts. In certain embodiments, such methods comprise contacting the fibroblast cells with a compound comprising a modified oligonucleotide and having a nucleobase sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

In certain embodiments, the fibroblast is an in vitro fibroblast. In certain embodiments, the fibroblast is a fibroblast in vivo. In certain embodiments, the contacting occurs in vitro. In certain embodiments, the contacting occurs in vivo. In certain embodiments, the contacting occurs ex vivo.

Certain additional therapies

Treatment of fibrosis may include more than one therapy. Thus, in certain embodiments, provided herein are methods for treating a subject having or suspected of having fibrosis, the method comprising administering at least one therapy in addition to administering a modified oligonucleotide having a nucleobase sequence complementary to a miRNA or a precursor thereof.

In certain embodiments, the methods provided herein comprise administering one or more additional agents. In certain embodiments, additional drugs include, but are not limited to, diuretics (e.g., spironolactone (sprionoctone), eplerenone, furosemide), inotropes (e.g., dobutamine, milrinone), digoxin, vasodilators, angiotensin II converting enzyme (ACE) inhibitors (e.g., captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril), angiotensin II receptor blockers (ARBs) (e.g., candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, eprosartan), calcium channel blockers, isosorbide dinitrate, hydralazine, nitrates (e.g., isosorbide mononitrate, isosorbide dinitrate), hydralazine, beta-blockers (e.g., carvedilol, metoprolol), and natriuretic peptides (e.g., nesiritide).

In certain embodiments, the additional therapy may be a drug that increases the body's immune system, including low doses of cyclophosphamide, thymic stimulin, vitamins and nutritional supplements (e.g., antioxidants, including vitamin A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea) and vaccines, such as Immune Stimulating Complexes (ISCOMs), that comprise a vaccine formulation that combines multimeric forms of the antigen with an adjuvant.

In certain such embodiments, additional therapies are selected to treat or ameliorate the side effects of one or more of the pharmaceutical compositions of the invention. Such side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathies. For example, elevated serum transaminase levels may indicate liver toxicity or abnormal liver function. For example, elevated bilirubin may indicate liver toxicity or liver function abnormality.

In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more additional agents are administered at the same time. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more additional agents are administered at different times. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other drugs are prepared together in a single formulation. In certain embodiments, one or more pharmaceutical compositions of the present invention and one or more other drugs are prepared separately.

Certain pharmaceutical compositions

In certain embodiments, compounds described herein comprising modified oligonucleotides complementary to mirnas or precursors thereof are formulated into pharmaceutical compositions for the treatment of fibrosis. In certain embodiments, compounds comprising modified oligonucleotides having nucleobase sequences complementary to mirnas or precursors thereof are made into pharmaceutical compositions for preventing fibrosis.

In certain embodiments, the pharmaceutical compositions of the present invention are administered in the form of dosage units (e.g., tablets, capsules, boluses (bolus), etc.). In certain embodiments, such pharmaceutical compositions comprise a modified oligonucleotide in a dose selected from the group consisting of 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg, 100mg, 105mg, 110mg, 115mg, 120mg, 125mg, 130mg, 135mg, 140mg, 145mg, 150mg, 155mg, 160mg, 165mg, 170mg, 175mg, 180mg, 185mg, 190mg, 195mg, 200mg, 205mg, 210mg, 215mg, 220mg, 225mg, 230mg, 235mg, 240mg, 245mg, 250mg, 255mg, 260mg, 265mg, 270mg, 280mg, 285mg, 290mg, 295mg, 300mg, 305mg, 310mg, 315mg, 320mg, 325mg, 330mg, 335mg, 340mg, 350mg, 355mg, 375mg, 370mg, 380mg, 390mg, 380mg, 150mg, 405mg, 410mg, 415mg, 420mg, 425mg, 430mg, 435mg, 440mg, 445mg, 450mg, 455mg, 460mg, 465mg, 470mg, 475mg, 480mg, 485mg, 490mg, 495mg, 500mg, 505mg, 510mg, 515mg, 520mg, 525mg, 530mg, 535mg, 540mg, 545mg, 550mg, 555mg, 560mg, 565mg, 570mg, 575mg, 580mg, 585mg, 590mg, 595mg, 600mg, 605mg, 610mg, 615mg, 620mg, 625mg, 630mg, 635mg, 640mg, 645mg, 650mg, 655mg, 660mg, 665mg, 670mg, 675mg, 680mg, 685mg, 690mg, 695mg, 700mg, 705mg, 710mg, 715mg, 720mg, 725mg, 730mg, 735mg, 740mg, 750mg, 745mg, 760mg, 775mg, 770mg, 800mg, and 800 mg. In certain such embodiments, the pharmaceutical composition of the invention comprises a dose of the modified oligonucleotide selected from the group consisting of 25mg, 50mg, 75mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 500mg, 600mg, 700mg, and 800 mg.

In certain embodiments, the medicament is a sterile lyophilized modified oligonucleotide reconstituted with a suitable diluent, such as sterile water for injection or sterile saline for injection. After dilution in saline, the reconstituted product is administered by subcutaneous injection or by intravenous infusion. The freeze-dried medicine product is composed of modified oligonucleotide, the modified oligonucleotide is prepared in water for injection or saline for injection, the pH value is adjusted to 7.0-9.0 by acid or alkali in the preparation process, and then freeze-drying is carried out. The lyophilized modified oligonucleotide may be 25-800mg of modified oligonucleotide. It is understood that this includes 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg and 800mg of the lyophilized modified oligonucleotide. The lyophilized pharmaceutical product can be packaged in a clear glass vial of type 2mLI (treated with ammonium sulfate), stoppered with a bromobutyl rubber stopper and then treated with aluminumAnd (6) sealing the top.

In certain embodiments, the compositions of the present invention may also contain other adjunct ingredients commonly used in pharmaceutical compositions, at levels determined in the art. Thus, for example, the compositions may contain additional compatible pharmaceutically active materials such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents, or may contain other materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavors, preservatives, antioxidants, opacifiers, thickeners, and stabilizers. However, such materials, when added, should not interfere too much with the biological activity of the ingredients of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with adjuvants which do not adversely interact with the oligonucleotides of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and/or aromatic substances and the like.

In certain embodiments, the pharmaceutical compositions of the invention comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, the excipient is selected from the group consisting of water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylases, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone.

In certain embodiments, the pharmaceutical compositions of the present invention are prepared using known techniques, including but not limited to mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

In certain embodiments, the pharmaceutical compositions of the present invention are liquids (e.g., suspensions, elixirs and/or solutions). In certain such embodiments, liquid pharmaceutical compositions are prepared using ingredients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.

In certain embodiments, the pharmaceutical compositions of the present invention are solids (e.g., powders, tablets, and/or capsules). In certain such embodiments, solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.

In certain embodiments, the pharmaceutical compositions of the present invention are formulated as a depot preparation. Certain such depot formulations generally have a longer onset of action than non-depot formulations. In certain embodiments, such formulations are administered by implantation (e.g., subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, the depot formulations are prepared using suitable polymeric materials and hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins or as sparingly soluble derivatives (e.g., sparingly soluble salts).

In certain embodiments, the pharmaceutical compositions of the present invention comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are used to prepare certain pharmaceutical compositions, including pharmaceutical compositions comprising hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethyl sulfoxide.

In certain embodiments, the pharmaceutical compositions of the invention comprise one or more tissue-specific delivery molecules designed to deliver one or more drugs of the invention into a particular tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with a tissue-specific antibody.

In certain embodiments, the pharmaceutical compositions of the present invention comprise a cosolvent system. Some of these co-solvent systems include, for example, benzyl alcohol, non-polar surfactants, water-soluble organic polymers, and an aqueous phase. In certain embodiments, such co-solvent systems can be used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a system comprising 3% (w/v) benzyl alcohol, 8% (w/v) of a non-polar surfactant, Polysorbate 80^TMAnd 65% (w/v) polyethylene glycol 300 in pure ethanol. The proportions of such co-solvent systems can vary over a considerable range without significantly altering their solubility and toxicity characteristics. In addition, the co-solvent component itself (identity) may also be varied: for example, other surfactants may be used in place of Polysorbate 80^TM(ii) a The size of the polyethylene glycol fraction can be varied; other biocompatible polymers may be substituted for polyethylene glycol, such as polyvinylpyrrolidone; and other sugars or polysaccharides may be substituted for glucose.

In certain embodiments, the pharmaceutical compositions of the present invention comprise a sustained release system. A non-limiting example of such a sustained release system is a semi-permeable matrix of a solid hydrophobic polymer. In certain embodiments, sustained release systems may release drugs over a period of hours, days, weeks, or months, depending on their chemical nature.

In certain embodiments, the pharmaceutical compositions of the present invention are prepared for oral administration. In certain such embodiments, the pharmaceutical composition is prepared by mixing one or more compounds comprising the modified oligonucleotide with one or more pharmaceutically acceptable carriers. Some of these carriers allow the pharmaceutical compositions to be formulated as tablets, pills, lozenges, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by a subject. In certain embodiments, pharmaceutical compositions for oral use are obtained by mixing an oligonucleotide and one or more solid excipients. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone (PVP). In certain embodiments, such mixtures are optionally milled, and optionally, adjuvants are added. In certain embodiments, the pharmaceutical composition is shaped to obtain tablet or lozenge cores. In certain embodiments, a disintegrating agent (e.g., cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate) is added.

In certain embodiments, a lozenge core with a coating is provided. In certain such embodiments, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gum, polyethylene glycol, and/or titanium dioxide, a varnish solution (lacquer solution), and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablet or lozenge coating.

In certain embodiments, the pharmaceutical composition for oral administration is a push-fit capsule made of gelatin. Certain of such push-fit capsules comprise one or more of the agents of the present invention in admixture with one or more fillers, such as lactose, binders (e.g., starch), and/or lubricants (e.g., talc or magnesium stearate), optionally stabilizers. In certain embodiments, the pharmaceutical composition for oral administration is a sealed soft capsule made of gelatin and a plasticizer (e.g., glycerol or sorbitol). In certain soft capsules, one or more drugs of the present invention are dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added.

In certain embodiments, the pharmaceutical composition is prepared for oral administration. Some of these pharmaceutical compositions are tablets or dragees formulated in a conventional manner.

In certain embodiments, the pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain such embodiments, the pharmaceutical composition comprises a carrier and is formulated in an aqueous solution (e.g., water) or a physiologically compatible buffered solution (e.g., Hanks 'solution, ringer's solution) or a physiological saline buffer. In certain embodiments, other ingredients (e.g., ingredients to aid in dissolution or to act as preservatives) are included. In certain embodiments, injectable suspensions are prepared using suitable liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are provided in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Some suitable solvents for injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, for example sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the drug substance for use in preparing highly concentrated solutions.

In certain embodiments, the pharmaceutical composition is prepared for mucosal administration. In certain such embodiments, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art.

In certain embodiments, the pharmaceutical composition is prepared for administration by inhalation. Some such pharmaceutical compositions are prepared for inhalation in the form of a spray from a nebulizer (pressurized pack) or nebulizer. Some such pharmaceutical compositions include a propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In certain embodiments, a compressed aerosol is used, and the dosage unit is determined by a valve delivering a certain dose. In certain embodiments, capsules and cartridges for use in an inhaler or insufflator may be formulated. Some such formulations comprise a powder mixture of the medicament of the invention and a suitable powder base, for example lactose or starch.

In certain embodiments, the pharmaceutical compositions are prepared for rectal administration, such as suppositories or retention enemas. Some such pharmaceutical compositions comprise known ingredients, such as cocoa butter and/or other glycerides.

In certain embodiments, the pharmaceutical composition is prepared for topical administration. Some of these pharmaceutical compositions comprise a mildly moist base, such as an ointment or cream. Exemplary suitable ointment bases include, but are not limited to, petrolatum plus volatile silicones, and lanolin and water-in-oil emulsions. Exemplary suitable cream bases include, but are not limited to, cold creams and hydrophilic ointments.

