OA17284A

OA17284A - Methods for treatment of Alport syndrome.

Info

Publication number: OA17284A
Application number: OA1201500104
Authority: OA
Inventors: Jeremy Duffield; Balkrishen Bhat; Deldre MACKENNA
Original assignee: Regulus Therapeutics Inc.
Priority date: 2012-10-09
Filing date: 2013-10-08
Publication date: 2016-04-29

Abstract

Provided herein are methods for the treatment of Alport Syndrome, using modified oligonucleotides targeted to miR-21. In certain embodiments, a modified oligonucleotide targeted to miR-21 improves kidney function and/or reduces fibrosis in subjects having Alport Syndrome. In certain embodiments, administration of a modified oligonucleotide targeted to miR-21 delays the onset of end-stage renal disease in a subject having Alport Syndrome. In certain embodiments, a modified oligonucleotide targeted to miR-21 delays the need for dialysis or kidney transplant in a subject having Alport Syndrome.

Description

Type IV collagen, a major component of the basement membrane, is a family of six alpha chains: alpha-1 collagen (Type IV), alpha-2 collagen (Type IV), alpha-3 collagen (Type IV), alpha-4 collagen (Type IV), alpha-5 collagen (Type IV), and alpha-6 collagen (Type IV). The alpha-3, alpha-4 and alpha-6 chains of collagen IV are fondamental components ofthe collagen network of the glomerular basement membrane (GBM), which performs the critical fonction of filtration of blood by the kidney.

Alport Syndrome is an inherited form of kidney disease in which an abnormal type of glomerular basement membrane (GBM) is produced, leading to interstitial fibrosis, glomerular sclerosis and eventual loss of kidney fonction. The disease is also frequently characterized by hearing defects and ocular anomalies. Alport Syndrome is caused by a mutation in Col4a3, Col4a4, or Col4a5, which encode the alpha3(IV), alpha4(IV), and alpha5(IV) chains of type IV collagen, respectively. Mutations in the Col4a5 gene on the X chromosome cause the X-linked form of Alport Syndrome, which accounts for 85% of ail cases of the disease. An autosomal récessive form is due to inheritance of mutations in each copy of either Col4a3 or Col4a4, each of which is located on chromosome 2. The rare autosomal dominant form is due to inheritance of a dominant-negative mutation in either the Col4a3 or Col4a4 gene. The X-linked form is more severe in males than in females, with most cases in males progressing to end-stage rénal disease (ESRD). The autosomal form is of similar severity in males and females. Most cases of the disease are due to an inherited mutation, but some cases are due to a de novo mutation in one of the Col4aA genes.

SUMMARY OF INVENTION

Provided here are methods for treating Alport Syndrome comprising administering to a subject having or suspected of having Alport Syndrome a modified oligonucleotide consisting of 12 to 25 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21. In certain embodiments, the subject has been diagnosed as having Alport Syndrome prior to administering the modified oligonucleotide, ln certain embodiments, the subject, prior to administration of the modified oligonucleotide, was determined to hâve an increased level of miR-21 in the kidney tissue of the subject. In certain embodiments, the subject, prior to administration of the modified oligonucleotide, was determined to hâve an increased level of miR-21 in the urine or biood of the subject.

In any of the embodiments provided herein, administration of a modified oligonucleotide complementary to miR-21, to a subject having or suspected of having Alport Syndrome, may improve kidney function; delay the onset of end stage rénal disease; delay time to dialysis; delay time to rénal transplant; and/or improve life expectancy in the subject.

In any of the embodiments provided herein, administration of a modified oligonucleotide complementary to miR-21, to a subject having or suspected of having Alport Syndrome may reduce hematuria; delay the onset of hematuria; reduce proteinuria; delay the onset of proteinuria; reduce kidney fibrosis; slow further progression of fibrosis; and/or hait further progression of fibrosis.

ln any of the embodiments provided herein, the subject may hâve a mutation selected from a mutation in the gene encoding the alpha 3 chain of type IV collagen, a mutation in the gene encoding the alpha 4 chain of type IV collagen, or a mutation in the gene encoding the alpha 5 chain of type IV collagen. In certain embodiments, the subject is male, ln certain embodiments, the subject is female. ln certain embodiments, the subject is identified as having hematuria, and/or proteinuria. ln certain embodiments, the subject has reduced kidney function. ln certain embodiments, the subject is in need of improved kidney function.

Any of the embodiments provided herein may comprise measuring biood urea nitrogen in the biood of the subject; measuring créatinine in the biood of the subject; measuring créatinine clearance in the subject; measuring proteinuria in the subject; measuring albumin:creatinine ratio in the subject; and/or measuring glomerular filtration rate in the subject.

Any of the embodiments provided herein may comprise measuring N-acetyl-p-Dglucosaminidase (NAG) protein in the urine of the subject; measuring neutrophil gelatinaseassociated lipocalin (NGAL) protein in the urine of the subject; measuring kidney injury molecule-1 (KIM-1) protein in the urine of the subject; measuring interleukin-18 (IL-18) protein in the urine of the subject; measuring monocyte chemoattractant protein (MCP1) levels in the urine of the subject; measuring connective tissue growth factor (CTGF) levels in the urine of the subject; measuring collagen IV (Col IV) fragments in the urine of the subject; measuring collagen III (Col III) fragments in the urine of the subject; and/or measuring podocyte protein levels in the urine of the subject, wherein the podocyte protein is selected from nephrin and podocin. Any of the embodiments provided herein may comprise measuring cystatin C protein in the blood of a subject; measuring β-trace protein (BTP) in the blood of a subject; and measuring 2-microglobulin (B2M) in the blood of a subject.

Any of the methods provided herein may improve one or more markers of kidney fonction in the subject, selected from reduced blood urea nitrogen in the subject; reduced créatinine in the blood of the subject; improved créatinine clearance in the subject; reduced proteinuria in the subject; reduced albumin:creatinine ratio in the subject; and/or improved glomerular filtration rate in the subject. Any of the methods provided herein may improve one or more markers of kidney fonction in the subject, selected from reduced NAG in the urine of the subject; reduced NGAL in the urine of the subject; reduced KIM-1 in the urine of the subject; reduced IL-18 in the urine of the subject; reduced MCP1 in the urine of the subject; reduced CTGF in the urine of the subject; reduced collagen IV fragments in the urine of the subject; reduced collagen lll fragments in the urine of the subject; and reduced podocyte protein Ievels in the urine of the subject, wherein the podocyte protein is selected from nephrin and podocin. Any of the methods provided herein may improve one or more markers of kidney fonction selected from reduced cystatin C protein in the blood of a subject, reduced β-trace protein (BTP) in the blood of a subject, and reduced 2-microglobulin (B2M) in the blood of a subject.

In any of the embodiments provided herein, the proteinuria is albuminuria. The albuminuria may be high normal albuminuria, microalbuminuria, or macroalbuminuria.

In certain embodiments, the Alport Syndrome is the X-linked form of Alport Syndrome. In certain embodiments, the Alport Syndrome is the autosomal form of Alport Syndrome.

Any of the embodiments provided herein may comprise administering at least one additional therapy selected from an angiotensin II converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), an anti-hypertensive agent, a vitamin D analog, an oral phosphate binder, dialysis, and kidney transplant. In any of these embodiments, the angiotensin Il converting enzyme (ACE) inhibitors is selected from captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril. In any of these embodiments, the angiotensin II receptor blockers (ARB) is selected from candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, and eprosartan. In any of these embodiments, an ACE inhibitor is selected from cilazapril, perindopril, and trandolapril.

In certain embodiments, an ACE inhibitor is administered at a dose ranging from 0.5 to 1 mg/m²/day, from 1 to 6 mg/m²/day, from 1 to 2 mg/m²/day, from 2 to 4 mg/m²/day, or from 4 to 8 mg/m²/day.

In certain embodiments, an ARB is administered at a dose ranging from 6.25 to 150 mg/m2/day. In any of these embodiments, an ARB is administered at a dose of 6.25 mg/m²/day, 10 mg/m²/day, 12.5 mg/m²/day, 18.75 mg/m²/day, 37.5 mg/m²/day, 50 mg/m²/day, or 150 mg/m²/day.

In certain embodiments, the at least one additional therapy is an aldostérone antagonsist. In certain embodiments, an aldostérone antagonist is spironolactone. In certain embodiments, spironolactone is administered at a dose ranging from 10 to 35 mg daily. In certain embodiments, spironolactone is administered at a dose of 25 mg daily.

In any of the embodiments provided herein, the nucleobase sequence of the modified oligonucleotide is at least 90% complementary, is at least 95% complementary, or is 100% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).

In any of the embodiments provided herein, the modified oligonucleotide consists of 8 to 30,12 to 25, or 15 to 25 linked nucleosides. In any of the embodiments provided herein, the modified oligonucleotide consists of 12,13,14,15,16,17,18,19, 20, 21, or 22 linked nucleosides. In any of the embodiments provided herein, the modified oligonucleotide consists of 15,16,17,18,19, 20, 21, or 22 linked nucleosides.

In any of the embodiments provided herein, the modified oligonucleotide comprises at least one modified nucleoside. The modified nucleoside may be selected from an S-cEt nucleoside, a 2’-O-methoxyethyl nucleoside, and an LNA nucleoside. The modified oligonucleotide may comprise at least one modified internucleoside linkage. Each internucleoside linkage of the modified oligonucleotide may be a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

In any of the embodiments provided herein, the modified oligonucleotide may hâve the structure 5’-A_ECsATCsAGTCsTGAUsAAGCsTA_E-3', (SEQ ID NO: 3) where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript Έ indicate 2’-MOE nucleosides: nucleosides followed by a subscript “S” indicate S-cEt nucleosides, and each internucleoside linkage is a phosphorothioate internucleoside linkage.

Provided herein is the use of a modified oligonucleotide consisting of 8 to 30,12 to 25, or 15 to 25 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21, for the treatment of Alport Syndrome.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Anti-miR-21 improves kidney function of Col4a3-/-mice. Blood urea nitrogen (BUN) at 9 weeks (A) and urinary albumin/creatinine ratio at weeks 3, 5, 7 and 9 (B). * indicates statistical significance.

Figure 2. Anti-miR-21 prevents glomerulosclerosis in Col4a3-/- mice. Glomerulosclerosis was evaluated using semi-quantitative sclerosis scores ranging from 0 (no sclerosis) to 4 (complété sclerosis) (n=10).

Figure 3. Anti-miR-21 reduces fibrosis in Col4a3-/- mice. (A) Quantification of Picrosirius (Sirius) red-stained fibrosis in kidney (n=6) and (B) Quantitative PCR of Collai transcripts normalized to GAPDH (n=6). * indicates statistical significance.

Figure 4. Anti-miR-21 reduces kidney injury ranking in Col4a3-/- mice. (A) Kidney injury rank score of rénal sections of 9 week old mice. (B) Proportion of glomerular crescents (n=5). (C) Quantification of tubule injury (n=5). * indicates statistical significance.

Figure 5 Anti-miR-21 reduces macrophage infiltration (A) and decreases myofibroblasts (B) in Col4a3-/- mice. (A) Quantification of F4/80 stain of rénal sections of 9 week old mice (n=5). (B) Quantification of aSMA stain of rénal sections of 9 week old mice (n=5). * indicates statistical significance.

Figure 6. Anti-miR-21 reduces reactive oxygen species in Col4a3-/- mice. (A) Quantification of hydrogen peroxide in urine of Col4a3-/- mice treated with anti-miR-21 or PBS control (n=8; * indicates statistical significance); (B) Quantification of DES in kidney tissue of Col4a3-/- mice treated with anti-miR-21 or PBS control, and in wild type mice (n=3 per group; ‘indicates statistical significance).

Figure 7. Anti-miR-21 improves podocyte number in Col4a3-/- mice. Quantification of number of WT1 -positive cells in the glomerulus of Col4a3-/- mice treated with anti-miR-21 or PBS control (n=3; p=0.005).

Figure 8. Anti-miR-21 increases lifespan of Col4a3-/- mice. (A) Weight of Col4a3-/- mice treated with anti-miR-21 or PBS control (p < 0.01 ); (B) Lifespan of Col4a3-/- mice treated with anti-miR-21 or PBS control (p < 0.001 ).

