Modified oligonucleotides for treating polycystic kidney disease

Abstract

Provided herein are methods for treating polycystic kidney disease, including autosomal dominant polycystic kidney disease, using modified oligonucleotides targeting miR-17.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 430,139, filed December 5, 2016, which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0003] Provided herein are compositions and methods for treating polycystic kidney disease. Background Art

[0004] Polycystic kidney disease is characterized by the accumulation of multiple fluid-filled cysts in the kidneys. These cysts are lined by a single layer of epithelial cells called the cyst epithelium. Over time, the cysts increase in size due to elevated cell proliferation and active secretion of fluid by the cyst epithelial cells. The enlarging cysts compress the surrounding normal tissues, leading to decreased kidney function. The disease eventually progresses to end-stage renal disease requiring dialysis or a kidney transplant. At this stage, the cysts may be surrounded by areas of fibrosis containing atrophic tubules.

[0005] Many genetic disorders can lead to polycystic kidney disease (PKD). Various forms of PKD are distinguished by inheritance, such as autosomal dominant or autosomal recessive inheritance; organ involvement and renal external phenotype manifestation; age of onset of end-stage renal disease, such as birth, childhood or adulthood; and potential gene mutations associated with the disease. See, e.g., Kurschat et al., 2014, Nature Reviews Nephrology, 10: 687-699. Summary of the invention

[0006] Embodiment 1. A compound comprising a modified oligonucleotide consisting of 9 linked nucleosides, wherein the modified oligonucleotide has the following nucleoside pattern in the 5' to 3' orientation:

[0007] N S N S N M N F N F N F N M N S N S

[0008] wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and all linkages are phosphorothioate linkages; and

[0009] The nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUU-3', wherein each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or a pharmaceutically acceptable salt thereof.

[0010] Embodiment 2. The compound of embodiment 1, wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence 5'-GCACUUU-3', wherein each cytosine is independently selected from non-methylated cytosine and 5-methylcytosine.

[0011] Embodiment 3. The compound of embodiment 1, wherein the nucleobase sequence of the modified oligonucleotide is 5'-AGCACUUUG-3', wherein each cytosine is independently selected from non-methylated cytosine and 5-methylcytosine.

[0012] Embodiment 4. The compound of any one of embodiments 1, 2 or 3, wherein each cytosine is a non-methylated cytosine.

[0013] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the compound consists of the modified oligonucleotide or a pharmaceutically acceptable salt thereof.

[0014] Embodiment 6. The compound of any one of Embodiments 1 to 5, wherein the pharmaceutically acceptable salt is a sodium salt.

[0015] Embodiment 7. A modified oligonucleotide having the structure:

[0016] or a pharmaceutically acceptable salt thereof.

[0017] Embodiment 8. The modified oligonucleotide according to embodiment 7, which is a pharmaceutically acceptable salt of the structure.

[0018] Embodiment 9. The modified oligonucleotide according to embodiment 7, which is a sodium salt of the structure.

[0019] Embodiment 10. A modified oligonucleotide having the structure:

[0020]

[0021] Embodiment 11. A pharmaceutical composition comprising the compound of any one of embodiments 1 to 6 or the modified oligonucleotide of any one of embodiments 7 to 10 and a pharmaceutically acceptable diluent.

[0022] Embodiment 12. The pharmaceutical composition of embodiment 11, wherein the pharmaceutically acceptable diluent is an aqueous solution.

[0023] Embodiment 13. The pharmaceutical composition of embodiment 12, wherein the aqueous solution is a saline solution.

[0024] Embodiment 14. A pharmaceutical composition comprising the compound of any one of embodiments 1 to 6 or the modified oligonucleotide of any one of embodiments 7 to 10, which is a lyophilized composition.

[0025] Embodiment 15. A pharmaceutical composition consisting essentially of a compound according to any one of embodiments 1 to 6 or a modified oligonucleotide according to any one of embodiments 7 to 10 in saline solution.

[0026] Embodiment 16. A method for inhibiting the activity of one or more members of the miR-17 family in a cell, comprising contacting the cell with a compound as described in any one of embodiments 1 to 6 or a modified oligonucleotide as described in any one of embodiments 7 to 10.

[0027] Embodiment 17. A method for inhibiting the activity of one or more members of the miR-17 family in a subject, comprising administering to the subject the pharmaceutical composition of any one of Embodiments 11 to 15.

[0028] Embodiment 18. The method of embodiment 17, wherein the subject suffers from a disease associated with miR-17. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1A-1B (A) Activity of RG4326 in miR-17 luciferase assay. (B) Activity of RG4326 in miR-17 family member luciferase assay.

[0030] Figure 2 PD marker fractions in IMCD3 cells after treatment with RG4326 or the control RG5124.

[0031] Figure 3A-3B .miPSA shows miR-17 target engagement in (A) kidneys of wild-type mice and (B) kidneys of RG4326-treated mice.