In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a modified oligonucleotide. In certain embodiments, a therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of disease or to prolong survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides of the invention are formulated as prodrugs. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of the modified oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active forms. For example, in some cases, the bioavailability of a prodrug may be higher (e.g., by oral administration) than the corresponding active form. In some cases, the solubility of the prodrug is improved compared to the corresponding active form. In certain embodiments, the prodrug is less soluble in water than the corresponding active form. In some cases, such prodrugs, where water solubility is detrimental to mobility, have good transport across cell membranes. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to the carboxylic acid upon administration. In some cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, prodrugs are created by modifying pharmaceutically active compounds such that the active compound is produced upon in vivo administration. Prodrugs can be designed to alter the metabolic stability or transport characteristics of a drug, mask side effects or toxicity, modify the taste of a drug, or alter other characteristics or properties of a drug. By understanding the pharmacokinetic processes and in vivo metabolism of drugs, one skilled in the art can design prodrugs of a compound once the pharmaceutical activity of the compound is known (see, e.g., Nogrady (1985) Medicinal Chemistry A biochemical approach, Oxford university Press, New York, pp. 388-.

Certain compounds

In certain embodiments, the methods provided herein comprise administering a compound comprising a modified oligonucleotide. In certain embodiments, the compound consists of a modified oligonucleotide.

In certain such embodiments, the compound comprises a modified oligonucleotide that hybridizes to a complementary strand, i.e., the compound comprises a double-stranded oligomeric compound. In certain embodiments, hybridization of the modified oligonucleotide to the complementary strand forms at least one blunt end. In certain such embodiments, hybridization of the modified oligonucleotide to the complementary strand forms blunt ends at each end of the double-stranded oligomeric compound. In certain embodiments, the end of the modified oligonucleotide comprises one or more additional linked nucleosides relative to the number of linked nucleosides of the complementary strand. In certain embodiments, one or more additional nucleosides are located at the 5' end of the modified oligonucleotide. In certain embodiments, one or more additional nucleosides are located at the 3' end of the modified oligonucleotide. In certain embodiments, at least one nucleobase of a nucleoside of the one or more additional nucleosides is complementary to the target RNA. In certain embodiments, each nucleobase of each one or more additional nucleosides is complementary to a target RNA. In certain embodiments, the end of the complementary strand comprises one or more additional linked nucleosides relative to the number of linked nucleosides of the modified oligonucleotide. In certain embodiments, one or more additional linking nucleosides is located at the 3' end of the complementary strand. In certain embodiments, one or more additional linking nucleosides are located at the 5' end of the complementary strand. In certain embodiments, two additional linked nucleosides are terminally linked. In certain embodiments, one additional nucleoside is terminally linked.

In certain embodiments, the compounds comprise modified oligonucleotides conjugated to one or more moieties, which increase the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotides. In certain such embodiments, the moiety is a cholesterol moiety or a lipid moiety. Additional moieties for conjugation include sugars, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In certain embodiments, the conjugate group is directly attached to the modified oligonucleotide. In certain embodiments, the conjugate group is attached to the modified oligonucleotide through a linking moiety selected from the group consisting of: amino, hydroxy, carboxylic acid, thiol, unsaturated bond (e.g., double or triple bond), 8-amino-3, 6-dioxaoctanoic Acid (ADO), succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), 6-aminocaproic acid (AHEX or AHA), substituted C₁-C₁₀Alkyl, substituted or unsubstituted C₂-C₁₀Alkenyl and substituted or unsubstituted C₂-C₁₀Alkynyl. In certain such embodiments, the substituent groupSelected from the group consisting of hydroxy, amino, alkoxy, carboxy, benzyl, phenyl, nitro, mercapto, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain such embodiments, the compounds comprise modified oligonucleotides having one or more stabilizing groups attached to one or both ends of the modified oligonucleotide to improve properties such as nuclease stability. Including a cap structure in the stabilizing group. These end modifications protect the modified oligonucleotide from exonuclease degradation and may facilitate delivery and/or localization within the cell. The cap may be present on either the 5 'end (5' cap) or the 3 'end (3' cap), or may be present on both ends. The cap structure includes, for example, inverted deoxy abasic caps (inverted deoxy abasic caps).

Suitable cap structures include 4 ', 5 ' -methylene nucleotides, 1- (. beta. -D-erythro-furanosyl) nucleotides, 4 ' -thio nucleotides, carbocyclic nucleotides, 1, 5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, dithiophosphate linkages, threo-pentofuranosyl nucleotides, acyclic 3 ', 4 ' -ring-opening nucleotides, acyclic 3, 4-dihydroxybutyl nucleotides, acyclic 3, 5-dihydroxypentyl nucleotides, 3 ' -3 ' -inverted nucleotide moieties, 3 ' -3 ' -inverted abasic moieties, 3 ' -2 ' -inverted nucleotide moieties, 3 ' -2 ' -inverted abasic moieties, 1, 4-butanediol phosphate, amino acid, 3 ' -phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3 ' -phosphorothioate, phosphorodithioate, bridged and unbridged methylphosphonate moieties, 5 ' -amino-alkyl phosphate, 1, 3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1, 2-aminododecyl phosphate, hydroxypropyl phosphate, 5 ' -5 ' -inverted nucleotide moieties, 5 ' -5 ' -inverted abasic moieties, 5 ' -phosphoramidate, 5 ' -phosphorothioate, 5 ' -amino, bridged and/or unbridged 5 ' -phosphoramidate, phosphorothioate and 5 ' -thiol moieties.

Certain nucleotide base sequences

Provided herein are methods for treating or preventing fibrosis. In certain embodiments, the method comprises administering a pharmaceutical composition comprising a modified oligonucleotide. In certain embodiments, the method comprises administering a compound comprising a modified oligonucleotide. In certain embodiments, the modified oligonucleotide has a sequence complementary to a miRNA or a precursor thereof. In certain embodiments, the miRNA is miR-21.

The nucleobase sequence of the mature miRNA and its corresponding stem-loop sequence described herein is that found in miRBase, a database of miRNA sequences and annotations for on-line search, seehttp:// microrna.sanger.ac.uk/. Entries in the miRBase sequence database represent the hairpin portion (stem-loop) of the predicted miRNA transcript, with information on the position and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor mirnas (pre-mirnas) and may in some cases include pre-mirnas and some flanking sequences of the putative original transcript. The miRNA nucleobase sequences described herein include any form of miRNA, including sequences disclosed in miRBase sequence database version 10.0 and sequences disclosed in any earlier version of the miRBase sequence database. Sequence database versions may lead to the renaming of certain mirnas. Sequence database versions may result in changes in the mature miRNA sequence. The compounds of the invention include modified oligonucleotides in the form of any nucleobase sequence complementary to a miRNA described herein.

It is to be understood that any nucleobase sequence described herein is not dependent on any modification of the sugar moiety, internucleoside linkage or nucleobase. It is also understood that a nucleobase sequence comprising U also includes the same nucleobase sequence in which the 'U' is replaced by a 'T' at one or more positions having a 'U'. Conversely, it is understood that a nucleobase sequence comprising a T also includes the same nucleobase sequence in which the 'T' is replaced by a 'U' at one or more positions having a 'T'.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to a miRNA or a precursor thereof, which means that the nucleobase sequence of the modified oligonucleotide is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the complement of the miRNA or a precursor thereof over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more nucleobases, or that both sequences hybridize under stringent hybridization conditions. Thus, in certain embodiments, the nucleobase sequence of the modified oligonucleotide may have one or more mismatched base pairs relative to its target miRNA or target miRNA precursor sequence and be capable of hybridizing to its target sequence. In certain embodiments, the modified oligonucleotide has a nucleobase sequence that is 100% complementary to a miRNA or a precursor thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is complementary to the full length of the miRNA.

In certain embodiments, miR-21 has the nucleobase sequence 5'-UAGCUUAUCAGACUGAUGUUGA-3' (SEQ ID NO: 1). In certain embodiments, the miR-21 stem-loop sequence has the nucleobase sequence 5'-UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA-3' (SEQ ID NO: 11).

In certain embodiments, the modified oligonucleotide has an amino acid sequence that is identical to a sequence as set forth in SEQ ID NO: 1, and the nucleotide base sequence of miR-21 is complementary.

In certain embodiments, the modified oligonucleotide has an amino acid sequence that is identical to a sequence as set forth in SEQ ID NO: 11, and a sequence complementary to the nucleotide base sequence of the miRNA stem-loop sequence shown in fig. 11. In certain embodiments, the modified oligonucleotide has an amino acid sequence that is identical to SEQ ID NO: 11 in the region from 8 to 29. In certain embodiments, the modified oligonucleotide has a nucleotide sequence identical to SEQ id no: 11, and nucleotide bases 46-66.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence comprising the nucleobase sequence 5'-UCAACAUCAGUCUGAUAAGCUA-3' (SEQ ID NO: 12).

In certain embodiments, the modified oligonucleotide has a sequence consisting of SEQ ID NO: 12, or a nucleotide sequence consisting of the nucleotide base sequence shown in the specification.

In certain embodiments, the modified oligonucleotide has a nucleobase sequence complementary to a nucleobase sequence comprising a pri-miR sequence of miR-21.

In certain embodiments, the modified oligonucleotide has an amino acid sequence identical to SEQ ID NO: 1, a nucleobase sequence complementary to a nucleobase sequence having at least 80% identity to a nucleobase sequence represented by 1. In certain embodiments, the modified oligonucleotide has an amino acid sequence identical to SEQ ID NO: 1, a nucleobase sequence complementary to a nucleobase sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity thereto.

In certain embodiments, the modified oligonucleotide has a sequence identical to SEQ ID NO: 11, and the nucleotide base sequence of the miR-21 stem-loop sequence has at least 80% of identity. In certain embodiments, the modified oligonucleotide has an amino acid sequence identical to SEQ ID NO: 11 has a nucleobase sequence complementary to a nucleobase sequence having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, or at least 98% identity to the nucleobase sequence of the miR-21 stem-loop sequence.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is fully complementary to a miRNA nucleobase sequence set forth herein, or a precursor thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has a mismatch relative to the nucleobase sequence of the mature miRNA or a precursor thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide has two mismatches relative to the nucleobase sequence of the miRNA or a precursor thereof. In certain such embodiments, the nucleobase sequence of the modified oligonucleotide has only two mismatches relative to the nucleobase sequence of the mature miRNA or a precursor thereof. In certain such embodiments, the mismatched nucleobases are contiguous. In certain such embodiments, the mismatched nucleobases are discontinuous.

In certain embodiments, the modified oligonucleotide consists of a number of linked nucleosides of equal length to the mature miRNA to which it is complementary.

In certain embodiments, the modified oligonucleotide has fewer linked nucleosides than the length of the mature miRNA to which it is complementary. In certain such embodiments, the modified oligonucleotide has one less ligated nucleotide than the mature miRNA to which it is complementary. In certain such embodiments, the modified oligonucleotide has one less nucleoside at the 5' end. In certain such embodiments, the modified oligonucleotide has one less nucleoside at the 3' end. In certain such embodiments, the modified oligonucleotide has two fewer nucleosides at the 5' end. In certain such embodiments, the modified oligonucleotide has two fewer nucleosides at the 3' end. A modified oligonucleotide having a plurality of linked nucleosides that is less than the length of a miRNA, wherein each nucleobase of the modified oligonucleotide is complementary to each nucleobase at a corresponding position of a miRNA is considered a modified oligonucleotide having a nucleobase sequence that is 100% complementary to a portion of the miRNA sequence.

In certain embodiments, the modified oligonucleotide has a greater number of linked nucleosides than the length of the miRNA to which it is complementary. In certain such embodiments, the nucleobase of the additional nucleoside is complementary to a nucleobase of the miRNA stem-loop sequence. In certain embodiments, the modified oligonucleotide has one more linked nucleotide than the length of the miRNA to which it is complementary. In certain such embodiments, the additional nucleoside is located at the 5' end of the modified oligonucleotide. In certain such embodiments, the additional nucleoside is located at the 3' end of the modified oligonucleotide. In certain embodiments, the modified oligonucleotide has one more linked nucleotide than the length of the miRNA to which it is complementary. In certain such embodiments, two additional nucleosides are located at the 5' end of the modified oligonucleotide. In certain such embodiments, two additional nucleosides are located at the 3' end of the modified oligonucleotide. In certain such embodiments, one additional nucleoside is located at the 5 'end and another additional nucleoside is located at the 3' end of the modified oligonucleotide.