Figure 9. Anti-miR-21 improves kidney function and increases lifespan of Col4a3-/- mice in a dose-responsive manner (n= 10-13 per treatment group). (A) Blood urea nitrogen at 7 weeks; (B) Lifespan of Col4a3-/- mice treated with anti-miR-21 at multiple doses, once weekly (QW) or twice weekly (BIW), or PBS control.

DETAILED DESCRIPTION

Unless defined otherwise, ail technical and scientific terms used herein hâve the same meaning as is commonly understood by one of skill in the arts to which the invention belongs. Unless spécifie définitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and médicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. In the event that there is a plurality of définitions for terms herein, those in this section prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical préparation, formulation and delivery, and treatment of subjects. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; and Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 18th édition, 1990; and which is hereby incorporated by référencé for any purpose. Where permitted, ail patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are incorporated by référencé in their entirety. Where référencé is made to a URL or other such identifier or address, it is understood that such identifiera can change and particular information on the internet can change, but équivalent information can be found by searching the internet. Référencé thereto évidences the availability and public dissémination of such information.

Before the présent compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the spécification and the appended claims, the singular forms “a,” “an” and “the include plural référents unless the context clearly dictâtes otherwise.

Définitions “Alport Syndrome means an inherited form of kidney disease in which an abnormal level of glomerular basement membrane (GBM) is produced, leading to interstitial fibrosis, glomerular sclerosis and eventual loss of kidney function. The disease is also frequently characterized by hearing defects and ocular anomalies.

“Hematuria” means the presence of red blood cells in the urine.

“Albuminuria” means the presence of excess albumin in the urine, and includes without limitation, normal albuminuria, high normal albuminuria, microalbuminuria and macroalbuminuria. Normally, the glomerular filtration permeability barrier, which is composed of podocyte, glomerular basement membrane and endothélial cells, prevents sérum protein from leaking into urine. Albuminuria may reflect injury of the glomerular filtration permeability barrier. Albuminuria may be calculated from a 24-hour urine sample, an ovemight urine sample or a spot-urine sample.

“High normal albuminuria” means elevated albuminuria characterized by (i) the excrétion of 15 to <30 mg of albumin into the urine per 24 hours and/or (ii) an albumin/creatinine ratio of

1.25 to <2.5 mg/mmol (or 10 to <20 mg/g) in males or 1.75 to <3.5 mg/mmol (or 15 to <30 mg/g) in females.

“Microalbuminuria” means elevated albuminuria characterized by (i) the excrétion of 30 to 300 mg of albumin into the urine per 24 hours and/or (ii) an albumin/creatinine ratio of 2.5 to <25 mg/mmol (or 20 to <200 mg/g) in males or 3.5 to <35 mg/mmol (or 30 to <300 mg/g) in females.

“Macroalbuminuria” means elevated albuminuria characterized by the excrétion of more than 300 mg of albumin into the urine per 24 hours and/or (ii) an albumin/creatinine ratio of >25 mg/mmol (or >200 mg/g) in males or >35 mg/mmol (or >300 mg/g) in females.

“Albumin/creatinine ratio” means the ratio of urine albumin (mg/dL) per urine créatinine (g/dL) and is expressed as mg/g. In certain embodiments, albumin/creatinine ratio may be calculated from a spot-urine sample and may be used as an estimate of albumin excrétion over a 24 hour period.

“Estimated glomerular filtration rate (eGFR) or “glomerular filtration rate (GFR) means a measurement of how well the kidneys are filtering créatinine, and is used as an estimate of how much blood passes through the glomeruli per minute. Normal results may range from 90-120 mL/min/1.73 m². Levels below 60 mL/min/1.73 m² for 3 or more months may be an indicator chronic kidney disease. Levels below 15 mL/min/1.73 m² may be an indicator of kidney failure.

• “Proteinuria” means the presence of an excess of sérum proteins in the urine. Proteinuria may be characterized by the excrétion of > 250 mg of protein into the urine per 24 hours and/or a urine protein to créatinine ratio of £ 0.20 mg/mg. Sérum proteins elevated in association with proteinuria include, without limitation, albumin.

“Blood urea nitrogen” or “BUN means a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea in the urea cycle as a waste product of the digestion of protein, and the urea is removed from the blood by the kidneys. Normal human adult blood may contain between 7 to 21 mg of urea nitrogen per 100 ml (7-21 mg/dL) of blood. Measurement of blood urea nitrogen is used as an indicator of rénal health. If the kidneys are not able to remove urea from the blood normally, a subject’s BUN rises.

“End stage rénal disease (ESRD)” means the complété or almost complété failure of kidney function.

“Impaired kidney function” means reduced kidney function, relative to normal kidney function.

“Fibrosis” means the formation or development of excess fibrous connective tissue in an organ or tissue. In certain embodiments, fibrosis occurs as a reparative or reactive process. In certain embodiments, fibrosis occurs in response to damage or injury. The term “fibrosis is to be understood as the formation or development of excess fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to a formation of fibrous tissue as a normal constituent of an organ or tissue.

“Slows further progression” means to reduce the rate at which a medical condition moves towards an advanced state.

“Halts further progression means to stop progression of a medical condition to an advanced state.

“Delay time to dialysis means to maintain sufficient kidney function such that the need for dialysis treatment is delayed.

“Delay time to rénal transplant” means to maintain sufficient kidney function such that the need for a kidney transplant is delayed.

“Improves life expectancy” means to lengthen the life of a subject by treating one or more symptoms of a disease in the subject.

“Anti-miR means an oligonucleotide having a nucleobase sequence complementary to a microRNA. In certain embodiments, an anti-miR is a modified oligonucleotide.

“Anti-miR-X” where “miR-X” désignâtes a particular microRNA, means an oligonucleotide having a nucleobase sequence complementary to miR-X. In certain embodiments, an anti-miR-X is fully complementary (i.e., 100% complementary) to miR-X. In certain embodiments, an anti-miR-X is at least 80%, at least 85%, at least 90%, or at least 95% complementary to miR-X. In certain embodiments, an anti-miR-X is a modified oligonucleotide.

“miR-21” means the mature miRNA having the nucleobase sequence UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO: 1).

“miR-21 stem-loop sequence means the stem-loop sequence having the nucleobase sequence UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGA UGGGCUGUCUGACA (SEQ ID NO: 2).

“Target nucleic acid means a nucleic acid to which an oligomeric compound is designed to hybridize.

“Targeting” means the process of design and sélection of nucleobase sequence that will hybridize to a target nucleic acid.

“Targeted to” means having a nucleobase sequence that will allow hybridization to a target nucleic acid.

“Modulation means a perturbation of function, amount, or activity. In certain embodiments, modulation means an increase in function, amount, or activity. In certain embodiments, modulation means a decrease in function, amount, or activity.

“Expression” means any functions and steps by which a gene’s coded information is converted into structures présent and operating in a cell.

“Nucleobase sequence” means the order of contiguous nucleobases in an oligomeric compound or nucleic acid, typically listed in a 5’ to 3' orientation, independent of any sugar, linkage, and/or nucleobase modification.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other in a nucleic acid.

“Nucleobase complementarity” means the ability of two nucleobases to pair noncovalently via hydrogen bonding.

“Complementary” means that one nucleic acid is capable of hybridizing to another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide capable of hybridizing to a target nucleic acid.

“Fully complementary means each nucleobase of an oligonucleotide is capable of pairing with a nucleobase at each corresponding position in a target nucleic acid. In certain embodiments, an oligonucleotide is fully complementary to a microRNA, i.e. each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. In certain embodiments, an oligonucleotide wherein each nucleobase has complementarity to a nucleobase within a région of a microRNA stem-loop sequence is fully complementary to the microRNA stem-loop sequence.

“Percent complementarity” means the percentage of nucleobases of an oligonucleotide that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligonucleotide that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

“Percent identity” means the number of nucleobases in a first nucleic acid that are identical to nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid. In certain embodiments, the first nucleic acid is a microRNA and the second nucleic acid is a microRNA. In certain embodiments, the first nucleic acid is an oligonucleotide and the second nucleic acid is an oligonucleotide.

“Hybridize” means the annealing of complementary nucleic acids that occurs through nucleobase complementarity.

“Mismatch means a nucleobase of a first nucleic acid that is not capable of WatsonCrick pairing with a nucleobase at a corresponding position of a second nucleic acid.

“Identical in the context of nucleobase sequences, means having the same nucleobase sequence, independent of sugar, linkage, and/or nucleobase modifications and independent of the methyl state of any pyrimidines présent.

“MicroRNA means an endogenous non-coding RNA between 18 and 25 nucleobases in length, which is the product of deavage of a pre-microRNA by the enzyme Dicer. Examples of mature microRNAs are found in the microRNA database known as miRBase (http://microrna.sanger.ac.uk/). In certain embodiments, microRNA is abbreviated as “microRNA” or“miR.” “Pre-microRNA or “pre-mïR means a non-coding RNA having a hairpin structure, which is the product of deavage of a pri-miR by the double-stranded RNA-specific ribonucléase known as Drosha.

“Stem-loop sequence means an RNA having a hairpin structure and containing a mature microRNA sequence. Pre-microRNA sequences and stem-loop sequences may overlap. Examples of stem-loop sequences are found in the microRNA database known as miRBase (http://microma.sanger.ac.uk/).

“Pri-microRNA or “pri-miR means a non-coding RNA having a hairpin structure that is a substrate for the double-stranded RNA-specific ribonucléase Drosha.

“microRNA precursor means a transcript that originates from a genomic DNA and that comprises a non-coding, structured RNA comprising one or more microRNA sequences. For example, in certain embodiments a microRNA precursor is a pre-microRNA. In certain embodiments, a microRNA precursor is a pri-microRNA.

“microRNA-regulated transcript” means a transcript that is regulated by a microRNA. “Seed sequence” means a nudeobase sequence comprising from 6 to 8 contiguous nucleobases of nucleobases 1 to 9 of the 5'-end of a mature microRNA sequence.

“Seed match sequence” means a nucleobase sequence that is complementary to a seed sequence, and is the same length as the seed sequence.

Oligomeric compound” means a compound that comprises a plurality of linked monomeric subunits. Oligomeric compounds included oligonucleotides.

Oligonucleotide” means a compound comprising a plurality of linked nucleosides, each of which can be modified or unmodified, independent from one another.

“Naturally occurring intemucleoside linkage” means a 3’ to 5’ phosphodiester linkage between nucleosides.

“Natural sugar” means a sugar found in DNA (2’-H) or RNA (2’-OH).

“Internucleoside linkage means a covalent linkage between adjacent nucleosides.

“Linked nucleosides” means nucleosides joined by a covalent linkage.

“Nucleobase” means a heterocyclic moiety capable of non-covalently pairing with another nucleobase.

“Nucleoside” means a nucleobase linked to a sugar moiety.

“Nucléotide means a nucleoside having a phosphate group covalently linked to the sugar portion of a nucleoside.

“Compound comprising a modified oligonucleotide consisting of” a number of linked nucleosides means a compound that includes a modified oligonucleotide having the specified number of linked nucleosides. Thus, the compound may include additional substituents or conjugates. Unless otherwise indicated, the compound does not include any additional nucleosides beyond those of the modified oligonucleotide.

“Modified oligonucleotide” means an oligonucleotide having one or more modifications relative to a naturally occurring terminus, sugar, nucleobase, and/or internucleoside linkage. A modified oligonucleotide may comprise unmodified nucleosides.

“Single-stranded modified oligonucleotide” means a modified oligonucleotide which is not hybridized to a complementary strand.

“Modified nucleoside” means a nucleoside having any change from a naturally occurring nucleoside. A modified nucleoside may hâve a modified sugar and an unmodified nucleobase. A modified nucleoside may hâve a modified sugar and a modified nucleobase. A modified nucleoside may hâve a natural sugar and a modified nucleobase. In certain embodiments, a modified nucleoside is a bicyclîc nucleoside. In certain embodiments, a modified nucleoside is a non-bicyclic nucleoside.

“Modified internucleoside linkage” means any change from a naturally occurring intemucleoside linkage.

“Phosphorothioate internucleoside linkage” means a linkage between nucleosides where one of the non-bridging atoms is a sulfur atom.

“Modified sugar moiety” means substitution and/or any change from a natural sugar.

“Unmodified nucleobase means the naturally occurring heterocyclic bases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

“5-methylcytosine” means a cytosine comprising a methyl group attached to the 5 position.

“Non-methylated cytosine” means a cytosine that does not hâve a methyl group attached to the 5 position.