[0032] Figures 4A-4C Efficacy of RG4326 in the Pkd2-KO model of PKD. Effects of treatment on (A) kidney weight-to-body weight ratio, (B) blood urea nitrogen (BUN) levels, and (C) cysticity index.

[0033] Figures 5A-5C Efficacy of RG4326 in the PCy model of PKD. Effects of treatment on (A) kidney weight-to-body weight ratio, (B) blood urea nitrogen (BUN) levels, and (C) cystic index. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Unless a specific definition is provided, the nomenclature and procedures and techniques used in analytical chemistry, synthetic organic chemistry, and medicine and pharmaceutical chemistry described herein are those well known and commonly used in the art. In the case where there are multiple definitions for the terms herein, the definitions in this section shall prevail. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of subjects. Some such techniques and procedures can be found in, for example, "Carbohydrate Modifications in Antisense Research", edited by Sangvi and Cook, American Chemical Society, Washington DC, 1994; and "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, Pa., 18th edition, 1990; and the documents are incorporated by reference for any purpose. Unless otherwise stated, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other disclosed materials mentioned in the entire disclosure of this article are incorporated by reference as a whole, where permitted. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change, and specific information on the internet can change, but equivalent information can be found by searching the internet. Such reference serves as a testimony to the availability and public dissemination of such information.

[0035] Before disclosing and describing the compositions and methods of the present invention, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, as used in this specification and the appended claims, the singular forms "a / kind", "an", and "the" include plural referents unless the context clearly indicates otherwise.

[0036] definition

[0037] "Polycystic kidney disease" or "PKD" is a cystic kidney disease characterized by the accumulation of multiple fluid-filled cysts in the kidneys. Multiple cysts form in at least one kidney, often leading to enlargement of one or more affected kidneys and progressive loss of kidney function.

[0038] "Markers of polycystic kidney disease" means medical parameters used to assess the severity of polycystic kidney disease, renal function and / or response to treatment in a subject with polycystic kidney disease. Non-limiting examples of markers of polycystic kidney disease include total kidney volume, hypertension, glomerular filtration rate, and renal pain.

[0039] "Renal function marker" means a medical parameter used to assess a subject's renal function. Non-limiting examples of renal function markers include glomerular filtration rate, blood urea nitrogen level, and serum creatinine level.

[0040] "Autosomal dominant polycystic kidney disease" or "ADPKD" is polycystic kidney disease caused by one or more genetic mutations in the PKD1 and / or PKD2 genes. 85% of ADPKD is caused by mutations in PKD1, located on chromosome 16, with the majority of the remaining ADPKD cases being caused by mutations in PKD2, located on chromosome 4.

[0041] "Autosomal recessive polycystic kidney disease" or "ARPKD" is polycystic kidney disease caused by one or more genetic mutations in the PKHD1 gene located on chromosome 6. Up to 50% of newborns with ARPKD die from complications of renal disease in utero, and approximately one-third of survivors develop end-stage renal disease (ESRD) within 10 years.

[0042] "Nephronophthisis" or "NPHP" refers to an autosomal recessive cystic kidney disease characterized by corticomedullary cysts, tubular basement membrane disruption, and tubulointerstitial nephropathy.

[0043] "Total kidney volume" or "TKV" is a measure of total kidney volume. Total kidney volume can be determined by magnetic resonance imaging (MRI), computed tomography (CT) scanning or ultrasound (US) imaging, and the volume is calculated by standard methods such as the ellipse volume equation (for ultrasound) or by quantitative stereology or border tracing (for CT / MRI).

[0044] “Height-adjusted total kidney volume” or “HtTKV” is a measure of total kidney volume per unit of height. Patients with HtTKV values ​​≥ 600 ml / m are predicted to develop stage 3 chronic kidney disease within 8 years.

[0045] "Renal pain" means clinically significant renal pain requiring sick leave, medical treatment (narcotics or analgesics of last resort), or invasive intervention.

[0046] "Worsening hypertension" means a change in blood pressure that requires initiation or increase in hypertension treatment.

[0047] "Fibrosis" means the formation or development of excess fibrous connective tissue in an organ or tissue. In certain embodiments, fibrosis occurs as a repair or reaction process. In certain embodiments, fibrosis occurs in response to damage or injury. The term "fibrosis" is understood to mean the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reaction process, which is contrary to the formation of fibrous tissue as a normal component of an organ or tissue.

[0048] "Hematuria" means the presence of red blood cells in the urine.

[0049] "Albuminuria" means the presence of excess albumin in the urine, and includes, but is not limited to, normoalbuminuria, hypernormoalbuminuria, microalbuminuria, and macroalbuminuria. Normally, the glomerular filtration permeability barrier, composed of podocytes, glomerular basement membrane, and endothelial cells, prevents serum proteins from leaking into the urine. Albuminuria may reflect damage to the glomerular filtration permeability barrier. Albuminuria may be calculated from a 24-hour urine sample, an overnight urine sample, or a spot urine sample.