In certain embodiments, a portion of the nucleobase sequence of the modified oligonucleotide is 100% complementary to the nucleobase sequence of the miRNA, but the modified oligonucleotide is not 100% complementary over its entire length. In certain such embodiments, the number of nucleosides of the modified oligonucleotide having 100% complementary portions is greater than the length of the miRNA. For example, a modified oligonucleotide consisting of 24 linked nucleosides, wherein the nucleobases of nucleosides 1 to 23 are each complementary to a corresponding position of a miRNA that is 23 nucleobases in length, has 23 nucleoside portions 100% complementary to the nucleobase sequence of the miRNA, and has about 96% overall complementarity to the nucleobase sequence of the miRNA.

In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 100% complementary to a portion of the nucleobase sequence of the miRNA. For example, a modified oligonucleotide consisting of 22 linked nucleosides in which the nucleobases of nucleosides 1 to 22 are each complementary to a corresponding position of a miRNA of 23 nucleobases in length is 100% complementary to the nucleobase portion of the nucleobase sequence of the 22 miRNAs. Such modified oligonucleotides have about 96% overall complementarity to the nucleobase sequence of the entire miRNA and 100% complementarity to the 22 nucleobase portion of the miRNA.

In certain embodiments, the portion of the nucleobase sequence of the modified oligonucleotide is 100% complementary to the portion of the nucleobase sequence of the miRNA or the precursor thereof. In certain such embodiments, each of the 15 consecutive nucleobases of the modified oligonucleotide is complementary to 15 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 16 consecutive nucleobases of the modified oligonucleotide is complementary to 16 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, the 17 consecutive nucleobases of the modified oligonucleotide are each complementary to 17 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 18 consecutive nucleobases of the modified oligonucleotide is complementary to 18 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 19 consecutive nucleobases of the modified oligonucleotide is complementary to 19 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 20 consecutive nucleobases of the modified oligonucleotide is complementary to 20 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 22 consecutive nucleobases of the modified oligonucleotide is complementary to 22 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, the 23 consecutive nucleobases of the modified oligonucleotide are each complementary to 23 consecutive nucleobases of the miRNA or precursor thereof. In certain such embodiments, each of the 24 consecutive nucleobases of the modified oligonucleotide is complementary to 24 consecutive nucleobases of the miRNA or precursor thereof.

Certain modified oligonucleotides

In certain embodiments, the modified oligonucleotide consists of 12-30 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 15-25 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 19-24 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 21-24 linked nucleosides.

In certain embodiments, the modified oligonucleotide consists of 12 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 13 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 14 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 15 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 16 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 19 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 20 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 21 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 22 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 23 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 24 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 25 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 26 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 27 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 28 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 29 linked nucleosides. In certain embodiments, the modified oligonucleotide consists of 30 linked nucleosides. In certain such embodiments, the modified oligonucleotide comprises a nucleotide sequence selected from SEQ ID NOs: 12.

Certain modifications

Modified oligonucleotides of the invention include one or more modifications to nucleobases, sugars and/or internucleoside linkages. Because of desirable properties such as increased cellular uptake, increased affinity for other oligonucleotide or nucleic acid targets, and increased stability in the presence of nucleases, modified nucleobases, sugars, and/or internucleoside linkages can be selected in unmodified form.

In certain embodiments, the modified oligonucleotides of the invention comprise one or more modified nucleosides. In certain such embodiments, the modified nucleoside is a stabilized nucleoside. An example of a stabilizing nucleoside is a sugar modified nucleoside.

In certain embodiments, the modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, sugar modified nucleosides can further include a natural or modified heterocyclic base moiety and/or a natural or modified internucleoside linkage, and can further include modifications independent of the sugar modification. In certain embodiments, the sugar modified nucleoside is a 2 ' -modified nucleoside in which the sugar ring is modified at the 2 ' carbon of the native ribose or 2 ' -deoxy-ribose.

In certain embodiments, the 2' -modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.

In certain embodiments, the bicyclic sugar moiety comprises a bridging group between the 2 '-carbon atom and the 4' -carbon atom. In certain such embodiments, the bridging group comprises 1-8 linked diradicals (biradicals). In certain embodiments, the bicyclic sugar moiety comprises 1-4 linked diradicals. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked diradicals. In certain embodiments, the bicyclic sugar moiety comprises 2 linked double free radicals. In certain embodiments, the linked diradicals are selected from the group consisting of-O-, -S-, -N (R)₁)-、-C(R₁)(R₂)-、-C(R₁)＝C(R₁)-、-C(R₁)＝N-、-C(＝NR₁)-、-Si(R₁)(R₂)-、-S(＝O)₂-, -S (═ O) -, -C (═ O) -, and-C (═ S) -; wherein each R₁And each R₂Independently H, hydroxy, C₁-C₁₂Alkyl, substituted C₁-C₁₂Alkyl radical, C₂-C₁₂Alkenyl, substituted C₂-C₁₂Alkenyl radical, C₂-C₁₂Alkynyl, substituted C₂-C₁₂Alkynyl, C₅-C₂₀Aryl, substituted C₅-C₂₀Aryl, heterocyclyl, substituted heterocyclyl, heteroaryl, substituted heteroaryl, C₅-C₇Alicyclic group, substituted C₅-C₇Alicyclic group, halogen, substituted oxy (-O-), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, mercapto, substituted mercapto, sulfonyl (S (═ O)₂-H), substituted sulfonyl, sulfoxyl (S (═ O) -H) or substituted sulfoxyl; and each substituent is independently halogen, C₁-C₁₂Alkyl, substituted C₁-C₁₂Alkyl radical, C₂-C₁₂Alkenyl, substituted C₂-C₁₂Alkenyl radical, C₂-C₁₂Alkynyl, toSubstituted C₂-C₁₂Alkynyl, amino, substituted amino, acyl, substituted acyl, C₁-C₁₂Aminoalkyl radical, C₁-C₁₂Aminoalkoxy, substituted C₁-C₁₂Aminoalkyl, substituted C₁-C₁₂Aminoalkoxy or a protecting group.

In some embodiments, the bicyclic sugar moiety is bridged between the 2 'and 4' carbon atoms by a diradical selected from the group consisting of: -O- (CH)₂)_p-、-O-CH₂-、-O-CH₂CH₂-, -O-CH (alkyl) -, -NH- (CH)₂)_p-, -N (alkyl) - (CH)₂)_p-, -O-CH (alkyl) -, - (CH (alkyl)) - (CH)₂)_p-、-NH-O-(CH₂)_p-, -N (alkyl) -O- (CH)₂)_p-or-O-N (alkyl) - (CH)₂)_p-, wherein p is 1, 2, 3, 4 or 5 and each alkyl group may be further substituted. In certain embodiments, p is 1, 2, or 3.

In certain embodiments, the 2 '-modified nucleoside includes a 2' -substituent selected from the group consisting of: halogen, allyl, amino, azido, SH, CN, OCN, CF₃、OCF₃O-, S-or N (R)_m) -an alkyl group; o-, S-or N (R)_m) -an alkenyl group; o-, S-or N (R)_m) -an alkynyl group; O-alkenyl-O-alkyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl, O-arylalkyl, O (CH)₂)₂SCH₃、O-(CH₂)₂-O-N(R_m)(R_n) Or O-CH₂-C(＝O)-N(R_m)(R_n) Wherein each R is_mAnd each R_nIndependently is H, an amino protecting group or a substituted or unsubstituted C₁-C₁₀An alkyl group. These 2' -substituents may be further substituted with one or more substituents independently selected from the group consisting of: hydroxy, amino, alkoxy, carboxyl, benzyl, phenyl, Nitro (NO)₂) Mercapto, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl, and alkynyl.

In certain embodiments, 2' -modificationsThe nucleoside includes a 2' -substituent selected from the group consisting of: F. NH (NH)₂、N₃、OCF₃、O-CH₃、O(CH₂)₃NH₂、CH₂-CH＝CH₂、O-CH₂-CH＝CH₂、OCH₂CH₂OCH₃、O(CH₂)₂SCH₃、O-(CH₂)₂-O-N(R_m)(R_n)、-O(CH₂)₂O(CH₂)₂N(CH₃)₂And N-substituted acetamides (O-CH)₂-C(＝O)-N(R_m)(R_n) Wherein each R is_mAnd each R_nIndependently is H, an amino protecting group or a substituted or unsubstituted C₁-C₁₀An alkyl group.

In certain embodiments, the 2 '-modified nucleoside comprises a 2' -substituent selected from the group consisting of: F. OCF₃、O-CH₃、OCH₂CH₂OCH₃、2′-O(CH₂)₂SCH₃、O-(CH₂)₂-O-N(CH₃)₂、-O(CH₂)₂O(CH₂)₂N(CH₃)₂And O-CH₂-C(＝O)-N(H)CH₃。

In certain embodiments, the 2 '-modified nucleoside comprises a 2' -substituent selected from the group consisting of: F. O-CH₃And OCH₂CH₂OCH₃。

In certain embodiments, the sugar modified nucleoside is a 4 '-mercapto-2' -modified nucleoside, in certain embodiments, the 4 '-mercapto-modified nucleoside has β -D-ribonucleoside wherein the 4' -O is replaced with 4 '-S, the 4' -mercapto-2 '-modified nucleoside is a 4' -mercapto-modified nucleoside wherein the 2 '-OH is replaced with a 2' -substituent, suitable 2 '-substituents include 2' -OCH₃、2′-O-(CH₂)₂-OCH₃And 2' -F.

In certain embodiments, the modified oligonucleotides of the invention comprise one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage comprises a phosphorus atom.

In certain embodiments, the modified oligonucleotides of the invention comprise at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified internucleoside linkage does not contain a phosphorus atom. In certain such embodiments, the internucleoside linkage is formed by a short chain alkyl internucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by a cycloalkyl internucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by a mixed heteroatom and alkylinternucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by a mixed heteroatom and cycloalkyl internucleoside linkage. In certain such embodiments, the internucleoside linkage is formed by one or more short chain heteroatom internucleoside linkages. In certain such embodiments, the internucleoside linkage is formed by one or more heterocyclic internucleoside linkages. In certain such embodiments, the internucleoside linkage has an amide backbone. In certain such embodiments, the internucleoside linkage has N, O, S and CH mixed₂And (4) component parts.

In certain embodiments, the modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, the modified oligonucleotide comprises one or more 5-methylcytosines. In certain embodiments, each cytosine of the modified oligonucleotide comprises a 5-methylcytosine.

In certain embodiments, the modified nucleobase is selected from the group consisting of 5-hydroxymethylcytosine, 7-deazaguanine, and 7-deazaadenine. In certain embodiments, the modified nucleobase is selected from the group consisting of 7-deaza-adenine, 7-deaza-guanine, 2-aminopyridine, and 2-pyridone. In certain embodiments, the modified nucleobase is selected from the group consisting of 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

In certain embodiments, the modified nucleobase comprises a polycyclic heterocycle. In certain embodiments, the modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, the modified nucleobase comprises a thiopheneAn oxazine derivative. In certain embodiments, a thiopheneThe oxazines may be further modified to form a nucleobase known in the art as a G-clamp ring (G-clamp).

Certain oligonucleotide motifs

Suitable motifs for use in the modified oligonucleotides of the invention include, but are not limited to, all modifications, consensus modifications, positional modifications, and gapmers. Modified oligonucleotides with all modification motifs (including consensus modification motifs) can be designed to target mature mirnas. Alternatively, modified oligonucleotides with all modification motifs (including consensus modification motifs) can be designed to target certain sites of pri-or pre-mirnas, blocking processing of miRNA precursors into mature mirnas. Modified oligonucleotides with all or a consensus of the modification motifs are potent inhibitors of miRNA activity.