“Modified nucleobase” means any nucleobase that is not an unmodified nucleobase. “Sugar moiety” means a naturally occurring furanosyl or a modified sugar moiety. “Modified sugar moiety” means a substituted sugar moiety or a sugar surrogate. “2-O-methyl sugar” or“2’-OMe sugar” means a sugar having a O-methyl modification at the 2’ position.

“2'-O-methoxyethyl sugar” or “2'-MOE sugar” means a sugar having a O-methoxyethyl modification at the 2’ position.

“2’-fluoro” or “2’-F” means a sugar having a fluoro modification of the 2’ position.

“Bicydic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including by not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicydic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2’-carbon and the 4’-carbon of the furanosyl. Nonlimiting exemplary bicydic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

“Locked nucleic acid (LNA) sugar moiety” means a substituted sugar moiety comprising a (CH₂)-0 bridge between the 4’ and 2’ furanose ring atoms.

“ENA sugar moiety” means a substituted sugar moiety comprising a (CH₂)₂-O bridge between the 4' and 2’ furanose ring atoms.

“Constrained ethyl (cEt) sugar moiety” means a substituted sugar moiety comprising a CH(CH₃)-0 bridge between the 4' and the 2' furanose ring atoms. In certain embodiments, the CH(CH₃)-0 bridge is constrained in the S orientation. In certain embodiments, the (CH₂)₂-O is constrained in the R orientation.

“S-cEt sugar moiety” means a substituted sugar moiety comprising an S-constrained CH(CH₃)-0 bridge between the 4' and the 2' furanose ring atoms.

“R-cEt sugar moiety” means a substituted sugar moiety comprising an R-constrained CH(CH₃)-0 bridge between the 4' and the 2' furanose ring atoms.

“2’-O-methyl nucleoside” means a 2’-modified nucleoside having a 2’-O-methyl sugar modification.

“2’-O-methoxyethyl nucleoside” means a 2’-modified nucleoside having a 2'-Omethoxyethyl sugar modification. A 2’-O-methoxyethyl nucleoside may comprise a modified or unmodified nucleobase.

“2’-fluoro nucleoside” means a 2’-modified nucleoside having a 2’-fluoro sugar modification. A 2'-fluoro nucleoside may comprise a modified or unmodified nucleobase.

“Bicyclic nucleoside” means a 2'-modified nucleoside having a bicyclic sugar moiety. A bicyclic nucleoside may hâve a modified or unmodified nucleobase.

“cEt nucleoside” means a nucleoside comprising a cEt sugar moiety. A cEt nucleoside may comprise a modified or unmodified nucleobase.

“S-cEt nucleoside” means a nucleoside comprising an S-cEt sugar moiety.

“R-cEt nucleoside” means a nucleoside comprising an R-cEt sugar moiety.

“β-D-deoxyribonucleoside” means a naturally occurring DNA nucleoside.

“β-D-ribonudeoside” means a naturally occurring RNA nucleoside.

“LNA nucleoside” means a nucleoside comprising a LNA sugar moiety.

“ENA nucleoside” means a nucleoside comprising an ENA sugar moiety.

“Subject means a human or non-human animal selected for treatment or therapy.

“Subject in need thereof means a subject that is identified as in need of a therapy or treatment.

“Subject suspected of having” means a subject exhibiting one or more clinical indicators of a disease.

“Administering” means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and selfadministering.

“Parentéral administration” means administration through injection or infusion. Parentéral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

“Subcutaneous administration means administration just below the skin.

“Intravenous administration” means administration into a vein.

“Administered concomitantly” refers to the co-administration of two or more agents in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Concomitant administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents need not manifest themselves at the same time. The effects need only be overlapping for a period of time and need not be coextensive.

“Duration” means the period of time during which an activity or event continues. In certain embodiments, the duration of treatment is the period of time during which doses of a pharmaceutical agent or pharmaceutical composition are administered.

“Therapy” means a disease treatment method. In certain embodiments, therapy includes, but is not limited to, chemotherapy, radiation therapy, or administration of a pharmaceutical agent.

“Treatment” means the application of one or more spécifie procedures used for the cure or amelioration of a disease. In certain embodiments, the spécifie procedure is the administration of one or more pharmaceutical agents.

“Ameliorate” means to lessen the severity of at least one indicator of a condition or disease. In certain embodiments, amelioration includes a delay or slowing in the progression of one or more indicators of a condition or disease. The severity of indicators may be determined by subjective or objective measures which are known to those skilled in the art.

“At risk for developing” means the state in which a subject is predisposed to developing a condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed with the condition or disease. In certain embodiments, a subject at risk for developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser extent required to be diagnosed with the condition or disease.

“Prevent the onset of” means to prevent the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

“Delay the onset of” means to delay the development of a condition or disease in a subject who is at risk for developing the disease or condition. In certain embodiments, a subject at risk for developing the disease or condition receives treatment similar to the treatment received by a subject who already has the disease or condition.

“Therapeutic agent means a pharmaceutical agent used for the cure, amelioration or prévention of a disease.

“Dose means a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual. In certain embodiments, a dose is administered as a slow infusion.

“Dosage unit means a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial containing lyophilized oligonucleotide. In certain embodiments, a dosage unit is a vial containing reconstituted oligonucleotide.

“Therapeutically effective amount refers to an amount of a pharmaceutical agent that provides a therapeutic benefit to an animal.

“Pharmaceutical composition” means a mixture of substances suitable for administering to an individual that includes a pharmaceutical agent. For example, a pharmaceutical composition may comprise a stérile aqueous solution.

“Pharmaceutical agent means a substance that provides a therapeutic effect when administered to a subject.

“Active pharmaceutical ingrédient means the substance in a pharmaceutical composition that provides a desired effect.

“Improved organ function means a change in organ fonction toward normal limits. In certain embodiments, organ function is assessed by measuring molécules found in a subject's blood or urine. For example, in certain embodiments, improved kidney function is measured by a réduction in blood urea nitrogen, a réduction in proteinuria, a réduction in albuminuria, etc.

“Acceptable safety profile” means a pattern of side effects that is within clinically acceptable limits.

“Side effect” means a physiological response attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, kidney function abnormalities, liver toxicity, rénal toxicity, central nervous system abnormalities, and myopathies. Such side effects may be detected directly or indirectly. For example, increased aminotransferase levels in sérum may indicate liver toxicity or liver function abnormality. For example, increased bilirubin may indicate liver toxicity or liver function abnormality.

“Subject compliance means adhérence to a recommended or prescribed therapy by a subject.

“Comply means the adhérence with a recommended therapy by a subject.

“Recommended therapy” means a treatment recommended by a medical professional for the treatment, amelioration, or prévention of a disease.

The term “blood as used herein, encompasses whole blood and blood fractions, such as sérum and plasma.

Overview

Alport Syndrome is an inherited form of kidney disease in which an abnormal level of glomerular basement membrane (GBM) is produced, leading to interstitial fibrosis, glomerular sclerosis and typically leads to end-stage rénal disease. In the management of Alport Syndrome, the primary goal for treatment is to maintain kidney function and prevent the onset of end-stage rénal disease (ESRD), which in turn improves life expectancy of subjects with Alport Syndrome.

Alport Syndrome is characterized by progressive fibrosis due to defects in GBM composition, thus improvements in GBM morphology and function are désirable. It is demonstrated herein that modified oligonucleotide targeted to miR-21 improves kidney function in an experimental model of Alport Syndrome. Additionally, glomerular sclerosis and fibrosis are reduced following anti-miR-21 treatment. It is further demonstrated herein that anti-miR-21 improves survival in an experimental model of Alport Syndrome. As such, these modified oligonucleotides targeted to miR-21 are useful for the treatment of Alport Syndrome.

Certain Uses ofthe Invention

Provided herein are methods for the treatment of Alport Syndrome, comprising administering to a subject having or suspected of having Alport Syndrome a modified oligonucleotide complementary to miR-21.

In certain embodiments, the subject has been diagnosed as having Alport Syndrome prior to administration of the modified oligonucleotide. Diagnosis of Alport Syndrome may be achieved through évaluation of parameters including, without limitation, a subject’s family history, clinical features (including without limitation proteinuria, albuminuria, hematuria, impaired GFR, deafness and/or ocular changes) and results of tissue biopsies. Kidney biopsies may be tested for the presence or absence of the type IV collagen alpha-3, alpha-4, and alpha-5 chains. Additionally, structural changes in the glomerulus can be detected by électron microscopy of kidney biopsy material. A skin biopsy may be tested for the presence of the type IV collagen alpha-5 chain, which is normally présent in skin and almost always absent from male subjects with the X-linked form of Alport Syndrome. Diagnosis of Alport Syndrome may also include screening for mutations in one or more of the Col4a3, Col4a4, or Col4a5 genes.

In certain embodiments, levels of miR-21 are increased in the kidney of a subject having Alport Syndrome. In certain embodiments, prior to administration, a subject is determined to hâve an increased level of miR-21 in the kidney. miR-21 levels may be measured from kidney biopsy material. In certain embodiments, prior to administration, a subject is determined to hâve an increased level of miR-21 in the urine or blood of the subject.

In certain embodiments, administration of a modified oligonucleotide complementary to miR-21 results in one or more clinically bénéficiai outcomes. In certain embodiments, the administration improves kidney function. In certain embodiments, the administration delays the onset of end-stage rénal disease. In certain embodiments, the administration delays time to dialysis. In certain embodiments, the administration delays time to rénal transplant. In certain embodiments, the administration improves life expectancy of the subject.

In certain embodiments, the administering reduces kidney fibrosis. In certain embodiments the administering slows further progression of kidney fibrosis. In certain embodiments, the administration halts further progression of kidney fibrosis. In certain embodiments, the administration reduces hematuria. In certain embodiments, the administration delays the onset of hematuria. In certain embodiments, the administration reduces proteinuria. In certain embodiments, the administration delays the onset of proteinuria.

The subject having or suspected of having Alport Syndrome may hâve a mutation in the gene encoding the alpha 3 chain of type IV collagen (Col4a3), a mutation in the gene encoding the alpha 4 chain of type IV collagen (Col4a4), or a mutation in the gene encoding the alpha 5 chain of type IV collagen (Col4a5). In certain embodiments, the subject is male. In certain embodiments, the subject is female.

In certain embodiments the subject has impaired kidney function. In certain embodiments, the subject is in need of improved kidney function. In certain embodiments, the subject is identified as having impaired kidney function. In certain embodiments, the subject is identified as having hematuria. In certain embodiments, the subject is identified as having proteinuria.

In any of the embodiments provided herein, a subject may be subjected to certain tests to evaluate kidney function. Such tests include, without limitation, measurement of blood urea nitrogen in the subject; measuring créatinine in the blood of the subject; measuring créatinine clearance in the blood of the subject; measuring proteinuria in the subject; measuring albumin:creatinine ratio in the subject; measuring glomerular filtration rate in the subject; and measuring urinary output in the subject.

In any of the embodiments provided herein, proteins présent in the urine or blood may be used to evaluate kidney fonction. Such tests of kidney fonction include, but are not limited to, measuring N-acetyl-p-D-glucosaminidase (NAG) protein in the urine of the subject; measuring neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject; measuring kidney injury molecule-1 (KIM-1) protein in the urine of the subject; measuring interieukin-18 (IL18) protein in the urine of the subject; measuring connective tissue growth factor (CTGF) levels in the urine of the subject; measuring monocyte chemoattractant protein 1 (MCP1) levels in the urine of the subject; measuring collagen IV (Col IV) fragments in the urine of the subject; measuring collagen III (Col III) fragment levels in the urine of the subject; measuring cystatin C protein in the blood of a subject; measuring β-trace protein (BTP) in the blood of a subject; and measuring 2-microglobulin (B2M) in the blood of a subject. In any of the embodiments provided herein, markers of podocyte injury can be measuring in the urine. Such proteins include nephrin and podocin. The proteins may be quantitated, for example, by enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA) using commercially available kits.

In any of the embodiments provided herein, the administration of a modified oligonucleotide targeted to miR-21 improves one or more markers of kidney fonction in the subject. Improvements in markers of kidney fonction include, without limitation: reduced blood urea nitrogen in the subject; reduced créatinine in the blood of the subject; improved créatinine clearance in the subject; reduced proteinuria in the subject; reduced albuminxreatinine ratio in the subject; improved glomerular filtration rate in the subject; and/or increased urinary output in the subject.