[0050] "High normoalbuminuria" means elevated albuminuria characterized by (i) 15 mg to <30 mg albumin excreted in the urine per 24 hours and / or (ii) an albumin / creatinine ratio of 1.25 mg / mmol to <2.5 mg / mmol (or 10 mg / g to <20 mg / g) in males or 1.75 mg / mmol to <3.5 mg / mmol (or 15 mg / g to <30 mg / g) in females.

[0051] "Microalbuminuria" means elevated albuminuria characterized by (i) 30 mg to 300 mg of albumin excreted in the urine per 24 hours and / or (ii) an albumin / creatinine ratio of 2.5 mg / mmol to <25 mg / mmol (or 20 mg / g to <200 mg / g) in males or 3.5 mg / mmol to <35 mg / mmol (or 30 mg / g to <300 mg / g) in females.

[0052] "Macroalbuminuria" means elevated albuminuria characterized by (i) greater than 300 mg of albumin excreted in the urine per 24 hours and / or (ii) an albumin / creatinine ratio >25 mg / mmol (or >200 mg / g) in males or >35 mg / mmol (or >300 mg / g) in females.

[0053] "Albumin / creatinine ratio" means the ratio of urine albumin (mg / dL) / urine creatinine (g / dL) and is expressed as mg / g. In certain embodiments, the albumin / creatinine ratio can be calculated from a spot urine sample and can be used as an estimate of albumin excretion over a 24-hour period.

[0054] "Glomerular filtration rate" or "GFR" means the flow rate of filtered fluid through the kidneys and is used as an indicator of renal function in a subject. In certain embodiments, the subject's GFR is determined by calculating an estimated glomerular filtration rate. In certain embodiments, the subject's GFR is directly measured in the subject using the inulin method.

[0055] "Estimated glomerular filtration rate" or "eGFR" means a measurement of how well the kidneys filter creatinine and is used to roughly estimate glomerular filtration rate. Because direct measurement of GFR is complicated, eGFR is often used in clinical practice. Normal results can be between 90-120 mL / min / 1.73 m 2 Less than 60mL / min / 1.73m for 3 months or longer 2 Levels below 15 mL / min / 1.73 m 2 Levels of β-catenin may be an indicator of kidney failure.

[0056] "Proteinuria" means the presence of excess serum protein in the urine. Proteinuria can be characterized by >250 mg of protein excreted into the urine per 24 hours and / or a urine protein to creatinine ratio >0.20 mg / mg. Elevated serum proteins associated with proteinuria include, but are not limited to, albumin.

[0057] "Blood urea nitrogen level" or "BUN level" means a measure of the amount of nitrogen in the form of urea in the blood. The liver produces urea as a waste product of protein digestion in the urea cycle, and urine is removed from the blood by the kidneys. Normal human adult blood can contain 7 mg to 21 mg of urea nitrogen per 100 ml of blood (7-21 mg / dL). Measurement of blood urea nitrogen levels is used as an indicator of kidney health. If the kidneys cannot remove urea from the blood normally, the subject's BUN level rises.

[0058] "Elevated" means an increase in a medical parameter that is considered clinically relevant. A health professional can determine whether the increase is clinically significant.

[0059] "End-stage renal disease (ESRD)" means complete or almost complete failure of kidney function.

[0060] "Quality of life" means the extent to which a subject's physical, mental, and social functioning is impaired by the disease and / or its treatment. A subject with polycystic kidney disease may have a reduced quality of life.

[0061] "Impaired renal function" means a decrease in renal function relative to normal renal function.

[0062] "Slowing the progression of" and "slowing progression" means reducing the rate at which a medical condition moves toward an advanced state.

[0063] "Delaying the time to dialysis" means maintaining adequate renal function, thereby delaying the need for dialysis treatment.

[0064] "Delaying time to kidney transplantation" means maintaining adequate kidney function, thereby delaying the need for a kidney transplant.

[0065] "Improving life expectancy" means extending the life of a subject by treating one or more symptoms of a disease in the subject.

[0066] "Subject" means a human or non-human animal selected for treatment or therapy.

[0067] “A subject in need thereof means a subject identified as being in need of therapy or treatment.

[0068] A "subject suspected of having" means a subject who exhibits one or more clinical indicators of a disease.

[0069] By "disease associated with miR-17" is meant a disease or condition that is modulated by the activity of one or more miR-17 family members.

[0070] "Administering" means providing a pharmaceutical agent or pharmaceutical composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0071] "Parenteral administration" means administration by injection or infusion.

[0072] Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

[0073] "Subcutaneous administration" means administration just beneath the skin.

[0074] "Intravenous administration" means administration into a vein.

[0075] "Concomitant administration" refers to the co-administration of two or more agents at the same time in any manner, wherein the pharmacological effects of both agents are manifested in the patient.