In certain embodiments, a fully modified oligonucleotide comprises sugar modifications on each nucleoside. In certain such embodiments, the plurality of nucleosides is a 2 '-O-methoxyethyl nucleoside and the remaining nucleosides are 2' -fluoro nucleosides. In certain such embodiments, each of the plurality of nucleosides is a 2' -O-methoxyethyl nucleoside and each of the plurality of nucleosides is a bicyclic nucleoside. In certain such embodiments, the fully modified oligonucleotide further comprises at least one modified internucleoside linkage. In certain such embodiments, each internucleoside linkage of the fully sugar-modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the fully sugar-modified oligonucleotide further comprises at least one phosphorothioate internucleoside linkage. In certain such embodiments, each internucleoside linkage of the fully sugar-modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a fully modified oligonucleotide is modified at each internucleoside linkage. In certain such embodiments, each internucleoside linkage of the fully modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a consistently modified oligonucleotide comprises the same sugar modification on each nucleoside. In certain such embodiments, each nucleoside of the modified oligonucleotide comprises a 2' -O-methoxyethyl sugar modification. In certain embodiments, each nucleoside of the modified oligonucleotide comprises a 2' -O-methyl sugar modification. In certain embodiments, each nucleoside of the modified oligonucleotide comprises a 2' -fluoro sugar modification. In certain such embodiments, the consistently modified oligonucleotides further comprise at least one modified internucleoside linkage. In certain such embodiments, each internucleoside linkage of the oligonucleotide that is consistently modified by a sugar is a modified internucleoside linkage. In certain embodiments, the oligonucleotide consistently modified by the sugar further comprises at least one phosphorothioate internucleoside linkage. In certain such embodiments, each internucleoside linkage of the oligonucleotide that is consistently modified by a sugar is a phosphorothioate internucleoside linkage.

In certain embodiments, a consistently modified oligonucleotide comprises the same internucleoside linkage modifications throughout. In certain such embodiments, each internucleoside linkage of the consistently modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotide comprises the same sugar modification on each nucleoside, and further comprises one or more internucleoside linkage modifications. In certain such embodiments, the modified oligonucleotide comprises 1 modified internucleoside linkage at the 5 'end and 1 modified internucleoside linkage at the 3' end. In certain embodiments, the modified oligonucleotide comprises 2 modified internucleoside linkages at the 5 'end and 2 modified internucleoside linkages at the 3' end. In certain embodiments, the modified oligonucleotide comprises 2 modified internucleoside linkages at the 5 'end and 3 modified internucleoside linkages at the 3' end. In certain embodiments, the modified oligonucleotide comprises 2 modified internucleoside linkages at the 5 'end and 4 modified internucleoside linkages at the 3' end. In certain such embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, the modified oligonucleotide is represented by formula III below:

(5’)QxQz¹(Qy)_nQz²Qz³Qz⁴Q-L(3’)

in certain such embodiments, the compound is represented by formula III. In certain embodiments, Q is a 2' -O-methyl modified nucleoside. In certain embodiments, x is a phosphorothioate. In certain embodiments, y is a phosphodiester. In certain embodiments, z¹、z²、z³And z⁴Each of which is independently a phosphorothioate or a phosphodiester. In certain embodiments, n is 6 to 17. In certain embodiments, L is cholesterol. In certain embodiments, n is 12 to 17.

In certain embodiments, x is

One of A and B is S and the other is O;

y is

z¹、z²、z³And z⁴Each of which isEach independently is x or y;

n＝6-17

l is

Wherein:

x is N (CO) R⁷Or NR⁷；

R¹、R³And R⁹Each of which is independently H, OH or-CH₂OR^bWith the proviso that R is¹、R³And R⁹At least one of which is OH, and R¹、R³And R⁹At least one of which is-CH₂OR^b；

R⁷Is R^dOr by NR^cR^dOr NHC (O) R^dSubstituted C₁-C₂₀An alkyl group;

R^cis H or C₁-C₆An alkyl group;

R^dis a glycosyl; or a steroid-like alcohol group optionally linked to at least one sugar group; and

R^bis composed of

One of A and B is S and the other is O.

In certain embodiments, R^dIs cholesterol. In certain embodiments, z¹、z²、z³And z⁴Each of which is

One of A and B is S and the other is O。

In certain embodiments, R¹is-CH₂OR^b. In certain embodiments, R⁹Is OH. In certain embodiments, R¹And R⁹Is in the trans form. In certain embodiments, R⁹Is OH. In certain embodiments, R¹And R³Is in the trans form. In certain embodiments, R³is-CH₂OR^b. In certain embodiments, R¹Is OH. In certain embodiments, R¹And R³Is in the trans form. In certain embodiments, R⁹Is OH. In certain embodiments, R³And R⁹Is in the trans form. In certain embodiments, R⁹Is CH₂OR^b. In certain embodiments, R¹Is OH. In certain embodiments, R¹And R⁹Is in the trans form. In certain embodiments, X is NC (O) R⁷. In certain embodiments, R⁷is-CH₂(CH₂)₃CH₂NHC(O)R^d。

In certain embodiments, the positionally modified oligonucleotide comprises a region to which nucleosides are linked, wherein each nucleoside of each region comprises the same sugar moiety, and wherein each nucleoside of each region comprises a sugar moiety that is different from the sugar moiety of an adjacent region.

In certain embodiments, the positionally modified oligonucleotide comprises at least 10 2' -fluoro modified nucleosides. Such position-modified oligonucleotides can be represented by the following formula I:

5′-T₁-(Nu₁-L₁)_n1-(Nu₂-L₂)_n2-Nu₂-(L₃-Nu₃)_n3-T₂-3', wherein:

each Nu₁And each Nu₃Independently a stabilizing nucleoside;

at least 10 Nu₂Is a 2' -fluoronucleoside;

L₁、L₂and L₃Each of which is independently an internucleoside linkage;

each T₁And each T₂Independently H, a hydroxyl protecting group, an optionally attached conjugate group or a capping group;

n₁from 0 to about 3;

n₂from about 14 to about 22;

n₃from 0 to about 3; and

with the proviso that if n is₁Is 0, then T₁Not H or a hydroxy protecting group, and if n₃Is 0, then T₂Not H or a hydroxy protecting group.

In certain such embodiments, n1 and n3 are each independently 1 to about 3. In certain embodiments, n1 and n3 are each independently 2 to about 3. In certain embodiments, n 1is 1 or 2 and n3 is 2 or 3. In certain embodiments, n1 and n3 are each 2. In certain embodiments, at least one of n1 and n3 is greater than zero. In certain embodiments, n1 and n3 are each greater than zero. In certain embodiments, one of n1 and n3 is greater than zero. In certain embodiments, one of n1 and n3 is greater than 1.

In certain embodiments, n₂Is 16-20. In certain embodiments, n₂Is 17-19. In certain embodiments, n₂Is 18. In certain embodiments, n₂Is 19. In certain embodiments, n₂Is 20.

In certain embodiments, from about 2 to about 8 Nu₂Nucleosides are stabilized nucleosides. In certain embodiments, from about 2 to about 6 Nu₂Nucleosides are stabilized nucleosides. In certain embodiments, from about 3 to about 4 Nu₂Nucleosides are stabilized nucleosides. In certain embodiments, 3 Nu₂Nucleosides are stabilized nucleosides.

In certain embodiments，Nu₂Each of the stabilized nucleosides is substituted with 2 to about 8 2' -fluoronucleosides and Nu₃The stabilizing nucleosides are separated. In certain embodiments, Nu₂Each of the stabilized nucleosides is substituted with 3 to about 8 2' -fluoronucleosides and Nu₃The stabilizing nucleosides are separated. In certain embodiments, Nu₂Each of the stabilized nucleosides is substituted with 5 to about 8 2' -fluoronucleosides and Nu₃The stabilizing nucleosides are separated.

In certain embodiments, the modified oligonucleotide comprises from 2 to about 6 Nu₂A stabilizing nucleoside. In certain embodiments, the modified oligonucleotide comprises 3 Nu₂A stabilizing nucleoside.

In certain embodiments, Nu₂Each of the stabilizing nucleosides is linked together in a contiguous sequence. In certain embodiments, Nu₂At least two of the stabilizing nucleosides are separated by at least one 2' -fluoronucleoside. In certain embodiments, Nu₂Each of the stabilizing nucleosides is separated by at least one 2' -fluoro nucleoside.

In certain embodiments, Nu₂At least two consecutive sequences of 2 '-fluoronucleosides are separated by at least one stabilizing nucleoside, wherein each consecutive sequence has the same number of 2' -fluoronucleosides.

In certain embodiments, T₁And T₂Each independently is H or a hydroxy protecting group. In certain embodiments, T₁And T₂At least one of which is 4, 4' -dimethoxytrityl. In certain embodiments, T₁And T₂At least one of which is an optionally attached conjugate group. In certain embodiments, T₁And T₂At least one of which is a capping group. In certain embodiments, the end capping group is a reverse phase deoxyabasic.

In certain embodiments, the positionally modified oligonucleotide comprises at least one modified internucleoside linkage. In certain such embodiments, each internucleoside linkage of the positionally modified oligonucleotide (oligonucleotide) is a modified internucleoside linkage. In certain embodiments, at least one internucleoside linkage of the positionally modified oligonucleotide is a phosphorothioate internucleoside linkage. In certain such embodiments, each internucleoside linkage of the positionally modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, the positional modification motif is represented by formula II below, which represents a modified oligonucleotide consisting of linked nucleosides:

T₁-(Nu₁)_n1-(Nu₂)_n2-(Nu₃)_n3-(Nu₄)_n4-(Nu₅)_n5-T₂wherein:

Nu₁and Nu₅Independently a 2' stabilizing nucleoside;

Nu₂and Nu₄Is a 2' -fluoronucleoside;

Nu₃is a 2' -modified nucleoside;

each of n1 and n5 is independently 0-3;

the sum of n2 plus n4 is between 10 and 25;

n3 is 0 to 5; and

each T₁And each T₂Independently H, a hydroxyl protecting group, an optionally attached conjugating group or a capping group.

In certain embodiments, the sum of n2 and n4 is 16. In certain embodiments, the sum of n2 and n4 is 17. In certain embodiments, the sum of n2 and n4 is 18. In certain embodiments, n 1is 2; n3 is 2 or 3; n5 is 2.

In certain embodiments, Nu₁And Nu₅Independently a 2' -modified nucleoside.

In certain embodiments, Nu₁Is O- (CH)₂)₂-OCH₃，Nu₃Is O- (CH)₂)₂-OCH₃、Nu₅O-(CH₂)₂-OCH₃，T₁Is H, T₂Is H.

In certain embodiments, the modified oligonucleotide complementary to a miRNA and consisting of 22 linked nucleosides has a formula II selected from table a, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, the modified oligonucleotide having formula II selected from table a has the amino acid sequence of SEQ ID NO: 12.

Modified oligonucleotides with gapmer motifs may have an inner region (internal region) consisting of linked 2 '-deoxynucleotides and an outer region (external region) consisting of linked 2' -modified nucleosides. Such gapmers can be designed to trigger RNase H cleavage of miRNA precursors. The internal 2' -deoxynucleoside region serves as a substrate for RNase H for cleavage of miRNA precursors targeted by the modified oligonucleotide. In certain embodiments, each nucleoside of each outer region comprises the same 2' -modified nucleoside. In certain embodiments, one outer region consists of the first 2 '-modified nucleoside and the other outer region consists of the second 2' -modified nucleoside.

Modified oligonucleotides with gapmer motifs may have sugar modifications on each nucleoside. In certain embodiments, the internal region consists of the first 2 '-modified nucleoside uniformly and each wing (wing) consists of the second 2' -modified nucleoside uniformly. In certain such embodiments, the inner region is uniformly composed of 2 '-fluoro nucleosides and each outer region is uniformly composed of 2' -O-methoxyethyl nucleosides.

In certain embodiments, each outer region of the gapmer consists of linked 2' -O-methoxyethyl nucleosides. In certain embodiments, each outer region of the gapmer consists of linked 2' -O-methyl nucleosides. In certain embodiments, each outer region of the gapmer consists of 2' -fluoro nucleosides. In certain embodiments, each outer region of the gapmer consists of linked bicyclic nucleosides.

In certain embodiments, each nucleoside of one outer region of the gapmer comprises a 2 '-O-methoxyethyl nucleoside and each nucleoside of the other outer region comprises a different 2' -modification. In certain such embodiments, each nucleoside of one outer region of the gapmer comprises a 2 '-O-methoxyethyl nucleoside and each nucleoside of the other outer region comprises a 2' -O-methyl nucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer comprises a 2 '-O-methoxyethyl nucleoside and each nucleoside of the other outer region comprises a 2' -fluoro nucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer comprises a 2 '-O-methyl nucleoside and each nucleoside of the other outer region comprises a 2' -fluoro nucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer comprises a 2' -O-methoxyethyl nucleoside and each nucleoside of the other outer region comprises a bicyclic nucleoside. In certain such embodiments, each nucleoside of one outer region of the gapmer comprises a 2' -O-methyl nucleoside and each nucleoside of the other outer region comprises a bicyclic nucleoside.