Certain Additional Thérapies

Treatments for Alport Syndrome or any of the conditions listed herein may comprise more than one therapy. As such, in certain embodiments provided herein are methods for treating a subject having or suspected of having Alport Syndrome comprising administering at least one therapy in addition to administering a modified oligonucleotide having a nucleobase sequence complementary to a miR-21.

In certain embodiments, the at least one additional therapy comprises a pharmaceutical agent.

ln certain embodiments, pharmaceutical agents include angiotensin II receptor blockers (ARB). ln certain embodiments, an angiotensin II receptor blocker is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, or eprosartan.

ln certain embodiments, pharmaceutical agents include angiotensin II converting enzyme (ACE) inhibitors. ln certain embodiments, an ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, or ramipril.

ln certain embodiments, a pharmaceutical agent is an anti-hypertensive agent. Antihypertensive agents are used to control biood pressure of the subject.

ln certain embodiments, a pharmaceutical agent is a vitamin D analog. Vitamin D analogs may be used to limit the production of parathyroid hormone in the subject.

ln certain embodiments, a pharmaceutical agent is an oral phosphate binderthat reduces dietary phosphate absorption.

ln certain embodiments, pharmaceutical agents include immunosuppressive agents, ln certain embodiments, an immunosuppressive agent is a corticosteroid, cyclophosphamide, or mycophenolate mofetil.

ln certain embodiments, a pharmaceutical agent is cyclosporine, an HMG-Coenzyme A inhibitor, a vasopeptidase inhibitor, or a TGF-beta-antagonist.

In certain embodiments, an additional therapy is gene therapy. In certain embodiments, the gene therapy provides a normal Col4a3 gene. In certain embodiments, the gene therapy provides a normal Col4a4 gene. ln certain embodiments, the gene therapy provides a normal Col4a5 gene.

ln certain embodiments, an additional therapy is dialysis. ln certain embodiments, an additional therapy is rénal transplant.

ln certain embodiments, pharmaceutical agents include anti-inflammatory agents. In certain embodiments, an anti-inflammatory agent is a steroidal anti-inflammatory agent, ln certain embodiments, a steroid anti-inflammatory agent is a corticosteroid. ln certain embodiments, a corticosteroid is prednisone, ln certain embodiments, an anti-inflammatory agent is a non-steroidal anti-inflammatory drug. ln certain embodiments, a non-steroidal antiinflammatory agent is ibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

ln certain embodiments, a pharmaceutical agent is a pharmaceutical agent that blocks one or more responses to fibrogenic signais.

ln certain embodiments, pharmaceutical agents include anti-diabetic agent. Antidiabetic agents include, but are not limited to, biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones.

Certain MicroRNA Nucleobase Sequences

The modified oligonucleotides described herein hâve a nucleobase sequence that is complementary to miR-21 (SEQ ID NO: 1), or a precursor thereof (SEQ ID NO: 2). In certain embodiments, each nucleobase of the modified oligonucleotide is capable of undergoing basepairing with a nucleobase at each corresponding position in the nucleobase sequence of miR21, or a precursor thereof. In certain embodiments the nucleobase sequence of a modified oligonucleotide may hâve one or more mismatched base pairs with respect to the nucleobase sequence of miR-21 or precursor sequence, and remains capable of hybridizing to its target sequence.

As the miR-21 sequence is contained within the miR-21 precursor sequence, a modified oligonucleotide having a nucleobase sequence complementary to miR-21 is also complementary to a région of the miR-21 precursor.

In certain embodiments, a modified oligonucleotide consists of a number of linked nucleosides that is equai to the length of miR-21.

In certain embodiments, the number of linked nucleosides of a modified oligonucleotide is less than the length of miR-21. A modified oligonucleotide having a number of linked nucleosides that is less than the length of miR-21, wherein each nucleobase ofthe modified oligonucleotide is complementary to each nucleobase at a corresponding position of miR-21, is considered to be a modified oligonucleotide having a nucleobase sequence that is fully complementary to a région ofthe miR-21 sequence. For example, a modified oligonucleotide consisting of 19 linked nucleosides, where each nucleobase is complementary to a corresponding position of miR-21 that is 22 nucleobases in length, is fully complementary to a 19 nucleobase région of miR-21. Such a modified oligonucleotide has 100% complementarity to a 19 nucleobase portion of miR-21, and is considered to be 100% complementary to miR-21.

In certain embodiments, a modified oligonucleotide comprises a nucleobase sequence that is complementary to a seed sequence, i.e. a modified oligonucleotide comprises a seedmatch sequence. In certain embodiments, a seed sequence is a hexamer seed sequence. In certain such embodiments, a seed sequence is nucleobases 1-6 of miR-21. In certain such embodiments, a seed sequence is nucleobases 2-7 of miR-21. In certain such embodiments, a seed sequence is nucleobases 3-8 of miR-21. In certain embodiments, a seed sequence is a heptamer seed sequence. In certain such embodiments, a heptamer seed sequence is nucleobases 1-7 of miR-21. In certain such embodiments, a heptamer seed sequence is nucleobases 2-8 of miR-21. In certain embodiments, the seed sequence is an octamer seed sequence. In certain such embodiments, an octamerseed sequence is nucleobases 1-8 of miR-

21. In certain embodiments, an octamer seed sequence is nucleobases 2-9 of miR-21.

In certain embodiments, a modified oligonucleotide has a nucleobase sequence having one mismatch with respect to the nucleobase sequence of miR-21, or a precursor thereof. In certain embodiments, a modified oligonucleotide has a nucleobase sequence having two mismatches with respect to the nucleobase sequence of miR-21, or a precursor thereof. In certain such embodiments, a modified oligonucleotide has a nucleobase sequence having no more than two mismatches with respect to the nucleobase sequence of miR-21, or a precursor thereof. In certain such embodiments, the mismatched nucleobases are contiguous. In certain such embodiments, the mismatched nucleobases are not contiguous.

In certain embodiments, the number of Iinked nucleosides of a modified oligonucleotide is greater than the length of miR-21. In certain such embodiments, the nucleobase of an additional nucleoside is complementary to a nucleobase of the miR-21 stem-loop sequence. In certain embodiments, the number of Iinked nucleosides of a modified oligonucleotide is one greater than the length of miR-21. In certain such embodiments, the additional nucleoside is at the 5’ terminus of an oligonucleotide. In certain such embodiments, the additional nucleoside is at the 3’ terminus of an oligonucleotide. In certain embodiments, the number of Iinked nucleosides of a modified oligonucleotide is two greater than the length of miR-21. In certain such embodiments, the two additional nucleosides are at the 5’ terminus of an oligonucleotide. In certain such embodiments, the two additional nucleosides are at the 3’ terminus of an oligonucleotide. In certain such embodiments, one additional nucleoside is located at the 5’ terminus and one additional nucleoside is located at the 3’ terminus of an oligonucleotide. In certain embodiments, a région ofthe oligonucleotide may be fully complementary to the nucleobase sequence of miR-21, but the entire modified oligonucleotide is not fully complementary to miR-21. For example, a modified oligonucleotide consisting of 24 Iinked nucleosides, where the nucleobases of nucleosides 1 through 22 are each complementary to a corresponding position of miR-21 that is 22 nucleobases in length, has a 22 nucleoside portion that is fully complementary to the nucleobase sequence of miR-21 and approximately 92% overall complementarity to the nucleobase sequence of miR-21.

Certain Modified Oligonucleotides

In certain embodiments, a modified oligonucleotide has the structure 5’AeCsATCsAGTCsTGAU_sAAGCsTAe-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’21

MOE nucleosides; nucleosides followed by a subscript “S indicate S-cEt nucleosides; and each internucleoside linkage is a phosphorothioate intemucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure 5’AeCsATCsAsGTCsUsGAUsAsAGCsUsAe-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’-MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt nucleosides; and each internucleoside linkage is a phosphorothioate intemucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure 5’^M0CeAsAsT_eCsUsA_eAeUsA_sA_eGeCsTeAs-3’ (SEQ ID NO: 4), where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt nucleosides; a superscript “Me indicates a 5-methyl group on the base ofthe nucleoside; and each internucleoside linkage is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide has the structure 5’A_eC_sA_eT_eCsA_eG_eT_eCsTGAUsAAGCsUsA_s-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’-MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt nucleosides; and each internucleoside linkage is a phosphorothioate intemucleoside linkage.

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

In certain embodiments, a modified oligonucleotide consists of 8 to 30 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 to 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 30 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 25 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 19 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 to 16 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 19 to 24 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 21 to 24 linked nucleosides.

In certain embodiments, a modified oligonucleotide consists of 8 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 9 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 10 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 11 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 12 linked nucleosides. in certain embodiments, a modified oligonucleotide consists of 13 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 14 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 15 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 16 linked nucleosides. in certain embodiments, a modified oligonucleotide consists of 17 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 18 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 19 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 20 linked nucleosides. in certain embodiments, a modified oligonucleotide consists of 21 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 22 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 23 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 24 linked nucleosides. In certain embodiments, a modified oligonucleotide consists of 25 linked nucleosides.

Certain Modifications

In certain embodiments, oligonucleotides provided herein may comprise one or more modifications to a nucleobase, sugar, and/or intemucleoside linkage, and as such is a modified oligonucleotide. A modified nucleobase, sugar, and/or intemucleoside linkage may be selected over an unmodified form because of désirable properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets and increased stability in the presence of nucleases.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides. In certain such embodiments, a modified nucleoside is a stabilizing nucleoside. An example of a stabilizing nucleoside is a sugar-modified nucleoside.

In certain embodiments, a modified nucleoside is a sugar-modified nucleoside. in certain such embodiments, the sugar-modified nucleosides can further comprise a natural or modified heterocyclic base moiety and/or a natural or modified intemucleoside linkage and may inciude further modifications independent from the sugar modification. In certain embodiments, a sugar modified nucleoside is a 2’-modified nucleoside, wherein the sugar ring is modified at the 2’ carbon from natural ribose or 2’-deoxy-ribose.

In certain embodiments, a 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.

Nucleosides comprising such bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4’-CH₂-O-2’) BNA; (B) β-D-Methyleneoxy (4*-CH₂-O-2·) BNA; (C) Ethyleneoxy (4’-(CH₂)₂-O-2’) BNA; (D) Aminooxy (4’-CH₂-O-N(R)-2’) BNA; (E) Oxyamino (4’CH₂-N(R)-O-2’) BNA; (F) Methyl(methyleneoxy) (4'-CH(CH₃)-O-2’) BNA (also referred to as constrained ethyl or cEt); (G) methylene-thio (4’-CH2-S-2’) BNA; (H) methylene-amino (4’-CH2N(R)-2 ) BNA; (I) methyl carbocyclic (4’-CH₂-CH(CH₃)-2’) BNA; (J) c-MOE (4'-CH₂-OMe-2’) BNA 10 and (K) propylene carbocyclic (4’-(CH₂)₃-2’) BNA as depicted below.

wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or ΟνΟ₁₂alkyl.

In certain embodiments, a 2’-modified nucleoside comprises a 2’-substituent group selected from F, OCF₃, O-CH₃, OCH₂CH₂OCH₃, 2’-O(CH₂)₂SCH₃, O-(CH₂)₂-ON(CH₃)₂, -O(CH₂)₂O(CH₂)₂N(CH₃)_2i and O-CH₂-C(=O)-N(H)CH₃.

In certain embodiments, a 2'-modified nucleoside comprises a 2'-substituent group selected from F, O-CH₃, and OCH₂CH₂OCH₃.

In certain embodiments, a sugar-modified nucleoside is a 4’-thio modified nucleoside. In certain embodiments, a sugar-modified nucleoside is a 4’-thio-2’-modified nucleoside. A 4’-thio modified nucleoside has a β-D-ribonucleoside where the 4'-0 replaced with 4'-S. A 4’-thio-2‘modified nucleoside is a 4’-thïo modified nucleoside having the 2’-OH replaced with a 2'substituent group. Suitable 2’-substituent groups include 2'-OCH₃, 2’-O-(CH₂)₂-OCH₃, and 2'-F.

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

In certain embodiments, a modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of a modified oligonucleotide is a phosphorothioate internucleoside linkage.