[0076] Concomitant administration does not require that the two agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of the two agents themselves do not need to manifest themselves simultaneously. The effects only need to overlap for a period of time and do not need to be co-extended.

[0077] "Duration" means a period of time over which an activity or event continues. In certain embodiments, the duration of treatment is a period of time over which multiple doses of a pharmaceutical agent or pharmaceutical composition are administered.

[0078] "Therapy" means a method of treating a disease. In certain embodiments, therapy includes, but is not limited to, administering one or more pharmaceutical agents to a subject suffering from a disease.

[0079] "Treatment" means applying one or more specific procedures for alleviating at least one indicator of a disease. In certain embodiments, the specific procedure is the administration of one or more agents. In certain embodiments, the treatment of PKD includes, but is not limited to, reducing total kidney volume, improving renal function, reducing hypertension, and / or reducing renal pain.

[0080] "Relieve" means to reduce the severity of at least one indicator of a condition or disease. In certain embodiments, relief includes delaying or slowing the progression of one or more indicators of a condition or disease. The severity of an indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0081] "At risk of developing" means a state in which a subject is susceptible to developing a condition or disease. In certain embodiments, a subject at risk of 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 of developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a lesser degree required to be diagnosed with the condition or disease.

[0082] "Preventing the onset of" means preventing the development of a disease or condition in a subject at risk of developing the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives treatment similar to that received by a subject already suffering from the disease or condition.

[0083] "Delaying the onset of" means delaying the development of a disease or condition in a subject at risk of developing the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives a treatment similar to that received by a subject already suffering from the disease or condition.

[0084] "Dose" means a specified amount of a medicament provided in a single administration. In certain embodiments, a dose can be administered as two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume that is not easily provided by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose can be administered as two or more injections to minimize injection site reactions in an individual. In certain embodiments, a dose is administered as a slow infusion.

[0085] "Dosage unit" means the form in which a medicament is provided. In certain embodiments, a dosage unit is a vial containing a lyophilized oligonucleotide. In certain embodiments, a dosage unit is a vial containing a reconstituted oligonucleotide.

[0086] A "therapeutically effective amount" refers to an amount of an agent that provides a therapeutic benefit to an animal.

[0087] "Pharmaceutical composition" means a mixture of substances, including a medicament, suitable for administration to an individual. For example, a pharmaceutical composition may comprise a sterile aqueous solution.

[0088] "Pharmaceutical agent" means a substance that provides a therapeutic effect when administered to a subject.

[0089] "Active pharmaceutical ingredient" means the substance in a pharmaceutical composition that provides the desired effect.

[0090] "Pharmaceutically acceptable salts" means physiologically and pharmaceutically acceptable salts of compounds provided herein, i.e., salts that retain the desired biological activity of the compound and do not have undesirable toxicological effects when administered to a subject. Non-limiting exemplary pharmaceutically acceptable salts of compounds provided herein include sodium salt forms and potassium salt forms. Unless specifically indicated otherwise, the terms "compound", "oligonucleotide" and "modified oligonucleotide" as used herein include pharmaceutically acceptable salts thereof.

[0091] "Saline solution" means a solution of sodium chloride in water.

[0092] "Improved organ function" means a change in organ function toward normal limits. In certain embodiments, organ function is assessed by measuring molecules present in the blood or urine of the subject. For example, in certain embodiments, improved renal function is measured by a decrease in blood urea nitrogen levels, a decrease in proteinuria, a decrease in albuminuria, etc.

[0093] By "acceptable safety profile" is meant a pattern of adverse effects that is within clinically acceptable limits.

[0094] "Side effects" means physiological responses other than the desired effects that are attributable to the treatment. In certain embodiments, side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal renal function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. Such side effects can be detected directly or indirectly. For example, an increase in transaminase levels in serum can indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin can indicate hepatotoxicity or abnormal liver function.

[0095] The term "blood" as used herein encompasses whole blood and blood fractions, such as serum and plasma.

[0096] "Anti-miR" means an oligonucleotide having a nucleobase sequence complementary to a microRNA. In certain embodiments, the anti-miR is a modified oligonucleotide.

[0097] "Anti-miR-17" means a modified oligonucleotide having a nucleobase sequence complementary to one or more miR-17 family members. In certain embodiments, anti-miR-17 is fully complementary to one or more miR-17 family members (i.e., 100% complementary). In certain embodiments, anti-miR-17 is at least 80%, at least 85%, at least 90%, or at least 95% complementary to one or more miR-17 family members.

[0098] "miR-17" means the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO: 1).

[0099] "miR-20a" means the mature miRNA having the nucleobase sequence 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' (SEQ ID NO: 2).

[0100] "miR-20b" refers to the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUCAUAGUGCAGGUAG-3' (SEQ ID NO: 3).

[0101] "miR-93" means the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUGUUCGUGCAGGUAG-3' (SEQ ID NO: 4).