In certain embodiments, the nucleosides of one outer region comprise two or more sugar modifications. In certain embodiments, the nucleosides of each outer region comprise two or more sugar modifications. In certain embodiments, at least one nucleoside of the outer region comprises a 2 '-O-methoxyethyl sugar and at least one nucleoside of the same outer region comprises a 2' -fluoro sugar. In certain embodiments, at least one nucleoside of the outer region comprises a 2' -O-methoxyethyl sugar and at least one nucleoside of the same outer region comprises a bicyclic sugar moiety. In certain embodiments, at least one nucleoside of the outer region comprises a 2' -O-methyl sugar and at least one nucleoside of the same outer region comprises a bicyclic sugar moiety. In certain embodiments, at least one nucleoside of the outer region comprises a 2 '-O-methyl sugar and at least one nucleoside of the same outer region comprises a 2' -fluoro sugar. In certain embodiments, at least one nucleoside of the outer region comprises a 2' -fluoro sugar and at least one nucleoside of the same outer region comprises a bicyclic sugar moiety.

In certain embodiments, each outer region of the gapmer consists of the same number of linked nucleosides. In certain embodiments, one outer region of the gapmer consists of a number of linked nucleosides that is different from the number of linked nucleosides of another outer region.

In certain embodiments, the outer region independently comprises 1-6 nucleosides. In certain embodiments, the outer region comprises 1 nucleoside. In certain embodiments, the outer region comprises 2 nucleosides. In certain embodiments, the outer region comprises 3 nucleosides. In certain embodiments, the outer region comprises 4 nucleosides. In certain embodiments, the outer region comprises 5 nucleosides. In certain embodiments, the outer region comprises 6 nucleosides. In certain embodiments, the internal region consists of 17-28 linked nucleosides. In certain embodiments, the internal region consists of 17-21 linked nucleosides. In certain embodiments, the inner region consists of 17 linked nucleosides. In certain embodiments, the inner region consists of 18 linked nucleosides. In certain embodiments, the inner region consists of 19 linked nucleosides. In certain embodiments, the inner region consists of 20 linked nucleosides. In certain embodiments, the inner region consists of 21 linked nucleosides. In certain embodiments, the inner region consists of 22 linked nucleosides. In certain embodiments, the inner region consists of 23 linked nucleosides. In certain embodiments, the inner region consists of 24 linked nucleosides. In certain embodiments, the inner region consists of 25 linked nucleosides. In certain embodiments, the inner region consists of 26 linked nucleosides. In certain embodiments, the inner region consists of 27 linked nucleosides. In certain embodiments, the inner region consists of 28 linked nucleosides.

Certain quantitative assays

The antisense inhibition of mirnas following administration of modified oligonucleotides can be assessed by various methods known in the art. In certain embodiments, these methods are used to quantitate miRNA levels in cells or tissues in vitro or in vivo. In certain embodiments, the change in miRNA levels is determined by microarray analysis. In certain embodiments, by several commercially available PCR assays, e.g.MicroRNA Assay (applied biosystems) to measure changes in miRNA levels. In certain embodiments, antisense inhibition of a miRNA is assessed by measuring the mRNA and/or protein level of the miRNA target. Antisense inhibition of mirnas generally results in increased mRNA and/or protein levels of the miRNA target.

Some experimental models

In certain embodiments, the invention provides methods of using and/or testing the modified oligonucleotides of the invention in experimental models. In certain embodiments, experimental models are employed to evaluate the efficacy of the modified oligonucleotides of the invention for treating fibrosis. The skilled person will be able to select and modify protocols for such experimental models to evaluate the agents of the invention.

The modified oligonucleotide can first be detected in cultured cells. Suitable cell types include cells associated with cell types requiring in vivo delivery of modified oligonucleotides. For example, suitable cell types for studying modified oligonucleotides for treating fibrosis include fibroblasts, cardiomyocytes, and astrocytes.

In certain embodiments, the extent to which the modified oligonucleotide inhibits the activity of a miRNA is evaluated in cultured cells. In certain embodiments, inhibition of miRNA activity can be assessed by measuring the level of miRNA. Alternatively, the level of a predicted or validated miRNA target may be determined. Inhibition of miRNA activity may result in an increase in mRNA and/or protein of the miRNA target. Furthermore, in certain embodiments, certain phenotypic outcomes may be determined. For example, suitable phenotypic outcomes include inhibition of cell proliferation, induction of cell death, and/or induction of apoptosis.

After identifying in vitro modified oligonucleotides that effectively inhibit the activity of mirnas, the modified oligonucleotides were further tested in an in vivo experimental model.

Suitable experimental models for testing drugs for the treatment of fibrosis, including drugs comprising modified oligonucleotides complementary to miR-122, include the pressure overload-induced hypertrophic model described herein.

Other experimental models for testing drugs for the treatment of fibrosis include, but are not limited to, the Methionine Choline Deficient (MCD) diet model (see, e.g., Yamaguchi et al, Hepatology, 2008, 47, 625-. db/db mice spontaneously develop obesity, diabetes and fatty liver. MCD diet feeding such mice for 4-8 weeks induced nonalcoholic steatohepatitis (NASH) and liver fibrosis. Modified oligonucleotides having nucleobases complementary to mirnas were tested in this model for their effect on liver fibrosis.

The following examples are provided in order to more fully illustrate certain embodiments of the invention. However, these examples should not be construed in any way as limiting the broad scope of the invention.

Examples

Example 1: role of miR-21 in heart disease

Microarray analysis of a transgenic mouse model of heart failure (heart suppressed overexpression of the heart β 1-adrenoreceptor (restricted overexpression of β 1-adrenergic receptors) (Engelhardt et al, 1999)) revealed a gradual deregulation of cardiac microRNA expression profiles with increasing disease severity (FIG. 1 a). Although miR-21 expression is moderate in normal myocardium, in failing hearts, this microRNA is one of the most strongly regulated microRNAs. Indeed, miR-21 is the only one of the most strongly upregulated micrornas in mice with end-stage heart failure (fig. 1 a). Quantitative northern blot analysis confirmed miR-21 up-regulation in mice and further revealed significant up-regulation in human heart failure (fig. 1b, c). The increased expression of the miR-21 precursor as assessed by northern blotting suggests a transcriptional mechanism. Therefore, the human miR-21 promoter was studied in more detail. miR-21 promoter regions were identified in several species, and are highly conserved (FIG. 4 a). Studies of human miR-21 expression (FIGS. 4a-c) demonstrate that it is transcriptionally regulated by two transcription factors, calcium/cAMP response element protein (CREB) and Serum Response Factor (SRF), which are normally activated during cardiac stress response. Deletion of CREB in the miR-21 promoter and mutation of the SRF binding site resulted in a significant decrease in miR-21 expression in response to serum stimulation, suggesting a major role for these two transcription factors in miR-21 regulation (fig. 4 c). In biased screening (biasted screen) to knock down several microRNAs ubiquitously expressed in zebrafish, the essential role of miR-21 in cardiac morphology and function was examined by a morpholino-based approach. Knockdown of miR-21 resulted in a great impairment of cardiac structure and function (fig. 5). As determined in the visual microscope, greater than 95% of the injected animals presented massive pericardial effusion (fig. 5a, insets, and b) and impaired ventricular function (fig. 5 c).

To investigate these essential functions of miR-21 in the mammalian heart in more detail, the inventors of the present invention regulated miR-21 expression in isolated cardiomyocytes. The inventors transfected synthetic miR-21 precursors as well as antisense miR-21 inhibitors, routinely achieving > 95% transfection efficiency for oligonucleotide delivery (FIG. 6 a). As determined by northern blot analysis, overexpression of miR-21 results in a robust increase in mature miR-21, whereas inhibition of miR-21 completely suppresses endogenous miR-21 expression (FIG. 6 b). However, neither the increase or inhibition of miR-21 levels in cardiomyocytes significantly affected the morphology, size or number of primary rat cardiomyocytes under resting conditions or under cardiomyocyte hypertrophic conditions (fig. 1 d). Cardiomyocyte-specific miR-21 transgenic mice overexpressing miR-21 25-fold more than wild-type littermate mice did not exhibit a clear cardiac phenotype with intact left ventricular myocardium structure and absent interstitial fibrosis (fig. 1e, bottom panel). Unlike the large increase in miR-21 expression in failing hearts, these data indicate that controlling miR-21 expression levels in isolated cardiomyocyte and cardiomyocyte-specific miR-21 transgenic mouse models fails to demonstrate the important function of miR-21 in hearts as demonstrated by observations in zebrafish and the large increase in miR-21 expression in failing hearts. Thus, the inventors of the present invention next investigated the possible role of miR-21 in cell types other than cardiomyocytes (e.g., cardiac fibroblasts). Using in situ hybridization, a weak miR-21 signal was detected in normal myocardium, whereas in failing myocardium, the hybridization signal was greatly increased. At high magnification, the hybridization signal is mainly confined to the microplasmic cells, presumably cardiac fibroblasts. Using a pre-plating procedure, the inventors of the present invention separated the neonatal rat heart into a cardiomyocyte fraction and a fibroblast fraction. The inventors did detect that endogenous miR-21 is predominantly expressed in cardiac fibroblasts (fig. 1 f).

Example 2: MiR-21 derepression of cardiac fibroblast ERK-signaling

MiR-21 expression is selectively increased in various human cancers (Lu et al, 2005; Iorio et al, 2005), and studies have shown that tumor growth and spread are affected by different mechanisms in different cells. Because miR-21 appears to target unique mRNA in a cell-type specific manner, the inventors of the present invention decided to investigate possible heart-specific miR-21 targets. Using bioinformatic methods, the inventors of the present invention performed screening of several microrna databases for potential miR-21 targets having seed sequences comprising the 3' UTR and matching flanking nucleotides, and focused the analysis on candidates reported to have cardiac expression. The following table 1 summarizes the results.

TABLE 1

Screening of theoretical miR-21 targets revealed 22 known potential target genes, 8 of which were shown to be expressed in cardiac tissue from previous studies. The combination of 3 different target prediction tools identified Spry1(sprouty1) as a very likely candidate. Sprouty1(SPRY1), a known inhibitor of the Ras/MEK/ERK pathway (Hanafusa et al, 2002, Casci et al, 1999), emerged as a possible target due to its high score and significant expression levels in the heart (Table 1). The 3' UTR of Spry1mRNA contains several predicted microRNA binding sites, only one of which corresponds to high upregulation of microRNAs during cardiac disease (miR-21, see FIG. 7). To determine the cell type in which Spry 1is expressed, the inventors of the present invention utilized an allele of Spry1 in which the lacZ gene replaces a portion of the Spry1 coding sequence. The assay used for lacZ expression showed significant staining in adult mouse hearts (fig. 2 a). Higher magnification determined the punctate pattern of Spry1 expression from mesenchymal fibroblasts (fig. 2a, bottom panel). Thus, miR-21 and its putative target Spry1 are co-expressed in cardiac fibroblasts, but not in the cardiomyocyte fraction. Consistent with these observations, no detectable downregulation of SPRY1 expression was found in transgenic mice that overexpress miR-21 in a cardiomyocyte-specific manner. In addition, cardiac CREB, a miR-21-expressed transcriptional activator (fig. 4), was localized only in fibroblasts. The inventors then tested the association of these findings with human disease. Analysis of left ventricular cardiac tissue samples from patients with end-stage heart failure due to idiopathic dilated cardiomyopathy did confirm increased miR-21 expression (fig. 1c) and significant suppression of SPRY1 protein expression (fig. 2 b). These findings were accompanied by activation of ERK-MAP kinase as evidenced by an increase in the phospho-ERK/ERK ratio (FIG. 2 b).

miR-21 function was then characterized in co-cultures of mesenchymal fibroblasts and cardiomyocytes to mimic the composition of intact cardiac tissue. The inventors of the present invention evaluated miR-21 function by transfection of synthetic miR-21 precursor molecules or inhibitors. Increased miR-21 induced strong repression of SPRY1 protein expression and increased ERK-MAP kinase activation (fig. 2 c). SiRNA-mediated silencing of Spry1 also resulted in activation of ERK-MAP kinase (FIG. 2 d). The inventors next evaluated whether miR-21-mediated derepression of fibroblast ERK-MAP kinase signaling was sufficient to affect fibroblast survival. Consistent with the potential role of ERK-MAP kinase signaling in myocardial fibrosis, the increase in miR-21 levels promoted cardiac fibroblast survival, while inhibition of endogenous miR-21 induced apoptotic cell death (fig. 2 e). The inventors of the present invention also found that regulation of SPRY1 expression based on miR-21 is critical for the secretory function of cardiac fibroblasts, as both overexpression of miR-21 and siRNA-mediated silencing of SPRY1 expression significantly increased fibroblast growth factor 2(FGF2) secretion into the supernatant (fig. 2 f). Thus, this study describes a new signaling pattern in failing hearts, where the re-expression of miR-21 during heart disease increases ERK-MAP kinase activity by inhibiting SPRY 1. In mammalian hearts, this mechanism can regulate fibroblast survival, thereby critically controlling the degree of interstitial fibrosis and cardiac remodeling.