In certain embodiments, a modified oligonucleotide comprises one or more modified nucleobases. In certain embodiments, a modified nucleobase is selected from 5-hydroxymethyl cytosine, 7-deazaguanine and 7-deazaadenine. In certain embodiments, a modified nucleobase is selected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. In certain embodiments, a modified nucleobase is selected from 5-substituted pyrimidines, 6azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5propynyluracil and 5-propynylcytosine.

In certain embodiments, a modified nucleobase comprises a polycyclic heterocycle. In certain embodiments, a modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, a modified nucleobase comprises a phenoxazine dérivative. In certain embodiments, the phenoxazine can be further modified to form a nucleobase known in the art as a G-clamp.

In certain embodiments, a modified oligonucleotide is conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. In certain such embodiments, the moiety is a cholestérol moiety. In certain embodiments, the moiety is a lipid moiety. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, the carbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In certain embodiments, a conjugate group is attached directly to an oligonucleotide. In certain embodiments, a conjugate group is attached to a modified oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), 8-amino-3,6dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxyiate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises a modified oligonucleotide having one or more stabilizing groups that are attached to one or both termini of a modified oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect a modified oligonucleotide from exonuclease dégradation, and can help in delivery and/or localization within a cell. The cap can be présent at the 5'-terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be présent on both termini. Cap structures include, for example, inverted deoxy abasic caps.

Certain Pharmaceutical Compositions

Provided herein are pharmaceutical compositions comprising oligonucleotides. In certain embodiments, a pharmaceutical composition provided herein comprises a compound comprising a modified oligonucleotide consisting of 15 to 25 linked nucleosides and having a nucleobase sequence complementary to miR-21. In certain embodiments, a pharmaceutical composition provided herein comprises a compound consisting of a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence complementary to miR-21. In certain embodiments, a pharmaceutical composition provided herein comprises a compound comprising a modified oligonucleotide consisting of 12 to 25 linked nucleosides and having a nucleobase sequence complementary to miR-21.

Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enterai, topical, suppository, through inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parentéral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the kidney).

In certain embodiments, a pharmaceutical composition is administered in the form of a dosage unit (e.g., tablet, capsule, bolus, etc.). In some embodiments, a pharmaceutical compositions comprises a modified oligonucleotide at a dose within a range selected from 25 mg to 800 mg, 25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800 mg, 100 mg to 700 mg, 150 mg to 650 mg, 200 mg to 600 mg, 250 mg to 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. In certain embodiments, such pharmaceutical compositions comprise a modified oligonucleotide in a dose selected from 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg, 140 mg, 145 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 270 mg, 270 mg, 280 mg, 285 mg, 290 mg, 295 mg, 300 mg, 305 mg, 310 mg, 315 mg, 320 mg, 325 mg, 330 mg, 335 mg, 340 mg, 345 mg, 350 mg, 355 mg, 360 mg, 365 mg, 370 mg, 375 mg, 380 mg, 385 mg, 390 mg, 395 mg, 400 mg, 405 mg, 410 mg, 415 mg, 420 mg, 425 mg, 430 mg, 435 mg, 440 mg, 445 mg, 450 mg, 455 mg, 460 mg, 465 mg, 470 mg, 475 mg, 480 mg, 485 mg, 490 mg, 495 mg, 500 mg, 505 mg, 510 mg, 515 mg, 520 mg, 525 mg, 530 mg, 535 mg, 540 mg, 545 mg, 550 mg, 555 mg, 560 mg, 565 mg, 570 mg, 575 mg, 580 mg, 585 mg, 590 mg, 595 mg, 600 mg, 605 mg, 610 mg, 615 mg, 620 mg, 625 mg, 630 mg, 635 mg, 640 mg, 645 mg, 650 mg, 655 mg, 660 mg, 665 mg, 670 mg, 675 mg, 680 mg, 685 mg, 690 mg, 695 mg, 700 mg, 705 mg, 710 mg, 715 mg, 720 mg, 725 mg, 730 mg, 735 mg, 740 mg, 745 mg, 750 mg, 755 mg, 760 mg, 765 mg, 770 mg, 775 mg, 780 mg, 785 mg, 790 mg, 795 mg, and 800 mg. In certain such embodiments, a pharmaceutical composition ofthe comprises a dose of modified oligonucleotide selected from 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 500 mg, 600 mg, 700 mg, and 800mg.

In certain embodiments, a pharmaceutical agent is stérile lyophilized modified oligonucleotide that is reconstituted with a suîtable diluent, e.g., stérile water for injection or stérile saline for injection. The reconstituted product is administered as a subcutaneous injection or as an intravenous infusion after dilution into saline. The lyophilized drug product consists of a modified oligonucleotide which has been prepared in water for injection, or in saline for injection, adjusted to pH 7.0-9.0 with acid or base during préparation, and then lyophilized. The lyophilized modified oligonucleotide may be 25-800 mg of an oligonucleotide. It is understood that this encompasses 25, 50, 75,100,125, 150,175, 200, 225, 250, 275, 300, 325, 350, 375, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, and 800 mg of modified lyophilized oligonucleotide. Further, in some embodiments, the lyophilized modified oligonucleotide is an amount of an oligonucleotide within a range selected from 25 mg to 800 mg, 25 mg to 700 mg, 25 mg to 600 mg, 25 mg to 500 mg, 25 mg to 400 mg, 25 mg to 300 mg, 25 mg to 200 mg, 25 mg to 100 mg, 100 mg to 800 mg, 200 mg to 800 mg, 300 mg to 800 mg, 400 mg to 800 mg, 500 mg to 800 mg, 600 mg to 800 mg, 100 mg to 700 mg,_150 mgto.650 mg, 200 mg to 600 mg, 250 mg to 550 mg, 300 mg to 500 mg, 300 mg to 400 mg, and 400 mg to 600 mg. The lyophilized drug product may be packaged in a 2 mL Type I, clear glass vial (ammonium sulfate-treated), stoppered with a bromobutyl rubber closure and sealed with an aluminum FLIP-OFF® overseal.

In certain embodiments, the pharmaceutical compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials usefui in physicallyformulating various dosage forms ofthe compositions of the présent invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfère with the biological activities ofthe components ofthe compositions ofthe présent invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the oligonucleotide(s) of the formulation.

Lipid moieties hâve been used in nucleic acid thérapies in a variety of methods. ln one method, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In another method, DNA complexes with mono- or polycationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. ln certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. ln certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, INTRALIPID is used to préparé a pharmaceutical composition comprising an oligonucleotide. Intralipid is fat émulsion prepared for intravenous administration. It is made up of 10% soybean oil, 1.2% egg yolk phospholipids, 2.25% glycerin, and water for injection. In addition, sodium hydroxide has been added to adjust the pH so that the final product pH range is 6 to 8.9.

In certain embodiments, a pharmaceutical composition provided herein comprise a polyamine compound or a lipid moiety complexed with a nucleic acid. In certain embodiments, such préparations comprise one or more compounds each individually having a structure defîned by formula (Z) or a pharmaceutically acceptable sait thereof, a b /X\ /X x r₂n nr₂ wherein each X^a and X^b, for each occurrence, is independently alkylene; n is 0,1, 2, 3, 4, or 5; each R is independently H, wherein at least n + 2 of the R moieties in at least about 80% ofthe molécules ofthe compound of formula (Z) in the préparation are not H; m is 1, 2, 3 or 4; Y is O, NR², or S; R¹ is alkyl, alkenyl, or alkynyl; each of which is optionally substituted with one or more substituents; and R² is H, alkyl, alkenyl, or alkynyl; each of which is optionally substituted each of which is optionally substituted with one or more substituents; provided that, if n = 0, then at least n + 3 of the R moieties are not H. Such préparations are described in PCT publication WO/2008/042973, which is herein incorporated by reference in its entirety for the disclosure of lipid préparations. Certain additional préparations are described in Akinc et al., Nature Biotechnology 26, 561 - 569 (01 May 2008), which is herein incorporated by reference in its entirety for the disclosure of lipid préparations.

In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, sait solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnésium stéarate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.

In certain embodiments, a pharmaceutical composition provided herein is prepared using known techniques, including, but not limited to mixing, dissolving, granulating, drageemaking, levigating, emulsifying, encapsulating, entrapping or tableting processes.

In certain embodiments, a pharmaceutical composition provided herein is a liquid (e.g., a suspension, élixir and/or solution). In certain of such embodiments, a liquid pharmaceutical composition is prepared using ingrédients known in the art, including, but not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.

In certain embodiments, a pharmaceutical composition provided herein is a solid (e.g., a powder, tablet, and/or capsule). In certain of such embodiments, a solid pharmaceutical composition comprising one or more oligonucleotides is prepared using ingrédients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrating agents.

In certain embodiments, a pharmaceutical composition provided herein is formulated as a depot préparation. Certain such depot préparations are typically longer acting than non-depot préparations. In certain embodiments, such préparations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. In certain embodiments, depot préparations are prepared using suitable polymeric or hydrophobie materials (for example an émulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble dérivatives, for example, as a sparingly soluble sait.

In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery Systems include, but are not limited to, liposomes and émulsions. Certain delivery Systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobie compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.

In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molécules designed to deliver the one or more pharmaceutical agents of the présent invention to spécifie tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, a pharmaceutical composition provided herein comprises a sustained-release system. A non-limiting example of such a sustained-release system is a semipermeable matrix of solid hydrophobie polymers. In certain embodiments, sustained-release Systems may, depending on their chemical nature, release pharmaceutical agents over a period of hours, days, weeks or months.

In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subeutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physïoiogically compatible buffers such as Hanks's solution, Ringerfs solution, or physiological saline buffer. In certain embodiments, other ingrédients are included (e.g., ingrédients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or émulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilie solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycérides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity ofthe suspension, such as sodium carboxym ethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the préparation of highly concentrated solutions.

In certain embodiments, a pharmaceutical composition provided herein comprises a modified oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease orto prolong the survival ofthe subject being treated. Détermination of a therapeutically effective amount is well within the capability of those skilled in the art.

In certain embodiments, one or more modified oligonucleotides provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easierto administerthan the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may hâve improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

In certain embodiments, a prodrug is produced by modifying a pharmaceutically active compound such that the active compound will be regenerated upon in vivo administration. The prodrug can be designed to alterthe metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamie processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs ofthe compound (see, e.g., Nogrady (1985) Médicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages 388-392).

Certain Additional Thérapies

Treatments for a disease associated with miR-21 may comprise more than one therapy. As such, in certain embodiments provided herein are methods for treating a subject having or suspected of having a disease associated with miR-21 comprising administering at least one therapy in addition to administering a modified oligonucleotide having a nucleobase sequence complementary to the microRNA.

In certain embodiments, pharmaceutical agents include anti-inflammatory agents. In certain embodiments, an anti-inflammatory agent is a stéroïdal anti-inflammatory agent. In certain embodiments, a steroid anti-inflammatory agent is a corticosteroid. In certain embodiments, a corticosteroid is prednisone. In certain embodiments, an anti-inflammatory agent is a non-steroidal anti-inflammatory drugs. In certain embodiments, a non-steroidal antiinflammatory agent is ibuprofen, a COX-I inhibitors, or a COX-2 inhibitors.

In certain embodiments, pharmaceutical agents include anti-diabetic agents. Antidiabetic agents include, but are not limited to, biguanides, glucosidase inhibitors, insulins, sulfonylureas, and thiazolidenediones.

In certain embodiments, pharmaceutical agents include, but are not limited to, diuretics (e.g. sprionolactone, eplerenone, furosemide), inotropes (e.g. dobutamine, milrinone), digoxin, vasodilators, angiotensin II converting enzyme (ACE) inhibitors (e.g. are captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril), angiotensin II receptor blockers (ARB) (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, an ACE inhibitor is selected from cilazapril, perindopril, and trandolapril.

In certain embodiments, an ACE inhibitor is administered at a dose of 0.025 to 0.1 mg/kg body weight. In certain embodiments, an ACE inhibitor is administered at a dose of 0.125 to 1.0 mg/kg bodyweight. In certain embodiments, an ACE inhibitor is administered at a dose ranging from 1 to 6 mg/m²/day. In certain embodiments, an ACE inhibitor is administered at a dose ranging from 1 to 2 mg/m²/day. In certain embodiments, an ACE inhibitor is administered at a dose ranging from 2 to 4 mg/m²/day. In certain embodiments, an ACE inhibitor is administered at a dose ranging from 0.5 to 1 mg/m²/day.