[0102] "miR-106a" means the mature miRNA having the nucleobase sequence 5'-AAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO: 5).

[0103] "miR-106b" refers to the mature miRNA having the nucleobase sequence 5'-UAAAGUGCUGACAGUGCAGAU-3' (SEQ ID NO: 6).

[0104] "miR-17 seed sequence" means the nucleobase sequence 5'-AAAGUG-3', which is present in every miR-17 family member.

[0105] "miR-17 family member" means a mature miRNA having a nucleobase sequence comprising the miR-17 seed sequence, and which is selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a and miR-106b.

[0106] "miR-17 family" means the following group of miRNAs: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b, each having a nucleobase sequence comprising the miR-17 seed sequence.

[0107] "Target nucleic acid" means a nucleic acid to which an oligomeric compound is designed to hybridize.

[0108] "Targeting" refers to the process of designing and selecting nucleobase sequences that will hybridize to a target nucleic acid.

[0109] "Targeting" means having a nucleobase sequence that will permit hybridization to a target nucleic acid.

[0110] "Modulate" means to interfere with a function, amount or activity. In certain embodiments, modulate means to increase a function, amount or activity. In certain embodiments, modulate means to decrease a function, amount or activity.

[0111] "Expression" means any function or step by which the coded information of a gene is converted into a structure that exists and functions in a cell.

[0112] "Nucleobase sequence" means the order of contiguous nucleobases in an oligomeric compound or nucleic acid, typically listed in 5' to 3' orientation without regard to any sugar, linkage, and / or nucleobase modifications.

[0113] "Consecutive nucleobases" means nucleobases that are immediately adjacent to each other in a nucleic acid.

[0114] "Nucleobase complementarity" refers to the ability of two nucleobases to pair non-covalently via hydrogen bonding.

[0115] "Complementary" means that one nucleic acid is capable of hybridizing to another nucleic acid or oligonucleotide. In certain embodiments, complementary means that an oligonucleotide is capable of hybridizing to a target nucleic acid.

[0116] "Complete complementarity" means that each nucleobase of an oligonucleotide can be paired with a nucleobase at each corresponding position in a target nucleic acid. In certain embodiments, an oligonucleotide is fully complementary to a microRNA (also referred to as 100% complementary), that is, each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. The modified oligonucleotide can be fully complementary to a microRNA and has a plurality of connected nucleosides less than the length of the microRNA. For example, an oligonucleotide with 16 connected nucleosides is fully complementary to a microRNA, wherein each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. In certain embodiments, an oligonucleotide with complementarity to a nucleobase in a region of a microRNA stem-loop sequence is fully complementary to the microRNA stem-loop sequence.

[0117] "Complementarity percentage" means the percentage of nucleobases in an oligonucleotide that are complementary to an equal length portion of a target nucleic acid. Complementarity percentage is calculated by dividing the number of nucleobases in the oligonucleotide that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

[0118] "Percent identity" means the number of nucleobases in the first nucleic acid that are identical to the nucleobases at the corresponding positions in the 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.

[0119] "Hybridization" means the annealing of complementary nucleic acids through nucleobase complementarity.

[0120] "Mismatch" means that a nucleobase in a first nucleic acid is not capable of Watson-Crick pairing with a nucleobase at a corresponding position in a second nucleic acid.

[0121] "Identical" in the context of nucleobase sequences means having the same nucleobase sequence regardless of sugar, linkage and / or nucleobase modifications and regardless of the methyl state of any pyrimidines present.

[0122] "microRNA" means an endogenous non-coding RNA with a length between 18 and 25 nucleobases, which is the product of the cleavage of precursor microRNA by enzyme Dicer. Examples of mature microRNAs can be found in the microRNA database called miRBase (microrna.sanger.ac.uk / ). In certain embodiments, microRNA is abbreviated as "miR".

[0123] "MicroRNA-regulated transcript" means a transcript regulated by a microRNA.

[0124] "Seed matching sequence" means a nucleobase sequence that is complementary to a seed sequence and has the same length as the seed sequence.

[0125] "Oligomeric compound" means a compound comprising a plurality of linked monomeric subunits. Oligomeric compounds include oligonucleotides.

[0126] "Oligonucleotide" means a compound comprising a plurality of linked nucleosides, each of which may independently of one another be modified or unmodified.

[0127] By "naturally occurring internucleoside linkage" is meant a 3' to 5' phosphodiester linkage between nucleosides.

[0128] "Natural sugar" means the sugar found in DNA (2'-H) or RNA (2'-OH).

[0129] "Internucleoside linkage" means a covalent linkage between adjacent nucleosides.

[0130] "Linked nucleosides" means nucleosides linked by a covalent linkage.

[0131] "Nucleobase" means a heterocyclic moiety capable of non-covalently pairing with another nucleobase.

[0132] "Nucleoside" means a nucleobase linked to a sugar moiety.