Example 3: therapeutic silencing of MiR-21 in vivo

To evaluate the function of miR-21 in vivo, miR-21 activity was inhibited in the normal background (setting) as well as in heart failure using a modified oligonucleotide complementary to miR-21 (miR-21 antagonist). A mouse model of hypertrophy induced by excessive pressure load was used as a model of human heart failure. In this model, reproducible cardiac stress is achieved by aortic arch constriction (TAC). This model is very similar to failing human heart due to its matching pattern of changes in both micrornas and mrnas in the global expression profile (Thum et al, 2007).

To determine the localization of antagomir-21 within the heart, Cy-3 labeled antagomir-21 was injected intravenously via a jugular vein catheter. Dark staining of the left ventricular myocardium was observed in general (FIG. 3a), indicating that modified oligonucleotides complementary to miR-21 achieve distribution in cardiac tissue. The miR-21 antagonists comprise a 2 ' -O-methyl sugar on each nucleoside, 2 phosphorothioate internucleoside linkages at the 5 ' most end of the oligonucleotide, 3 phosphorothioate internucleoside linkages at the 3 ' most end of the oligonucleotide, and cholesterol linked by a hydroxyprolinol (hydroxyprolinol) linker. The miR-21 antagonist has the amino acid sequence of SEQ id no: 1 or SEQ ID NO: 12. Untreated (sham) mice or mice subjected to TAC surgery were treated with antagomir-21 or control oligonucleotide for 3 consecutive days at a dose of 80 mg/kg. Treatment with antagomir-21 potently suppressed elevated cardiac miR-21 expression for up to 3 weeks, as determined by northern blot (fig. 3b) and real-time PCR analysis (fig. 8). This treatment completely reversed TAC-induced downregulation of SPRY1 and ERK-MAP kinase activation during stress overload to levels observed in sham operated mice (fig. 3 c). In untreated mice, interstitial fibrosis and heart weight increased significantly at 3 weeks post TAC, but was greatly reduced by antagomir-21 treatment (fig. 3d, e). In fact, the collagen content of the myocardium was essentially normal by antagomir-21 treatment. Furthermore, where the heart weight doubled 3 weeks after TAC, antagomir-21 treatment prevented hypertrophy. Treatment with antagomir-21 did not cause significant changes in cardiac weight or interstitial fibrosis in sham operated mice, indicating that miR-21 antagonism had no detectable effect on normal heart or significant cardiotoxicity of antagomir-21 treatment. Global transcriptome analysis revealed various dysregulated gene normalization after TAC surgery and antagomir-21 treatment (fig. 3 f). Specifically, genes that are greatly up-regulated during cardiac fibrosis, such as collagen 1 α 1, collagen 3 α 1, biglycan, fibromodulin, or connective tissue growth factor, are reduced by 42%, 39%, 44%, 38%, and 37%, respectively, following specific inhibition by miR-21 (lower panel of fig. 3 f). In a further study, cardiac function was assessed by echocardiography. Left ventricular end-diastolic diameter (left ventricular end-Diastolic diameter) increased significantly 3 weeks after TAC, with a decrease in fractional shortening (FIG. 3g), as commonly observed in human heart failure. Antagomir-21 treatment prevented left ventricular dilatation and substantially normalized the fractional shortening parameter to the level observed in sham operated animals when compared to controls (figure 3 g). Similar results were observed with antagomir-21 treatment in an isoproterenol-induced heart disease model.

Additional experiments were also performed in which mice were treated with antagomir-21 3 weeks after left ventricle overload. During this period, the animals showed marked left ventricular hypertrophy, fibrosis and impaired cardiac function. After this 3 week period, mice were treated with antagomir-21 and observed for a further 3 weeks. The animals treated with the control showed a progressive impairment of the left ventricular function with interstitial fibrosis and cardiac hypertrophy, whereas the animals treated with antagomir-21 showed a significant reduction of the impairment of the cardiac function and a regression of the cardiac hypertrophy and fibrosis.

These data demonstrate the key role of miR-21 and SPRY1 derived from fibroblasts in the heart. Abnormal expression of miR-21 in cardiac fibroblasts inhibits SPRY1 protein expression, resulting in increased ERK-MAP kinase activity. This in turn increases cardiac fibroblast survival, thereby increasing interstitial fibrosis and cardiac remodeling that are characteristic of failing hearts. This model (summarized in fig. 3h) points out a major role in cardiac fibroblast activation in myocardial diseases. Antagonizing miR-21 in a murine model of cardiac disease prevents deterioration of structure and function. Accordingly, the present invention provides methods for treating fibrosis comprising administering a modified oligonucleotide complementary to miR-21. The invention also provides methods for treating fibrosis associated with heart disease comprising administering a modified oligonucleotide complementary to miR-21.

Example 4: mir-21 modulation of extracellular signal-regulated kinase MAPK pathway

Malignant transformation from normal cells to cancer cells requires several oncogenic properties, such as uncontrolled cell division, resistance to programmed cell death (apoptosis), invasion and angiogenesis. Genetic variation often leads to widespread constitutive activation of downstream signal transduction pathways, such as the mitogen-activated protein (MAP) kinase cascade including c-RAF-1, MEK-1, ERK1/2, p38, and JNK, among others. The mitogen-activated protein kinase (MAPK) cascade is a key signal transduction pathway involved in regulating normal cell proliferation, survival and differentiation. Aberrant regulation of the MAPK cascade has consequences for cancer and other human diseases. The extracellular signal-regulated kinase (ERK) MAPK pathway has been the subject of intensive research, in particular, leading to the development of pharmacological inhibitors for the treatment of cancer. It has been well established that in normal cells, receptor tyrosine kinases such as the EGF receptor and Platelet Derived Growth Factor Receptor (PDGFR) regulate activation of the kinase ERK1/2 by activation of Ras, which in turn activates the RAF-1/MEK-1/ERK1/2 cascade. Because this signal transduction pathway is hyperactivated in a variety of human cancers, inhibitors of the receptor tyrosine kinases, Ras, c-RAF-1, and MEK-1 have been, and are in various stages of, development. Thus, it is feasible to treat cancer by administering an effective amount of an inhibitor of the RAF-1/MEK-1/P-ERK 1/2 pathway.

Sprouty (spry) is a family of intracellular proteins that are endogenous modulators of the receptor tyrosine kinase pathway (e.g., the Ras/MAP kinase pathway). Mammalian species express 4 isoforms of Sprouty, which act as inhibitors of growth factor-induced cell differentiation, migration and proliferation. The inventors of the present invention identified sprouty-1 as inhibiting ERK phosphorylation, which may lead to modulation of tumor formation. The inventors propose that upregulation of miR-21 inhibits sprouty-1 and thus activates ERK in a variety of human cancers, leading to increased and accelerated tumor formation. Antagonism of miR-21 (e.g. by antagomir-21) is therefore able to prevent and/or reduce tumour formation and/or progression.

Example 5: experimental procedure

Expression analysis (micro RNA array, Affymetrix Gene chip analysis, northern blotting, real-time PCR)

microRNAs (Castoldi et al, 2007) and global mRNA expression profiles were generated from RNA preparations of murine left ventricular myocardium. micro-RNA deregulation was confirmed by northern blotting and stem-loop specific real-time PCR. For oligonucleotide arrays and spotted microRNA arrays, the R software package from the Bioconductor protocol as described previously (Thim et al, 2007) was applied (www.bioconductor.org) Data analysis was performed.

MiR-21 promoter analysis

24 hours after isolation, cells were transfected with 1. mu.g of reporter plasmid using established lipofection methods (Lipofectamine, Invitrogen, USA). After 12 hours, the medium was changed to FCS-free medium. After 24 hours, the cells were treated with 5% FCS for 8 hours, while the other groups were cultured without FCS. Luciferase activity was measured in cell lysates using the dual luciferase Kit (DualLuciferase Kit) (Promega, Germany) according to the manufacturer's instructions.

Cardiomyocyte isolation, culture and transfection assays

Neonatal cardiomyocytes were isolated as described in the previous literature (Merkle et al, 2007). Cardiomyocyte size was determined from digitally recorded images using the Axio Vision LE4.1 software package (Carl Zeiss Vision GmbH, Jena, Germany). Cardiomyocyte cultures were transfected with precursors and inhibitors of miR-21 (Ambion, USA) or siRNA against sprouty1 (Promega, Germany). The inventors of the present invention also performed experiments with cardiac fibroblasts and fibroblast/cardiomyocyte co-cultures using appropriate culture conditions.

Zebra fish maintenance, microinjection of morpholino antisense oligonucleotides and determination of cardiac function

Standard morpholino modified oligonucleotides are used for mature dre-miR-21(MO-1 ═ 5'-GCCAACACCAGTCTGATAAGCTA-3'), and also multiply-blocked (multi-blocking) morpholino modified oligonucleotides are used to interfere with various steps and functions in miR-21 processing (MO-2 ═ 5'-TGTAACAGCCAACACCAGTCTGATAAGCTAT-3'). Standard control oligonucleotides (MO-control) (GENETOOLS, LLC) were injected at the same concentrations as the negative controls. Morpholinos were microinjected into wild type zebrafish single cell stage embryos and the overall morphology, especially cardiac function, was assessed at several time points during development. Pictures and movies were recorded and ventricular shortening scores at 48, 72, 80, 96 and 120 hours post-fertilization (hpf) were measured essentially as described in the literature (Rottbauer et al, 2005).

Mouse model of cardiac hypertrophy and failure

Aortic coarctation was performed conventionally. β 1-adrenergic receptor transgenic mice (TG4 line) have been described in detail previously (Engelhardt et al, 1999).

Human heart sample

The inventors of the present invention have studied heart tissues of patients undergoing heart transplantation due to end-stage heart failure caused by dilated cardiomyopathy, and compared them with samples of healthy adult hearts. Immediately after transplantation, tissue sections were removed from the left ventricle, excised tissue was snap frozen in liquid nitrogen and stored at-80 ℃ until analysis.

Micro RNA target prediction method

Potential microRNA targets were identified using the microRNA database and the target prediction tools miRBase (http:// microrna. sanger. ac. uk /), PicTar (http:// polar. bio. nyu. edu /), and TargetScan (http:// www.targetscan.org/index. html).

Western blotting and analysis of cardiac fibrosis

Protein lysates were prepared from transplanted hearts or co-cultures as described in the literature (Buitrago et al, 2005) and expression of SPRY1, ERK1/2, ERK1/2 and G.beta.was assayed as described herein. For morphological and fibrotic analyses, hearts were fixed in 4% formalin and embedded in paraffin. Tissue sections (5 μm) from LV were stained with hematoxylin and eosin or with picorius red. The picrorius red sections were analyzed using a NikonECLIPSE 50i microscope equipped with filters to provide circular polarized illumination. Tissue images were acquired with a 20X objective, recorded with a refrigerated digital camera (DS-5Mc, Nikon) and analyzed using SigmaScan pro5.0 image analysis software (SPSS inc., USA). The collagen content was calculated as a percentage of the area of each image (expressed in pixels).

Alpha MHC-miR-21 transgenic mice

Transgenic mice overexpressing miR-21 are generated by prokaryotic injection of fertilized oocytes of FVB/N mice with a transgenic construct containing the mature miR-21 sequence under the control of the murine alpha-myosin heavy chain (alpha MHC) promoter, flanked by 154bp upstream and 136bp downstream of the native precursor sequence.