In certain embodiment, ramipril is administered at a dose ranging from 1 to 6 mg/m²/day. In certain embodiments, ramipril is administered at a dose ranging from 1 to 2 mg/m²/day. In certain embodiment, enalapril is administered at a dose ranging from 2 to 4 mg/m²/day. In certain embodiment, lisinopril is administered at a dose ranging from 4 to 8 mg/m²/day. In certain embodiment, benazepril is administered at a dose ranging from 4 to 8 mg/m²/day. In certain embodiment, fosinopril is administered at a dose ranging from 4 to 8 mg/m²/day. In certain embodiment, quinapril is administered at a dose ranging from 4 to 8 mg/m²/day. In certain embodiment, cilazapril is administered at a dose ranging from 1 to 2 mg/m²/day. In certain embodiment, perinpril is administered at a dose ranging from 1 to 2 mg/m²/day. In certain embodiment, trandolapril is administered at a dose ranging from 0.5 to 1 mg/m²/day.

• In certain embodiments, an ARB is administered at a dose ranging from 6.25 to 150 mg/m2/day.

In certain embodiments, an ARB is administered at a dose of 6.25 mg/m²/day. In certain embodiments, an ARB is administered at a dose of 10 mg/m²/day. In certain embodiments, an ARB is administered at a dose of 12.5 mg/m²/day. In certain embodiments, an ARB is administered at a dose of 18.75 mg/m²/day.

In certain embodiments, an ARB is administered at a dose of 37.5 mg/m²/day. In certain embodiments, an ARB is administered at a dose of 50 mg/m²/day. In certain embodiments, an ARB is administered at a dose of 150 mg/m²/day.

In certain embodiments, losartan is administered at a dose of 12.5 mg/m²/day. In certain embodiments, losartan is administered at a dose of 12.5 mg/m²/day. In certain embodiments, candesartan is administered at a dose of 6.25 mg/m²/day. In certain embodiments, irbestartan is administered at a dose of 37.5 mg/m²/day. In certain embodiments, telmisartan is administered at a dose of 10 mg/m²/day. In certain embodiments, valsartan is administered at a dose of 18.75 mg/m²/day. In certain embodiments, espresartan is administered at a dose of 150 mg/m²/day.

In certain embodiments, a pharmaceutical agent is an aldostérone antagonsist. In certain embodiments, an aldostérone antagonist is spironolactone. In certain embodiments, spironolactone is administered at a dose ranging from 10 to 35 mg daily. In certain embodiments, spironolactone is administered at a dose of 25 mg daily.

In certain embodiments, pharmaceutical agents include heparinoids. In certain embodiments, a heparinoid is pentosan polysulfate.

In certain embodiments, a pharmaceutical agent is a pharmaceutical agent that blocks one or more responses to fibrogenic signais.

In certain embodiments, a pharmaceutical agent is an anti-connective tissue growth factor therapy. In certain embodiments, an anti-CTGF therapy is a monoclonal antibody against CTGF.

In certain embodiments, an additional therapy may be a pharmaceutical agent that enhances the bod/s immune system, including low-dose cyclophosphamide, thymostimulin, vitamins and nutritional suppléments (e.g., antioxidants, including vitamins A, C, E, betacarotene, zinc, sélénium, glutathione, coenzyme Q-10 and echinacea), and vaccines, e.g., the immunostimulating complex (ISCOM), which comprises a vaccine formulation that combines a multimeric présentation of antigen and an adjuvant.

In certain embodiments, the additional therapy is selected to treat or ameliorate a side effect of one or more pharmaceutical compositions of the présent invention. Such side effects include, without limitation, injection site reactions, liver fonction test abnormalities, kidney fonction abnormalities, liver toxicity, rénal toxicity, central nervous system abnormalities, and myopathies. For example, increased aminotransferase levels in sérum may indicate liver toxicity or liver fonction abnormality. For example, increased bilirubin may indicate liver toxicity or liver fonction abnormality.

Further examples of additional pharmaceutical agents include, but are not limited to, immunoglobulins, including, but not limited to intravenous immunoglobulin (IVlg); analgésies (e.g., acetaminophen); salicylates; antibiotics; antivirals; antifungal agents; adrenergic modifiera; hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin, progestin, somatostan, and thyroid hormones); immunomodulators; muscle relaxants; antihistamines; osteoporosis agents (e.g., biphosphonates, calcitonin, and estrogens); prostaglandins, antineoplastic agents; psychotherapeutic agents; sédatives; poison oak or poison sumac products; antibodies; and vaccines.

Certain Kits

The présent invention also provides kits, ln some embodiments, the kits comprise one or more compounds of the invention comprising a modified oligonucleotide, wherein the nucleobase sequence of the oligonucleotide is complementary to the nudeobase sequence of miR-21. The compounds complementary to miR-21 can hâve any of the nudeoside patterns described herein. ln some embodiments, the compounds complementary to miR-21 can be présent within a vial. A plurality of vials, such as 10, can be présent in, for example, dispensing packs, ln some embodiments, the vial is manufactured so as to be accessible with a syringe. The kit can also contain instructions for using the compounds complementary to miR-21.

ln some embodiments, the kits may be used for administration of the compound complementary to miR-21 to a subject. ln such instances, in addition to compounds complementary to miR-21, the kit can further comprise one or more of the following: syringe, alcohol swab, cotton bail, and/or gauze pad. ln some embodiments, the compounds complementary to miR-21 can be présent in a pre-filled syringe (such as a single-dose syringes with, for example, a 27 gauge, % inch needle with a needle guard), rather than in a vial. A plurality of pre-filled syringes, such as 10, can be présent in, for example, dispensing packs. The kit can also contain instructions for administering the compounds complementary to miR21.

Certain Experimental Models ln certain embodiments, the présent invention provides methods of using and/or testing modified oligonucleotides of the présent invention in an experimental model. Those having skill in the art are able to select and modify the protocols for such experimental models to evaluate a pharmaceutical agent of the invention.

Generally, modified oligonucleotides are first tested in cultured cells. Suitable cell types include those that are related to the cell type to which delivery of a modified oligonucleotide is desired in vivo. For example, suitable cell types for the study of the methods described herein include primary or cultured cells.

ln certain embodiments, the extent to which a modified oligonucleotide interfères with the activity of miR-21 is assessed in cultured cells. ln certain embodiments, inhibition of microRNA activity may be assessed by measuring the levels of the microRNA. Altematively, the level of a predicted or validated microRNA-regulated transcript may be measured. An inhibition of microRNA activity may resuit in the increase in the miR-21-regulated transcript, and/or the protein encoded by miR-21-regulated transcript. Further, in certain embodiments, certain phenotypic outcomes may be measured.

Several animal models are available to the skilled artisan for the study of miR-21 in models of human disease. For example, inhibltors of miR-21 may be studied in an experimental model of Alport Syndrome, for example CoI4a3 knockout mice (CoMa/f' mice). The severity of the disease in the mouse model dépends upon the genetic background of the mouse carrying the Col4a3 mutation. For example, the onset and progression of the disease are generally more rapid on the 129X1/SvJ relative to the C57BL/6J background. Accordingly, the genetic background of the Col4a3⁷' mouse may be selected to vary the onset and progression of disease. Additional models include canine models of X-linked, autosomal récessive or autosomal dominant Alport Syndrome. See, for example, Kashtan, Nephrol. Dial. Transplant, 2002,17:1359-1361.

Certain Quantitation Assays

The effects of antisense inhibition of miR-21 following the administration of modified oligonucleotides may be assessed by a variety of methods known in the art. In certain embodiments, these methods are be used to quantitate microRNA levels in cells or tissues in vitro or in vivo. In certain embodiments, changes in microRNA levels are measured by microarray analysis. In certain embodiments, changes in microRNA levels are measured by one of several commercially available PCR assays, such as the TaqMan® MicroRNA Assay (Applied Biosystems). In certain embodiments, antisense inhibition of miR-21 is assessed by measuring the mRNA and/or protein level of a target of miR-21. Antisense inhibition of miR-21 generally results in the increase in the level of mRNA and/or protein of a target of the microRNA.

Target Engagement Assay

Modulation of microRNA activity with an anti-miR or microRNA mimic may be assessed by measuring target engagement. In certain embodiments, target engagement is measured by microarray profiling of mRNAs. The sequences of the mRNAs that are modulated (either increased or decreased) by the anti-miR or microRNA mimic are searched for microRNA seed sequences, to compare modulation of mRNAs that are targets of the microRNA to modulation of mRNAs that are not targets of the microRNA. In this manner, the interaction of the anti-miR with miR-21, or miR-21 mimic with its targets, can be evaluated. In the case of an anti-miR, mRNAs whose expression levels are increased are screened for the mRNA sequences that comprise a seed match to the microRNA to which the anti-miR is complementary.

EXAMPLES

The following examples are presented in order to more fully illustrate some embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

Those of ordinary skill in the art will readily adopt the underlying principles of this discovery to design various compounds without departing from the spirit ofthe current invention.

Example 1: Anti-miR-21 in a model of Alport Syndrome

Col4a3f mice on the 129sv genetic background spontaneously develop severe kidney disease similar to human Alport Syndrome. As such, Col4a3f mice are used as an experimental model of Alport Syndrome.

Modified oligonucleotides complementary to miR-21 (anti-miR-21 compounds) were tested in the Col483^ model of Alport Syndrome. Wild-type mice were used as control mice.

The structure ofthe anti-miR-21 compound is 5’-AeCsATCsAGTCsTGAUsAAGCsTAe-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-Ddeoxyribonucleosides; nucleosidesfollowed by a subscript Έ indicate 2-MOE nucleosides; nucleosides followed by a subscript “S indicate S-cEt nucleosides. Each intemucleoside linkage is a phosphorothioate intemucleoside linkage.

At 3 weeks of âge, mice were genotyped to identify Col4a3-/- mice. From 3 weeks of âge to 9 weeks of âge, sex-matched littermates of mice were treated with anti-miR-21 or PBS. AntimiR-21 was administered subcutaneously at a dose of 25 mg/kg, twice per week. Treatment groups were: (1) wild-type mice, PBS administration, n = 8; (2) Col4a3-/- mice, PBS administration, n = 12; (3) Col4a3-/- mice, anti-miR-21 administration, n = 12. Wild type littermates of Col4a3-/- mice were used as the wild-type control mice. An ovemight urine sample (approximately 16 hours) was collected weekly. Plasma and kidneys were harvested at the end of week 9. Fluid and tissue samples were analyzed to détermine changes in kidney function, kidney damage and glomerular sclerosis and interstitial fibrosis.

Endpoints in blood or urine included measurement of blood urea nitrogen (BUN), albuminuria, albumin/creatinine ratio, glomerular filtration rate. Histological analysis included évaluation of glomerular sclerosis, interstitial fibrosis, injury to the tubules, macrophage infiltration, and presence of myofibroblasts.

Blood urea nitrogen (BUN) was measured at week 9. Statistical significance was calculated by the Mann Whitney test. As shown in Figure 1 A, a statistically significant réduction in BUN was observed in animais treated with anti-miR-21, relative to PBS-treated control animais at the end of the study. The reduced BUN was observed overall (Figure 1 A), as well as in male mice only (approximately 90 mg/dL compared to approximately 25 mg/dL in control male mice) and female mice only (approximately 70 mg/dL compared to approximately 25 mg/dL in control female mice) not shown). The BUN in Col4a3+/+ mice was approximately 12.5 mg/kL (within normal limits; not shown). BUN is a blood marker of kidney function. Higher BUN correlates with poorer kidney function. A réduction in BUN is an indicatorof reduced kidney injury and damage and improved function.

Albuminurie was assessed by measuring albumin in urine samples, collected over 16 hours at a frequency of once weekly, by ELISA and by normalizing to urinary créatinine excrétion. Ail analyses were performed atthe same time atthe end ofthe study. As shown in Figure 1B, Col4a3-/- mice develop severe albuminuria. However, mice treated with anti-miR-21 developed much less albuminuria as detected by a réduction in urinary albumin to créatinine ratio. This réduction was observed by week 7 and persisted to week 9. Wild type littermates of Col4a3-/- mice exhibited no albuminuria, as expected. Albuminuria is a sensitive measure of glomerular and tubular damage. A réduction in albumin to créatinine ratio indicates a réduction in glomerular and/or tubular disease.