[0133] "Nucleotide" means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[0134] "Compounds comprising a modified oligonucleotide consisting of a plurality of linked nucleosides" means compounds comprising a modified oligonucleotide having a specified number of linked nucleosides. Thus, the compound may include additional substituents or conjugates. Unless otherwise indicated, the modified oligonucleotide does not hybridize with a complementary strand, and the compound does not include any additional nucleosides beyond those of the modified oligonucleotide.

[0135] "Modified oligonucleotide" means a single-stranded oligonucleotide having one or more modifications relative to naturally occurring termini, sugars, nucleobases and / or internucleoside linkages. A modified oligonucleotide may contain unmodified nucleosides.

[0136] "Modified nucleosides" means nucleosides with any changes compared to naturally occurring nucleosides. Modified nucleosides can have modified sugars and unmodified nucleobases. Modified nucleosides can have modified sugars and modified nucleobases. Modified nucleosides can have natural sugars and modified nucleobases. In certain embodiments, the modified nucleosides are bicyclic nucleosides. In certain embodiments, the modified nucleosides are non-bicyclic nucleosides.

[0137] "Modified internucleoside linkage" means any change from a naturally occurring internucleoside linkage.

[0138] "Phosphorothioate internucleoside linkage" means a linkage between nucleosides in which one of the non-bridging atoms is a sulfur atom.

[0139] "Modified sugar moiety" means a substitution and / or any change compared to the natural sugar.

[0140] By "unmodified nucleobase" is meant 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).

[0141] "5-methylcytosine" means a cytosine comprising a methyl group attached to the 5 position.

[0142] "Unmethylated cytosine" means a cytosine that does not have a methyl group attached to the 5-position.

[0143] "Modified nucleobase" means any nucleobase that is not an unmodified nucleobase.

[0144] "Sugar moiety" means a naturally occurring furanosyl or a modified sugar moiety.

[0145] By "modified sugar moiety" is meant a substituted sugar moiety or sugar surrogate.

[0146] "2'-O-methyl sugar" or "2'-OMe sugar" means a sugar with an O-methyl modification at the 2' position.

[0147] "2'-O-methoxyethyl sugar" or "2'-MOE sugar" means a sugar with an O-methoxyethyl modification at the 2' position.

[0148] "2'-Fluoro" or "2'-F" means a sugar having a fluorine modification at the 2' position.

[0149] " bicyclic sugar moiety " means the sugar moiety (including but not limited to furanosyl) of the modification comprising 4 to 7 rings, the sugar moiety of the modification comprises the bridging of two atoms connecting the 4 to 7 rings to form the second ring, thereby producing a bicyclic structure. In certain embodiments, the 4 to 7 rings are sugar rings. In certain embodiments, the 4 to 7 rings are furanosyl. In certain such embodiments, the bridging connects the 2'-carbon and 4'-carbon of furanosyl. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt and R-cEt.

[0150] "Locked nucleic acid (LNA) sugar moiety" means a nucleic acid comprising (CH 2)-O-bridged substituted sugar moiety.

[0151] "ENA sugar moiety" means a moiety comprising (CH 2 ) 2 -O bridged substituted sugar moiety.

[0152] "Constrained ethyl (cEt) sugar moiety" means a sugar moiety comprising CH (CH 3 )-O-bridged substituted sugar moieties. In certain embodiments, CH(CH 3 )-O bridges are constrained to be in the S orientation. In certain embodiments, CH(CH 3 )-O is constrained to be in the R orientation.

[0153] "S-cEt sugar moiety" means a moiety comprising an S-constrained CH (CH 3 )-O-bridged substituted sugar moiety.

[0154] "R-cEt sugar moiety" means a moiety comprising an R-constrained CH (CH 3 )-O-bridged substituted sugar moiety.

[0155] "2'-O-methyl nucleoside" means a 2'-modified nucleoside having a 2'-O-methyl sugar modification.

[0156] "2'-O-methoxyethyl nucleoside" means a 2'-modified nucleoside having a 2'-O-methoxyethyl sugar modification. The 2'-O-methoxyethyl nucleoside may contain a modified or unmodified nucleobase.

[0157] "2'-fluoro nucleoside" means a 2'-modified nucleoside having a 2'-fluoro sugar modification. A 2'-fluoro nucleoside may contain a modified or unmodified nucleobase.

[0158] "Bicyclic nucleoside" means a 2'-modified nucleoside having a bicyclic sugar moiety. Bicyclic nucleosides can have modified or unmodified nucleobases.

[0159] "cEt nucleoside" means a nucleoside comprising a cEt sugar moiety. The cEt nucleoside may comprise a modified or unmodified nucleobase.

[0160] "S-cEt nucleoside" means a nucleoside comprising an S-cEt sugar moiety.

[0161] "R-cEt nucleoside" means a nucleoside comprising an R-cEt sugar moiety.