X-gal staining of myocardium from Spry-lacZ mice

Hearts were collected from Spry1-lacZ +/-mice, and fixed in PBS containing 2% formaldehyde and 0.05% glutaraldehyde for 2 hours. Subsequently, the heart was placed in a flask containing 0.01% sodium deoxycholate, 0.02% Nonidet P-40(Nonidet P-40), 2mM MgCl₂And 2mM EGTA in PBS for 4 times up to 30 minutes for β -galactosidase activity assay, hearts were placed in PBS containing 0.5mg/ml X-gal, 10mM K₃Fe(CN)₆And 10mM, K₄Fe(CN)₆Is incubated at 37 ℃. For the entire slide analysis, the hearts were transferred to 30% glycerol and digital images were taken with a Nikon digital camera DXM1200F and ACT-1 software. For histological analysis, hearts were dehydrated in isopropanol, cleared in xylene and transferred to paraffin. 10 μm paraffin sections were made using standard protocols and recorded.

Injection and detection of modified oligonucleotides

Jugular vein catheters were permanently inserted into male C57/B16 mice (10-12 weeks old) prior to TAC surgery. The modified oligonucleotide was injected daily for 3 days at 24 hours post TAC via jugular vein catheter at 80 mg/kg/day. The positive control, which was an effective delivery (Cy 3-labeled modified oligonucleotide), was injected into the catheter once at 80mg/kg, and 3 hours later, the heart was removed, fixed, and observed for Cy3 staining by a fluorescence microscope.

Fibroblast apoptosis and FGF2 production

Annexin V positive fibroblasts were measured by FACS analysis (annexin-V-FLUOS kit, Roche Diagnostics GmbH, Mannheim, Germany) after treatment with miR-21 precursors, inhibitors or corresponding controls. Enzyme-linked immunosorbent assay (ELISA) was performed to quantify FGF2 concentration in the supernatant of MiR-21-regulated cardiac fibroblasts. FGF2 was assayed using the Quantikine FGF basic immunoassay kit (R & D SYSTEMS, Minneapolis, USA) according to the manufacturer's instructions.

Statistical analysis

Mean data are expressed as mean ± SEM. Statistical analysis was performed using Prism software (GraphPad, san diego, CA) or StatView (SAS Institute inc., Cary, USA) software packages. ANOVA was applied followed by Bonferroni test and student's t test, as appropriate. Differences were considered significant when P < 0.05, indicated by asterisks.^*P is less than 0.05, and p is less than 0.05,^**p is less than 0.01, and,^***p is < 0.005.

RNA isolation, real-time RT-PCR and northern blotting

For total RNA extraction from frozen tissue or cell cultures, RNeasy mini kit (Qiagen, Hilden, Germany) was used according to the manufacturer's instructions. miRNAs were isolated by TRIZOL (Invitrogen, Karlsruhe, Germany) or miRNA isolation kit (mirVana, Ambion, USA). The integrity of the isolated RNA was confirmed by denaturing agarose gel electrophoresis or capillary electrophoresis (Bioanalyzer 2100; Agilent) as described in the literature (Thum and Borlak, 2004). For real-time PCR, the inventors of the present invention used the iCycleriQTM real-time PCR detection system (BioRad, Germany).

The inventors of the present invention used a target-specific stem-loop structure and a reverse transcription primer, and performed quantitative determination of miR-21 expression after reverse transcription using a specific TaqMan hybridization probe (TaqMan miR-21 microRNA assay, applied biosystems, Foster City, USA). Small RNA molecule U6B micronucleus (RNU6B) was amplified as a control (TaqMan microRNA assay control, applied biosystems, Foster City, USA). All miRNA samples were obtained from isolates containing the same total RNA concentration.

For northern blot analysis, 3 μ g of total RNA was loaded onto 15% acrylamide, 6M urea and TBE gels together with appropriate DNA labels. After electrophoresis, RNA was transferred to a nylon membrane (QiabraneNylon, Qiagen) using a semi-dry transfer apparatus (transfer). The membrane was then prehybridized in hybridization buffer (ULTRAhyb-Oligo hybridization buffer, Ambion, USA) at 65 ℃ for 1 hour. LNA oligonucleotides (mircurY LNA array detection probes; Exiqon), previously labeled with T4 kinase (Exiqon) and 32P-ATP, were then added to the buffer and the membranes were allowed to hybridize overnight at 42 ℃. Subsequently, the blot was washed 3 times for 3 minutes at room temperature (in 0.2 × SSC), followed by one wash at 42 ℃ for 15 minutes. The membrane was then exposed to a phosphoimager (phosphoimager).

Micro RNA expression analysis

For microarray microRNA expression analysis, the inventors of the present invention purified microRNAs separately using a flashPage fractionator system (Ambion, USA). microRNAs obtained from 8. mu.g total RNA were labeled with the dye Cy3(Molecular Probes, Carlsbad, Calif.) using the mirVana microRNA labeling kit (Ambion, USA) according to the manufacturer's instructions. Each sample was hybridized to a different array. The microRNA microarray hybridization, microRNA purification and enrichment, labeling and microarray hybridization steps were performed as described in the Ambion mirVana guidelines (www.ambion.com/techlib/prot /) or as described in literature 2. Data acquisition was performed using ScanAlyze software (Eisen-Lab, Lawrence Berkeley National Lab (LBNL), Berkeley, USA). Alternatively, 5 μ g of total RNA was labeled with Cy 3-conjugated RNA linker (Dharmacon, USA) and ligated to microarray platform for miRNA genome-wide profiling [ miChip; capture probes immobilized on the array corresponded to (211 human, 51 mouse) unique human mirnas deposited in miRbase version 6.1 ] hybridization. Hybridization signal intensities were obtained using an Axon scanner (4000B, Molecular Dynamics) with the same photomultiplier tube setup. Further analysis was performed using Genepix 6(Molecular Dynamics) and Excel software. Specific TaqMan RT-PCR analysis and RNA blot analysis confirmed the expression of miR-21.

Global transcriptome analysis

For transcriptome analysis, reverse transcription, second strand synthesis and clearing of double stranded cDNA (clean) were performed starting from 2 μ g total RNA following the Affymetrix protocol (One-Cycle cDNA synthesis kit, Affymetrix, USA) (n-4 sham post-operative control heart, n-4 TAC and placebo treated left ventricle; n-4 TAC and miR21 antagonist treated left ventricle). Synthesis of biotin-labeled cRNA was carried out using IVT labeling kit (Affymetrix, USA). The cRNA concentration was determined and the distribution of cRNA fragment sizes was examined by gel electrophoresis. Mu.g of fragmented cRNA was used for hybridization on the mouse genome 4302.0 gene chip (Affymetrix, USA). Data analysis of the R software package was studied using the array from the Bioconductor protocol (www.bioconductor.org). Variance stability (variancestationarization) normalizes the resulting signal intensity. All data sets were mass-tested using limma (linear model for microarray analysis) software package and statistically analyzed to select differentially expressed genes.

Heart co-culture experiment and micro RNA/siRNA transfection procedure

Cardiomyocytes from neonatal rats were isolated and cultured as described in the literature (Merckle et al, 2007). For the analysis of the modulation of miR-21 on cardiomyocyte size, pure cardiomyocyte cultures were used by adding a pre-seeding step that excluded contamination of the major non-cardiomyocytes (e.g. fibroblasts). Over 95% of the cultured cardiomyocytes stained positive for actin, indicating high purity of the cell culture. Synthesis of miR (pre negative control #2, Ambion; 50nmol/L, 72 hours), miR-21 precursor molecule (pre-MiR, Ambion; 50nmol/L, 72 hours) or miR-21 antagonist (anti-miR, Ambion; 50nmol/L, 72 hours) was performed by a liposome-based method (Lipofectamine, Invitrogen, USA; see 6 for details). Cardiomyocytes were cultured with low FCS (0.1%, control conditions) or with high FCS (5.0%) for 48 hours to induce cardiomyocyte hypertrophy. For cell size determination, the surface area of neonatal cardiomyocytes (72 hours post-transfection) in 96-well plates (seeding density 40,000 cells/well) was calculated using the AxioVison Rel 4.4 software package (Carl Zeiss GmbH, Jena, Germany). Data are presented as mean ± SEM.

To simulate in vivo cardiac conditions, the inventors of the present invention used a co-culture system of cardiomyocytes and cardiac fibroblasts, omitting the pre-seeding step as described above. Here, the inventors of the present invention studied in detail the expression of Spry-1 and Erk activation after transfection of synthetic miR (50nmol/L, 72 hours), miR-21 precursor molecule (50nmol/L, 72 hours) or miR-21 antagonist (50nmol/L, 72 hours). In separate experiments, synthetic siRNA or a mixture of specific siRNAs against Spry1 (3 different microRNAs, 16.7nmol/L each) were transfected into co-cultures. Transfection efficiency was monitored by real-time PCR measurements (TaqMan microRNA assay, Applied Biosystems) and northern blotting.

Micro RNA target prediction tool

Potential miR-21 binding sites in human and murine genomic 3' UTR sequences were scanned using the MiRanda algorithm (Griffiths-Jones et al, 2006). Subsequently, Karlin-Altschul normalization was performed (miRBase Targets version 4.0; http:// microrna. sanger. ac. uk/Targets/v4 /). In detail, the inventors of the present invention utilized the miRBase database (version 4, Sanger Institute; USA) and applied the algorithm "high number of conserved species; > 6 ")," low p value; < 0.001 "and a high miRBase score; (> 15), potential miR-21 targets were sorted. This step indicates that 22 known genes are possible miR-21 targets, and previous studies have indicated that 8 of them are expressed in cardiac tissue based on their GEO expression profiles (http:// www.ncbi.nlm.nih.gov/GEO /) (see Table 1). Using the PicTar miRNA database 9, 5 genes were additionally predicted to be miR-21 targets (Krek et al, 2005) (http:// PicTar. bio. nyu. edu /). Using the TargetScan miRNA target prediction software (Whitehead Institute for biological Research, USA, version 4.0; 7 months 2007; http:// www.targetscan.org /), the inventors of the present invention determined that only two targets have a target with an 8-mer seed-matched (8-mer seed matrices) conserved site for miR-21. Spry1 was then studied in more detail.

Western blotting method

Protein lysates from transplanted hearts or cultured fibroblasts/cardiomyocytes were prepared as described in the literature (Buitrago et al, 2005). The extract (20-50. mu.g protein/lane) was mixed with the loading buffer and separated on a 10% SDS-polyacrylamide gel under reducing conditions. Electrotransfer of proteins to PVDF membranes (Bio-Rad) bands were detected using a chemiluminescence assay (ECL Plus, Amersham). the inventors of the present invention used a primary antibody directed against sprouty-1(Santa Cruz, sc-30048, dilution 1: 250-1: 500), Erk1/2(CellSignaling, #9102, dilution 1: 1000), Erk1/2(Cell Signaling, #9101, dilution 1: 1000) and G- β (Santa Cruz Biotechnology, sc-378, dilution 1: 1000) and a suitable secondary antibody (anti-mouse HRP, #7076, Cell dilution 1: 10000; anti-rabbit HRP (Cell Signaling, # 74, Cell dilution 1: 10000).

In vivo TAC model and administration of modified oligonucleotides

The inventors used male C57BL/6 mice (10-12 weeks old, 25g) from Charles River Laboratories (Sulzfeld, Germany). Aortic Coarctation (TAC) was performed using a 7-0 suture and a 27 gauge needle wrapped twice around the aorta. The needle was then withdrawn, resulting in approximately 80% narrowing of the aorta. During the same procedure, the jugular catheter is implanted by standard surgical procedures. The sequence is as follows: antagomir-21, 5 '-oUsooCsoAoCoAoUoCoAoCoAoGoUoGoUoAoGsoGsUsoAs-Chol-3'. The nucleotides used for the synthesis were 2 ' -OMe modified (subscript ' o '). Subscript's' represents a phosphorothioate linkage; "Cy 3" represents a Cy3 dye label at the 5' end of the oligonucleotide; "Chol" refers to cholesterol linked by hydroxyprolinol linkages. Treatment was initiated 24 hours after TAC and animals received injections of PBS or miR-21 antagonist via implanted jugular vein catheters (for 3 consecutive days, the jugular vein was injected with PBS or miR-21 antagonist at a dose of 80mg/kg body weight, 0.2 ml/injection). As a positive control, an effective cardiac delivery Cy-3-labeled modified oligonucleotide (antagomir-181a) (80mg/kg) was injected into a jugular vein catheter, and 3 hours later, the heart was removed, fixed, and observed for Cy3 staining by a fluorescence microscope.