Alport Sydrome is also characterized by progressive development of glomerulosclerosis and significant interstitial kidney fibrosis that occurs as inappropriate glomerular leakage occurs. Accordingly, glomerulosclerosis was assessed by blinded scoring of glomeruli for sclerotic lésions (loss of capillary loop+fibrosis or hyalinosis). Thirty glomeruli were scored sequentially from each mouse by a blinded observer. The score was from 0-4 where 0 = normal; 1 = <25% ofthe glomerulus affected bysclerosis; 2 = 25-50% ofthe glomerulus isaffected bysclerosis; 3 = 50-75% of the glomerulus is affected by sclerosis; 4 = 75-100% ofthe glomerulus is affected by sclerosis. The proportion of glomeruli with no disease was much higher in mice treated with anti-miR-21 and the proportion of glomeruli with moderately or severely affected glomeruli (score 2-4) was significantly higher in the mice treated with the PBS (Figure 2). Glomeruli were also scored in wild type littermates (WT) of Col43a-/- mice. Interstitial fibrosis was measured morphometrically in whole sagittal sections stained with Picrosirius red from PBS-treated and anti-miR-21 Col4a3-/- animais. As shown in Figure 3A, a statistically significant réduction in interstitial fibrosis was observed in the anti-miR-21 treated Col4a3-/- mice. In addition, quantitative PCR for the transcripts for the major pathological matrix protein Collagen Ia(1 ) (Collai) showed that the kidney tissue from anti-miR-21 treated Co/4a3-/-mice showed much less production of this pathological collagen (Figure 3B).

Rénal tissue injury was assessed in paraffin-embedded and paraformaldéhyde (4%)fixed tissue sections stained by the periodic acid-Schiff (PAS) reaction, initially the kidney sections were ranked for overall injury based on tubule and glomerular injury and inflammation. Damage was assessed based on a variety of factors including tubule dilation, loss of brush border, cellular infiltration, glomerular inflammation, interstitial edema and cellular necrosis. Kidney sections were ranked in a blinded fashion for overall injury and given a kidney injury rank score. The kidney sections from Col4a3 -/- mice showed a significantly lower kidney injury rank score, which is indicative of less kidney injury (Figure 4A). To analyze this in more detail, the glomeruli were assessed by a blinded observer for the proportion that had glomerular crescents. The crescent is a prolifération of cells within Bowman’s capsule, is defined by & 2 layers of cells within Bowman’s space. The crescent is a well-established marker of glomerular injury. In Col4a3-/- mice that received anti-miR-21 the proportion of glomeruli with crescents was approximately 44%, whereas in mice that received the PBS control treatment, the proportion of glomeruli with crescents was approximately 19% (Figure 4B). in Col4a3+/+ littermates, the proportion of glomeruli with crescents was less than 5% (not shown). The tubules of the nephrons of the kidney are also a site for damage. The tubule damage was assessed by overlaying a grid over sequential images covering the whole sagittal section of each kidney. In a blinded fashion, damage of the tubules was assessed in each square of the grid. Tubular damage was assessed based on the presence of tubule dilation/flattening, loss of brush border, cellular infiltration, and cellular necrosis. The presence of these features results in a positive score for that square on the grid. An overall score is applied to each image which is the % of squares that has tubule damage. This is averaged for ail the images from that kidney. The average score for each kidney is then subjected to statistical analysis. As is shown, the tubule injury score was significantly lower in the Col4a3-/- mice treated with anti-miR-21, relative to the Col4a3-/- receiving PBS (Figure 4C). The tubule injury score in Col4a3+/+ littermates was less than 10% (not shown).

Additional histological analysis of kidney samples was performed to evaluate macrophage infiltration, endothélial stability, and the myofibroblast déposition. As judged by F4/80 staining, macrophage infiltration was reduced in anti-miR-21 treated Col4a3-/- mice compared to PBS-treated Col4a3-/- control mice (Figure 5A). Immunocytochemical staining for CD31 demonstrated an improvement in endothélial stability in anti-miR-21 treated Col4a3-/mice compared to PBS-treated Col4a3-/- control mice (not shown). Détection of alpha-SMA revealed a réduction in myofibroblast déposition in anti-miR-21 treated Col4a3-/- mice compared to PBS-treated Col4a3-/- control mice (Figure 5B). In Col4a3+/+ mice, alpha-SMA staining was approximately 5% (not shown).

Reactive oxygen species (ROS) are a byproduct of normal cellular metabolism. During cellular stress, excess ROS can cause lipid peroxidation of cell and organelle membranes, resulting in disruption of the structural integrity and capacity for cell transport and energy production. In the kidney, ROS produced during cellular stress can cause rénal injury. To assess whether the génération of ROS was reduced following inhibition of miR-21 in Col4a3-/mice, urinary hydrogen peroxide levels were measured in anti-miR-21 and PBS-treated mice. Urinary hydrogen peroxide levels were signifîcantly reduced in mice that received anti-miR-21 (Figure 6A). In Col4a3+/+ mice, urinary hydrogen peroxide levels were less than 5 μΜ (not shown). Further, immunocytochemical staining of kidney tissue with dihydroethidium (DHE), which is a measure of ROS, demonstrated a réduction in ROS in the kidney tissue of anti-miR21 treated Col4a3-/· mice compared to PBS-treated control mice (Figure 6B). In Col4a3+/+ mice, less than 10% DHE staining was observed (not shown). These data demonstrate réduction of ROS in both the urine and kidney tissue in Col4a3-/- mice treated with anti-miR-21. Accordingly, one mechanism by which anti-miR-21 may reduce kidney injury may include a réduction in the génération of reactive oxygen species.

Immunoblotting of protein in the kidneys of Col4a3-/- mice treated with anti-miR-21 revealed an increase in the amount of MPV17L protein in the kidney, relative to Col4a3-/- mice. MPV17L is a mitochondrial inner membrane protein that is implicated in the metabolism of reactive oxygen species, and protects against oxidative stress. Accordingly, the reduced génération of ROS following anti-miR-21 treatment may occur at least in part due to increased MPV17L levels.To further explore the mechanistic effects of anti-miR-21, PPAR-alpha protein was measured by immunoblotting of the kidneys of Col4a3-/- mice treated with PBS or anti-miR21 treatment. Anti-miR-21 treatment increased PPAR-alpha protein, suggesting a stimulation of metabolic pathways.

Podocytes are highly specialized épithélial cells that are an essential component of the glomerular filtration barrier. Podocyte loss can lead to proteinuria, and in some disease states to glomerularsclerosis. To evaluate whether podocyte number was affected by inhibition of miR-21 in Col4a3-/- mice, podocyte number was measured in anti-miR-21 and PBS-treated mice. Podocyte number was signifîcantly increased in Col4a3-/- mice that received anti-miR-21, relative to PBS-treated mice, and was comparable to the podocyte number observed in wild type littermates of Col4a3-/· mice (Figure 7). Accordingly, one mechanism by which anti-miR-21 may reduce kidney injury in a model of Alport Syndrome is by preventing or reducing podocyte loss.

A similar study was conducted using the following anti-miR-21 compounds:

anti-miR-21 compound #1 (above): 5’-AeCsATCsAGTC_sTGAUsAAGCsTAe-3’ (SEQ ID NO:3) anti-miR-21 compound #2: 5’-AeCsATCsA_sGTC_sU_sGAU_sAsAGCsUsAe-3’ (SEQ ID NO:

3) :

anti-miR-21 compound #3: s'-^CeAsAsTeCsUsAeAeUsAsAeGeCsTeAs-S’ (SEQ ID NO:

4) ; and anti-miR-21 compound #4: 5’-AeCsAeTeCsAeGeTeC_sTGAUsAAGCsUsAs-3’ (SEQ ID NO: 3); where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’-MOE nucleosides: nucleosides followed by a subscript “S” indicate S-cEt nucleosides; and a superscript “Me” indicates a 5-methyl group on the base of the nucleoside. Each intemucleoside linkage is a phosphorothioate intemucleoside linkage.

Each compound was administered to three-week old Col4a3-/- mice at a dose of 25 mg/kg, twice weekly, for nine weeks. Control groups included Col4a3-/- mice treated with PBS, and wild-type littermates of Col4a3-/- mice. Each treatment group contained 10 to 12 mice. For compounds #1,2, and 4, endpoints were evaluated as described above and included BUN, urinary albumin to créatinine ratio, kidney injury (PAS staining), glomerulosclerosis, and proportion of glomeruli with crescents. For compound #3, endpoints included BUN, urinary albumin to créatinine ratio, and collagen gene expression (as a measure of fibrosis), evaluated as described above.

Consistent with the results described above, anti-miR-21 compound #1 improved ail endpoints evaluated. The efficacy of both anti-miR-21 compounds #2 was similar to that of compound #1, with improvements observed in BUN, urinary albumin to créatinine ratio, kidney injury, extent of glomerulosclerosis, and percentage of glomeruli with crescents. The efficacy of compound #3 was similar to that of compound #1, with improvements in BUN, urinary albumin to créatinine ratio, and Collai expression. Anti-miR-21 compound #4, whiie less efficacious than compounds the other compounds tested, still resulted in improvements in BUN, kidney injury, extent of glomerulosclerosis, and percentage of glomeruli with crescents.

Taken together, these data iliustrate that in a model of Alport Sydrome, anti-miR-21 treatment attenuated the loss of kidney function and development of albuminuria. Glomerulosclerosis and interstitial fibrosis were markedly attenuated and proximal tubules were preserved. As anti-miR-21 prevents progressive loss of kidney function in the Col4a3-/- mouse, and atténuâtes both glomerular and tubulo-interstitial disease, anti-miR-21 is a therapeutic agent for human Alport Syndrome.

Example 2: Elévation of miR-21 in a model of Alport Syndrome

To evaluate the dysrégulation of miR-21 in an experimental model of Alport Syndrome, miR-21 levels were measured in kidney tissue harvested from mice. RNA was isolated from whole kidney and miR-21 was measured by quantitative PCR. In Col4a3-/- mice, miR-21 levels were elevated approximately three-fold relative to miR-21 levels in wild-type mice.

Accordingly, a subject receiving treatment for Alport Syndrome may be identified as having elevated miR-21 in kidney biopsy material, urine, or blood, prior to administration of the treatment.

Example 3: Survival studies in a model of Alport Syndrome

Wild-type mice generally live for 2 to 3 years (or 730 to 1095 days). In Col4a3-/- mice on a 129X1/SvJ background, end-stage rénal failure can occur as early as 2 months of âge. In Col4a3-/-on a C57BL/6J background, end-stage rénal failure can occur as early as 6 months of âge. Regardless ofthe background, the lifespan of Col4a3-/- mice is significantly shorter than that of wild-type mice. As such, Col4a3-/- mice, on any genetic background, can be serve as a model for end-stage rénal failure in Alport Syndrome and can be used to evaluate the effects of candidate therapeutic agents on life expectancy.

Mice are genotyped to identify Col4a3-/· mice. Anti-miR-21 is administered subcutaneously at a dose of ranging from 10 to 25 mg/kg, once or twice per week for up to one year. PBS may be administered as a controi treatment. Ovemight urine samples (approximately 16 hours) are collected on a weekly or monthly schedule throughout the study. Age of each mouse at death is recorded. Plasma and kidneys are collected at death oratthe end ofthe study. Fluid and tissue samples are anaiyzed to détermine changes in kidney function, glomerularsclerosis, and fibrosis.

Fluid and tissue samples are anaiyzed to détermine changes in kidney function, kidney damage and glomerular sclerosis and interstitial fibrosis. Endpoints in blood or urine include measurement of blood urea nitrogen (BUN), albuminuria, albumin/creatinine ratio, glomerular filtration rate. Histological analysis includes évaluation of glomerular sclerosis, interstitial fibrosis, injury to the tubules, macrophage infiltration, and presence of myofibroblasts.

Delay in the onset of end-stage rénal failure and increased life expectancy in anti-miR21 treated mice, relative to PBS-treated control mice, is observed, suggesting that anti-miR-21 is a therapeutic agent that can increase the life expectancy of subjects with Alport Syndrome.

Anti-miR-21lncreases survival in a model of Alport Syndrome-single dose study

To evaluate the effects of anti-miR-21 on survival in an experimental model of Alport Syndrome, anti-miR-21 compound was administered to Col4a3-/- mice.

The structure ofthe anti-miR-21 compound is 5’-AeCsATCsAGTC_sTGAUsAAGCsTAe-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-Ddeoxyribonucleosides; nucleosides followed by a subscript Έ indicate 2’-MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt nucleosides. Each internucleoside linkage is a phosphorothioate internucleoside linkage.