[0162] "β-D-deoxyribonucleoside" means a naturally occurring DNA nucleoside.

[0163] "β-D-ribonucleoside" means a naturally occurring RNA nucleoside.

[0164] "LNA nucleoside" means a nucleoside comprising an LNA sugar moiety.

[0165] "ENA nucleoside" means a nucleoside comprising an ENA sugar moiety.

[0166] Overview

[0167] Polycystic kidney disease (PKD) is a hereditary form of nephropathy in which fluid-filled cysts develop in the kidneys, leading to adrenal insufficiency and usually to end-stage renal disease. Some PKDs are also characterized by kidney enlargement. Excessive proliferation of cysts is a hallmark pathological feature of PKD. In the management of PKD, the main goal of treatment is to manage symptoms such as hypertension and infection, maintain renal function, and prevent the onset of end-stage renal disease (ESRD), thereby improving the life expectancy of PKD subjects.

[0168] The miR-17 family members of the miR-17-92 cluster of microRNAs are upregulated in the mouse PKD model. Genetic deletion of the miR-17-92 cluster in the mouse PKD model reduces renal cyst growth, improves renal function and prolongs survival (Patel et al., PNAS, 2013; 110(26): 10765-10770). In the PKD experimental model, it has been shown that inhibition of miR-17 with research tool compounds reduces kidney weight-to-body weight ratio and improves renal function. In addition, miR-17 inhibition also curbs the proliferation and cyst growth of primary cultures derived from human donor cysts.

[0169] In order to identify inhibitors of one or more miR-17 family members that are sufficiently effective, safe and convenient for PKD subjects, about 200 modified oligonucleotides containing a nucleobase sequence complementary to the miR-17 seed sequence were designed, which have different lengths and chemical compositions. The length range of the compound is 9 to 20 connected nucleosides, and the number, type and position of the chemical modification of the compound are different. As pharmacology, pharmacokinetic behavior and safety cannot be simply predicted based on the chemical structure of the compound. In a series of experiments designed to eliminate compounds with unfavorable properties, the characteristics of the compound are evaluated in vitro and in vivo, including efficacy, efficacy, pharmacokinetic behavior, safety and metabolic stability. As described herein, each of nearly 200 compounds is first tested in several in vitro assays (e.g., efficacy, toxicology, metabolic stability) to identify compounds suitable for further testing in more complex in vivo assays (e.g., pharmacokinetic profiles, efficacy, toxicology). This screening process identifies the candidate agent RG4326 for the treatment of PKD. As shown herein, changes in the type and position of sugar moieties result in significant effects on the properties of the tested compounds, including potency and tissue distribution. RG4326 was selected as a candidate agent because it exhibited the most suitable pharmacodynamic, safety, and pharmacokinetic profiles relative to other compounds of the same length and nucleobase sequence but with different sugar modification patterns.

[0170] Certain compounds of the present invention

[0171] Provided herein are compounds comprising a modified oligonucleotide consisting of 9 linked nucleosides, wherein the modified oligonucleotide has the following nucleoside pattern in the 5' to 3' orientation:

[0172] N S N S N M N F N F N F N M N S N S

[0173] Wherein the nucleoside with the subscript "M" behind is a 2'-O-methyl nucleoside, the nucleoside with the subscript "F" behind is a 2'-fluoro nucleoside, and the nucleoside with the subscript "S" behind is an S-cEt nucleoside; and wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUU-3', wherein each cytosine is a non-methylated cytosine or a 5-methylcytosine; or a pharmaceutically acceptable salt thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 5'-AGCACUUUG-3', wherein each cytosine is a non-methylated cytosine or a 5-methylcytosine. In certain embodiments, each cytosine is a non-methylated cytosine. In some embodiments, each linkage is independently selected from a phosphodiester linkage and a thiophosphate linkage. In some embodiments, all linkages are thiophosphate linkages.

[0174] This article provides the structure of A S G S C M A F C F U F U M U S G S A compound wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and each cytosine is a non-methylated cytosine or a 5-methylcytosine; or a pharmaceutically acceptable salt thereof. In certain embodiments, each cytosine is a non-methylated cytosine. In some embodiments, each linkage is independently selected from a phosphodiester linkage and a phosphorothioate linkage. In some embodiments, all linkages are phosphorothioate linkages.

[0175] This article provides the structure of A S G S C M A F C F U F U M U S G S A compound wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and each cytosine is a non-methylated cytosine; or a pharmaceutically acceptable salt thereof. In some embodiments, each linkage is independently selected from a phosphodiester linkage and a phosphorothioate linkage. In some embodiments, all linkages are phosphorothioate linkages.