Cardiac function analysis

After 3 weeks TAC, mice were anesthetized with isoflurane and heart size and function were analyzed by 15MHz pulsed doppler echocardiography. The hearts were then removed, the atria were removed and weighed and subjected to further analysis. Echocardiography studies were performed with shallow anesthesia with spontaneous breathing using isoflurane. Two independent echocardiographers, experienced in rodent imaging and blinded to the experimental group, performed echocardiographs, running a Toshiba Power Vision 6000 equipped with a 15MHz transducer. Left parasternal short axis view in 2D recorded at papillary muscle level. Correct rotation of the probe is judged by the smooth appearance of the Left Ventricular (LV) cavity after changing the angle and the cranial-caudal transducer movement. The LV end-diastolic area was calculated by manual tracing of the inner border of the heart followed by area measurement using the Nice software package (Toshiba Medical Systems). A cursor was placed in the middle of the LV cavity to record a contemporaneous transverse M-mode trace. The shortening score was calculated as described in the literature (Collins et al, 2001).

Detection of cardiac fibrosis

Mouse hearts were fixed in 4% buffered formalin and embedded in paraffin. 5 μm pimcrospirius red sections were analysed using a Nikon ECLIPSE 50i microscope equipped with filters to provide circular polarized illumination (Whittaker et al, 1994). The lower filter (lower filter) was placed on the microscope field iris aperture ring while the upper filter was constructed from a combination of quarter wave plates placed under a polarizer aligned linearly so that its transmission axis was at 45 ° to the fast axis of the plate. The two filters are crossed, that is to say arranged so that the background in the field of view is as dark as possible. Tissue images were acquired with a 20X objective, recorded with a refrigerated digital camera (DS-5Mc, Nikon), and analyzed using sigmasscanpro 5.0 image analysis software (SPSS inc., USA). The original (circularly polarized) image is each accurately resolved into its cyan, yellow, magenta and black components (using the auto-function CYMK provided by image analysis). The black component is subtracted from the polarized image. Before subtraction, the brightness of the black component was adjusted appropriately to ensure exclusion of interstitial spaces and non-collagenous components rather than the thinnest collagen fibers (confirmed by inspection). And then, carrying out final color separation on the deducted image into components of chroma, saturation and brightness (HSV) by adopting an automatic function HSV provided by image software. A histogram of chrominance frequencies, which contains 256 colors, is obtained from a sharp 8-bit chrominance image. The following chromaticity definitions were used: red 2-9 and 230-256, orange 10-38, yellow 39-51, green 52-128. The chromaticity range 129-. A number of pixels were measured for the chromaticity red, orange, yellow and green ranges, expressed as a percentage of the total number of collagen pixels, which in turn was expressed as a percentage of the total number of pixels in the image.

Fibroblast apoptosis and FGF-2 production

Cardiac fibroblasts obtained by the pre-seeding step during the isolation of cardiac myocytes were cultured until confluent. Cell purity > 95% was determined by staining with fibroblast marker anti-rat prolyl-4-hydroxylase (Acris Antibodies, AF 5110-1; data not shown). Annexin V positive fibroblasts were determined by FACS analysis (annexin-VFLUOS kit, Roche Diagnostics GmbH, Mannheim, Germany) after treatment with miR-21 precursors, inhibitors or corresponding controls (see above). Enzyme-linked immunosorbent assay (ELISA) was performed to quantify FGF-2 concentration in miR-21-regulated cardiac fibroblast supernatant. FGF-2 assays were performed using Quantikine FGF basic immunoassay kit (R & D SYSTEMS, Minneapolis, USA) according to the manufacturer's instructions.

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Claims (70)

1. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the preparation of a medicament for treating fibrosis in a subject having or suspected of having fibrosis, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

2. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the manufacture of a medicament for preventing fibrosis in a subject at risk of developing fibrosis, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

3. The use of claim 1 or 2, wherein the fibrosis is liver fibrosis, heart fibrosis, kidney fibrosis, lung fibrosis, skin fibrosis, age-related fibrosis or spleen fibrosis.

4. The use of claim 1 or 2, wherein the subject has at least one cardiac disease or disorder.

5. The use of claim 4, wherein the heart disease or disorder is selected from the group consisting of cardiac hypertrophy, hypertensive heart failure, diastolic heart failure, systolic heart failure, heart-related storage disease, cardiomyopathy, constrictive pericarditis, coronary artery disease, acute myocardial infarction, chronic myocardial infarction, right heart failure, arrhythmia, myocarditis-related fibrosis, and heart valve disease.

6. The use of claim 5, wherein the cardiomyopathy is selected from the group consisting of dilated cardiomyopathy, obstructive hypertrophic cardiomyopathy, ileus hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and diabetic cardiomyopathy.

7. The use of claim 5, wherein the heart valve disease is selected from the group consisting of mitral valve stenosis, aortic valve stenosis, tricuspid valve stenosis, and pulmonary valve stenosis.

8. The use according to claim 5, wherein the heart valve disease is selected from mitral insufficiency, aortic insufficiency, tricuspid insufficiency and pulmonary insufficiency.

9. The use of claim 1 or 2, wherein the medicament is formulated for intravenous administration, subcutaneous administration, intra-arterial administration, or intracardiac administration.

10. The use of claim 1, wherein the medicament is for improving cardiac weight gain, left ventricular dilation, or fractional shortening reduction.

11. The use of claim 2, wherein the medicament is for preventing cardiac weight gain, left ventricular dilation, or fractional shortening decrease.

12. The use of claim 1 or 2, wherein the medicament is for improving cardiac function.

13. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the preparation of a medicament for treating fibrosis in a subject having or suspected of having fibrosis, wherein the subject has at least one liver disease or disorder and the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

14. The use of claim 13, wherein the at least one liver disease or disorder is chronic liver injury.

15. The use of claim 13, wherein the at least one liver disease or condition is a hepatitis virus infection.

16. The use of claim 15, wherein the hepatitis infection is a hepatitis c infection.

17. The use of claim 13, wherein at least one liver disease or condition is non-alcoholic steatohepatitis.

18. The use of claim 13, wherein the at least one liver disease or condition is cirrhosis.

19. The use of any one of claims 13 and 14-18, wherein the medicament is for intravenous administration or subcutaneous administration.

20. The use of any one of claims 13 and 14-18, wherein the medicament is for improving liver function.

21. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the manufacture of a medicament for treating fibrosis in a subject having or suspected of having fibrosis, wherein the subject has at least one pulmonary disease or disorder, and wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

22. The use of claim 21, wherein the at least one pulmonary disease or condition is chronic obstructive pulmonary disease.

23. The use of claim 21 or 22, wherein the medicament is for pulmonary administration.

24. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the manufacture of a medicament for treating fibrosis in a subject having or suspected of having fibrosis, wherein the subject has at least one other disease or disorder, and wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

25. The use of claim 24, wherein the at least one other disease or condition is pulmonary hypertension.

26. The use of claim 24, wherein the at least one other disease or condition is a vascular-related disease.

27. The use of claim 26, wherein the vascular-related disorder is selected from the group consisting of arterial stiffness, mediaclerosis, and arteriosclerosis.

28. The use of claim 24, wherein the at least one other disease or condition is intestinal sclerosis.

29. The use of claim 24, wherein the at least one other disease or disorder is systemic scleroderma.

30. The use of claim 24, wherein the at least one other disease or condition is selected from retroperitoneal fibrosis, proliferative fibrosis, nephrogenic systemic fibrosis, congestive fibrosis, mediastinal fibrosis, myelofibrosis, post vasectomy pain syndrome, rheumatoid arthritis.

31. The use of any one of claims 24 and 25-30, wherein the medicament is for intravenous administration, subcutaneous administration, intraarterial administration, intracardiac administration, or pulmonary administration.

32. The use of claim 1, 13, 21 or 24, wherein the medicament is for ameliorating fibrosis.

33. The use of claim 1, 13, 21 or 24, wherein the medicament is for slowing further progression of fibrosis.

34. The use of claim 1, 13, 21 or 24, wherein the medicament is for stopping further progression of fibrosis.

35. The use of claim 1, 13, 21 or 24, wherein the medicament is for reducing fibrosis.

36. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the preparation of a medicament for treating a fibroproliferative disorder in a subject having or suspected of having the fibroproliferative disorder, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

37. A method for inhibiting fibroblast proliferation in vitro, comprising contacting fibroblasts with a modified oligonucleotide consisting of 15-24 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21, thereby inhibiting fibroblast proliferation.

38. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the preparation of a medicament for inhibiting fibroblast proliferation in a subject, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

39. A method for stimulating apoptosis in fibroblasts in vitro, comprising contacting fibroblasts with a modified oligonucleotide consisting of 15-24 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21, thereby stimulating apoptosis in fibroblasts.

40. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the manufacture of a medicament for stimulating apoptosis of fibroblasts in a subject, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

41. A method for increasing Sprouty1 protein in fibroblasts in vitro, comprising contacting fibroblasts with a modified oligonucleotide consisting of 15-24 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21, thereby stimulating Sprouty1 protein expression.

42. Use of a modified oligonucleotide consisting of 15-24 linked nucleosides in the preparation of a medicament for increasing Sprouty1 protein in fibroblasts in a subject, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.

43. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein said modified oligonucleotide consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 linked nucleosides.

44. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the nucleobase sequence of the modified oligonucleotide consists of the nucleobase sequence of SEQ ID NO: 12 consists of at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleobases.

45. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the modified oligonucleotide is conjugated to a ligand.

46. The use or method of claim 45, wherein the ligand is cholesterol.

47. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein at least one internucleoside linkage is a modified internucleoside linkage.

48. The use or method of claim 47, wherein each internucleoside linkage is a modified internucleoside linkage.

49. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage.

50. The use or method of claim 49, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.

51. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein at least one nucleoside comprises a modified sugar.

52. The use or method of claim 51, wherein each of the plurality of nucleosides comprises a modified sugar.

53. The use or method of claim 51, wherein each nucleoside comprises a modified sugar.

54. The use or method of claim 53, wherein each nucleoside comprises a 2' -O-methoxyethyl sugar.

55. The use or method of claim 51, wherein each of the plurality of nucleosides comprises a 2 '-O-methoxyethyl sugar and each of the plurality of nucleosides comprises a 2' -fluoro sugar.

56. The use or method of claim 51, wherein each modified sugar is independently selected from a 2 ' -O-methoxyethyl sugar, a 2 ' -fluoro sugar, a 2 ' -O-methyl sugar, or a bicyclic sugar moiety.

57. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein at least one nucleoside comprises a modified nucleobase.

58. The use or method of claim 57, wherein the modified nucleobase is a 5-methylcytosine.

59. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein at least one nucleoside comprises a cytosine, wherein the cytosine is a 5-methylcytosine.

60. The use or method of claim 59, wherein each cytosine is a 5-methylcytosine.

61. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 1 has 100% complementarity.

62. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 1 has full-length complementarity.

63. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the nucleobase sequence of the modified oligonucleotide has a sequence that is complementary to the nucleobase sequence of SEQ ID NO: 11 has 100% complementarity.

64. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein said miR-21 nucleobase sequence consists of the nucleobase sequence of SEQ ID NO: 1 in the sequence listing.

65. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the modified oligonucleotide is formulated as a pharmaceutical composition.

66. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the modified oligonucleotide is a single-stranded modified oligonucleotide.

67. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein said administering reduces collagen content.

68. The use or method of any one of claims 1, 2, 13, 21, 24, and 36-42, wherein the modified oligonucleotide is an antisense oligonucleotide.

69. Use of an antisense oligonucleotide complementary to miR-21 in the manufacture of a medicament for the treatment and/or prevention of fibrosis.

70. A method for screening a pharmaceutically active compound for the treatment and/or prevention of fibrosis or a predisposition therefor, said method comprising the steps of:

a. providing a sample containing miR-21;

b. contacting a candidate substance with a sample;

c. determining the effect of the candidate substance on the sample;

wherein a decrease in miR-21 activity is indicative of a pharmaceutically-active compound.