Col4a3+/1 mice (hétérozygotes) on a 129X1/SvJ background were crossed to generate Col4a3-/- mice. At 3 weeks of âge, mice were genotyped to identify Col4a3-/- mice. Treatment groups were: (1) Col4a3+/+ mice (wild-type littermates), PBS administration, twice weekly, n = 12; (2) Col4a3-/- mice, PBS administration, twice weekly, n = 12; (3) Col4a3-/- mice, 25 mg/kg anti-miR-21 administration subcutaneously, twice weekly, n = 12. Treatments were administered twice weekly, from week 3 through week 16. Animal weights were measured weekly, and lifespan was recorded.

As expected, Col4a3-/- mice experienced weight loss beginning at around 9 weeks of âge, and death occurred between 9 and 11 weeks of âge. As shown in Figure 8A, anti-miR-21 increased peak body weight and significantly delayed weight loss (p < 0.01). As shown in Figure 8B, anti-miR-21 significantly increased lifespan (p < 0.001). Thus, treatment with anti-miR-21 not only delayed the weight loss, but importantly improved survival of Col4a3-/- mice.

Anti-miR-21 increases survival in a model of Alport Syndrome-dose response study

To evaluate the dose-responsive effects of anti-miR-21 on survival in an experimental model of Alport Syndrome, several doses of anti-miR-21 compound were administered to Col4a3-/- mice.

The structure ofthe anti-miR-21 compound is 5’-AeCsATCsAGTCsTGAU_sAAGCsTAe-3’ (SEQ ID NO: 3), where nucleosides not followed by a subscript indicate β-Ddeoxyribonucleosides; nucleosides followed by a subscript “E indicate 2’-MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt nucleosides. Each internucleoside linkage is a phosphorothioate internucleoside linkage.

At 3 weeks of âge, mice were genotyped to identify Col4a3-/- mice. Treatment groups were:

(1) Col4a3-/- mice, PBS administration once weekly, n = 13;

(2) Col4a3-/- mice, 12.5 mg/kg anti-miR-21 administration, once weekly, n = 12;

(3) Col4a3-/- mice, 25 mg/kg anti-miR-21 administration, once weekly, n = 13;

(4) Col4a3-/- mice, 50 mg/kg anti-miR-21 administration, once weekly, n = 12;

(5) Col4a3-/- mice, 25 mg/kg anti-miR-21 administration, twice weekly, n = 12;

Treatments were administered starting on day 24. Animal weights were measured weekly, and lifespan was recorded. At week 7, blood was collected for measurement of BUN.

As shown in Figure 9A, a réduction in BUN was observed in animais treated with antimiR-21, relative to PBS-treated control animais. Although a réduction in BUN was observed, it was not strongly dose-responsive, perhaps due to the fact that the disease was more severe in the Col4a3-/- mice used for this experiment (the mice were obtained from a different vendor than the Col4a3-/- mice described in the previous examples). The observed réduction in BUN is an indicator of reduced kidney injury and damage and improved function.

As shown in Figure 9B, treatment with anti-miR-21 increased the lifespan of Col4a3-/~ mice in a dose responsive manner. The increased lifespan was observed for both twice weekly and once weekly treatments. The médian survival was as follows: PBS, 62 days; 12.5 mg/kg anti-miR-21 once weekly (QW), 72.5 days; 25 mg/kg anti-miR-21 once weekly (QW), 77 days; 50 mg/kg anti-miR-21 once weekly (QW), 89 days; 25 mg/kg anti-miR-21 twice weekly (BIW), 82.5 days.

Delay in the onset of kidney dysfunction and increased life expectancy in anti-miR-21 treated mice, relative to PBS-treated control mice, was observed, suggesting that anti-miR-21 is a therapeutic agent that can increase the life expectancy of subjects with Alport Syndrome.

Example 4: Anti-mIR distribution in the kidney of Col4a3-/- mice

Oligonucleotides, including anti-miR compounds, are known to distribute to several cell types within the kidney. As reported by Chau et al., Sci Transi Med., 2012,121ra18, following administration of a Cy3-labeled anti-miR to either normal mice or mice subjected to kidney injury (unilatéral urétéral obstruction, a model of interstitial fibrosis), the greatest fluorescence intensity in the kidney was in proximal tubule epithelium. The endothélium, pericytes, myofibroblasts, and macrophages also ail contained détectable amounts of Cy3-labeled anti-miR. However, the glomerulus, in particular podocytes, did not appear to take up significant amounts of anti-miR consistent with the known distribution of chemically modified oligonucleotides (Masarjian et al., Oligonucleotides, 2004,14, 299-310).

To investigate the distribution of anti-miR in a mouse model of Alport Syndrome, Cy3labeled anti-miR compound was administered to two different groups of Col4a3-/- mice, one at 6 weeks of âge (n = 3) and one at 8 weeks of âge (n = 4) and to one group of wild type mice at 8 weeks of âge (n = 3). Two days following administration ofthe anti-miR compound, animais were sacrificed and kidneys were harvested and processed for histological analysis.

Sections of kidney tissue were co-labeled with antibodîes spécifie to several different cellular markers to identify anti-miR uptake in particular cell types. Staining was performed for alpha-SMA (a myofibroblast marker), PDGFR-beta (a pericyte/myofibroblast marker), CD31 (an endothélial cell marker), F4/80 (a macrophage marker), and GP38 (a podocyte marker). As expected, anti-miR compound was taken up into the proximal tubule epithelium, pericytes, myofibroblasts, and macrophages. In contrast to previous observations in normal mice and mice with interstitial fibrosis, in the Col4a3-/- mice anti-miR was taken up into the glomerulus, inciuding into podocytes.

As described herein, the efficacy observed following anti-miR-21 administration in an experimental model of Alport Syndrome is accompanied by improvements not only in interstitial fibrosis surrounding tubules but also fibrosis in the glomeruli (known as glomerulosclerosis). These data suggest that those improvements may be directly related to anti-miR-21 effects in the glomeruli, in addition to or instead of feedback from an improved tubule structure and function.

Claims

What is claimed is:

1. A method of treating Alport Syndrome comprising administering to a subject having or suspected of having Alport Syndrome a modified oligonucleotide consisting of 12 to 25 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21.
2. The method of claim 1 wherein the subject has been diagnosed as having Alport Syndrome prior to administering the modified oligonucleotide.
3. The method of claim 1 wherein the subject, prior to administration of the modified oligonucleotide, was determined to hâve an increased level of miR-21 in the kidney, urine or blood of the subject.
4. The method of any of the above claims, wherein the administering:

a. improves kidney function;

b. delays the onset of end stage rénal disease;

c. delays time to dialysis;

d. delays time to rénal transplant; and/or

e. improves life expectancy.
5. The method of any of the above claims, wherein the administering:

a. reduces hematuria;

b. delays the onset of hematuria;

c. reduces proteinuria;

d. delays the onset of proteinuria;

e. reduces kidney fibrosis;

f. slows further progression of fibrosis; and/or

g. halts further progression of fibrosis.
6. The method of any of the above claims, wherein the subject has a mutation selected from a mutation in the gene encoding the alpha 3 chain of type IV collagen, a mutation in the gene encoding the alpha 4 chain of type IV collagen, or a mutation in the gene encoding the alpha 5 chain of type IV collagen.
7. The method of any of the above claims, wherein the subject is male.
8. The method of any of the above claims, wherein the subject is female.
9. The method of any of the above claims, wherein the subject is identified as having hematuria, and/or proteinuria.
10. The method of any of the above claims, wherein the subject has reduced kidney function.
11. The method of any of the above claims, wherein the subject is in need of improved kidney function.
12. The method of any of the above claims, comprising:

a. measuring blood urea nitrogen in the blood of the subject;

b. measuring créatinine in the blood of the subject;

c. measuring créatinine clearance in the subject;

d. measuring proteinuria in the subject;

e. measuring albumin:creatinine ratio in the subject;

f. measuring glomerular filtration rate in the subject;

g. measuring cystatin C in the subject;

h. measuring β-trace protein (BTP) in the blood of the subject;

i. measuring 2-microglobulin in the blood of the subject;

j. measuring N-acetyl^-D-glucosaminidase (NAG) protein in the urine ofthe subject;

k. measuring neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject;

l. measuring kidney injury molecule-1 (KIM-1) protein in the urine ofthe subject;

m. measuring interleukin-18 (IL-18) protein in the urine of the subject;

n. measuring monocyte chemoattractant protein (MCP1 ) levels in the urine of the subject;

o. measuring connective tissue growth factor (CTGF) levels in the urine of the subject;

p. measuring collagen IV fragments in the urine of the subject;

q. measuring collagen III fragments in the urine of the subject; and/or

r. measuring podocyte protein levels in the urine ofthe subject, wherein the podocyte protein is selected from nephrin and podocin.
13. The method of any ofthe above claims, wherein the administering improves one or more markers of kidney function in the subject, selected from:

a. reduced blood urea nitrogen in the subject;

b. reduced créatinine in the blood of the subject;

c. improved créatinine clearance in the subject;

d. reduced proteinuria in the subject;

e. reduced albumin'.creatinine ratio in the subject;

f. improved glomerular filtration rate in the subject;

g. reduced cystatin C in the blood of the subject;

h. reduced β-trace protein (BTP) in the blood of the subject;

i. reduced 2-microglobulin (B2M) in the blood of a subject;

j. reduced NAG protein in the urine ofthe subject;

k. reduced NGAL protein in the urine ofthe subject;

l. reduced KIM-1 protein in the urine ofthe subject;

m. reduced IL-18 protein in the urine ofthe subject;

n. reduced monocyte chemoattractant protein (MCP1) levels in the urine of the subject;

o. reduced connective tissue growth factor (CTGF) levels in the urine of the subject;

p. reduced collagen IV fragments in the urine of the subject;

q. reduced collagen III fragments in the urine of the subject; and/or

r. reduced podocyte protein levels in the urine of the subject, wherein the podocyte protein is selected from nephrin and podocin.
14. The method of any of claims 5,12, or 13, wherein the proteinuria is albuminuria.
15. The method of claim 14 wherein the albuminuria is high normal albuminuria, microalbuminuria, or macroalbuminuria.
16. The method of any of the above claims, wherein the Alport Syndrome is the X-linked form of Alport Syndrome.
17. The method of any of the above claims, wherein the Alport Syndrome is the autosomal form of Alport Syndrome.
18. The method of any of the above claims, comprising administering at least one additional therapy selected from an angiotensin II converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), an anti-hypertensive agent, a vitamin D analog, an oral phosphate binder, dialysis, and kidney transplant.
19. The method of claim 17 wherein the angiotensin II converting enzyme (ACE) inhibitors is selected from captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril.
20. The method of claim 17 wherein the angiotensin II receptor blockers (ARB) is selected from candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, and eprosartan.
21. The method of any of the above claims, wherein the nucleobase sequence of the modified oligonucleotide is at least 90% complementary, is at least 95% complementary, or is 100% complementary to the nucleobase sequence of miR-21 (SEQ ID NO: 1).
22. The method of any of the above claims, wherein the modified oligonucleotide consists of 15 to 25 linked nucleosides.
23. The method of any of the above claims, wherein the modified oligonucleotide consists of 15,16, 17, 18,19, 20, 21, or 22 linked nucleosides.
24. The method of any of the above claims, wherein the modified oligonucleotide comprises at least one modified nucleoside.
25. The method of claim 21, wherein the modified nucleoside is selected from an S-cEt nucleoside, a 2’-O-methoxyethyl nucleoside, and an LNA nucleoside.
26. The method of any of the above claims, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
27. The method of any of the above claims, wherein each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage.
28. The method of claim 23 or 24, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.
29. The method of any of the above claims, wherein the modified oligonucleotide has the structure 5’-AeCsATCsAGTC_sTGAUsAAGCsTAe-3’, where nucleosides not followed by a subscript indicate β-D-deoxyribonucleosides; nucleosides followed by a subscript “E” indicate 2’-MOE nucleosides; nucleosides followed by a subscript “S” indicate S-cEt

5 nucleosides, and each intemucleoside linkage is a phosphorothioate intemucleoside linkage.
30. Use of a modified oligonucleotide consisting of 12 to 25 linked nucleosides, wherein the nucleobase sequence of the modified oligonucleotide is complementary to miR-21, for the treatment of Alport Syndrome.