[0176] Provided herein are compounds comprising a modified oligonucleotide consisting of 9 linked nucleosides, wherein the modified oligonucleotide has the following nucleoside pattern in the 5' to 3' orientation:

[0177] N S N S N M N F N F N F N M N S N S

[0178] Wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and all linkages are phosphorothioate linkages; and wherein the nucleobase sequence of the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUU-3', wherein each cytosine is a non-methylated cytosine or a 5-methylcytosine; or a pharmaceutically acceptable salt thereof. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 5'-AGCACUUUG-3', wherein each cytosine is a non-methylated cytosine or a 5-methylcytosine. In certain embodiments, each cytosine is a non-methylated cytosine.

[0179] This article provides the structure of A S G S C M A F C F U F U M U S G S A compound wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, each cytosine is a non-methylated cytosine or a 5-methylcytosine, and all linkages are phosphorothioate linkages; or a pharmaceutically acceptable salt thereof. In certain embodiments, each cytosine is a non-methylated cytosine.

[0180] This article provides the structure of A S G S C M A F C F U F U M U S G SA compound wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, each cytosine is a non-methylated cytosine, and all linkages are phosphorothioate linkages; or a pharmaceutically acceptable salt thereof.

[0181] Provided herein is a modified oligonucleotide named RG4326, wherein the structure of the modified oligonucleotide is:

[0182] Provided herein are pharmaceutically acceptable salts of modified oligonucleotides RG4326. Thus, in some embodiments, the modified oligonucleotides have the following structure:

[0183] Or a pharmaceutically acceptable salt thereof. A non-limiting exemplary pharmaceutically acceptable salt of RG4326 has the following structure:

[0184]

[0185] In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide comprises fewer cationic counterions (such as Na + ) (i.e., some of the phosphorothioate and / or phosphodiester linkages are protonated). In some embodiments, the pharmaceutically acceptable salt of RG4326 comprises less than 8 cationic counterions (such as, Na + ) / molecule of RG4326. That is, in some embodiments, the pharmaceutically acceptable salt of RG4326 may contain an average of 1, 2, 3, 4, 5, 6, or 7 cationic counterions / molecule of RG4326, with the remaining phosphorothioate groups being protonated.

[0186] Some uses of the present invention

[0187] Provided herein are methods for inhibiting the activity of one or more members of the miR-17 family in a cell, comprising contacting the cell with a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0188] Provided herein are methods for inhibiting the activity of one or more members of the miR-17 family in a subject, comprising administering to the subject a pharmaceutical composition provided herein. In certain embodiments, the subject suffers from a disease associated with one or more members of the miR-17 family.

[0189] Provided herein is a method for treating polycystic kidney disease (PKD), comprising administering a compound provided herein to a subject in need thereof, comprising a nucleobase sequence complementary to the miR-17 seed sequence. In certain embodiments, the subject suffers from polycystic kidney disease. In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD) and nephronophthisis (NPHP). In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD).

[0190] In certain embodiments, the subject suffers from a disease characterized by a variety of non-renal indicators, and also characterized by polycystic kidney disease. Such diseases include, for example, Joubert syndrome and related conditions (JSRD), Meckel syndrome (Meckel syndrome, MKS) or Bardet-Biedl syndrome (Bardet-Biedlsyndrome, BBS). Therefore, provided herein is a method for treating polycystic kidney disease (PKD), including administering a compound provided herein to a subject, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject suffers from Joubert syndrome and related conditions (JSRD), Meckel syndrome (MKS) or Bardet-Biedl syndrome (BBS). Provided herein is a method for treating polycystic kidney disease (PKD), including administering a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject is suspected of suffering from Joubert syndrome and related conditions (JSRD), Meckel syndrome (MKS) or Bardet-Biedl syndrome (BBS).

[0191] In certain embodiments, polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in PKD1 or PKD2 genes. ADPKD is a progressive disease in which cyst formation and kidney enlargement lead to renal insufficiency and 50% of patients eventually lead to end-stage renal disease at the age of 60. ADPKD patients may need lifelong dialysis and / or renal transplantation. ADPKD is the most common genetic cause of renal failure. Excessive proliferation of cysts is a hallmark pathological feature of ADPKD. In the management of PKD, the main goal of treatment is to maintain renal function and prevent the onset of end-stage renal disease (ESRD), thereby improving the life expectancy of PKD subjects. The total renal volume of ADPKD patients usually increases steadily, increasing in association with a decline in renal function. Provided herein is a method for treating ADPKD, comprising administering a compound provided herein to a subject suffering from or suspected of suffering from ADPKD, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0192] In certain embodiments, polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). ARPKD is caused by mutations in the PKHD1 gene and is the cause of chronic kidney disease in children. The typical ARPKD renal phenotype is kidney enlargement; however, ARPKD has a significant impact on other organs, especially the liver. Patients with ARPKD progress to end-stage renal disease and require kidney transplantation at the age of 15. Provided herein is a method for treating ARPKD, comprising administering a compound provided herein to a subject suffering from or suspected of having ARPKD, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0193] In certain embodiments, polycystic kidney disease is nephronophthisis (NPHP). Nephronophthisis is an autosomal recessive cystic kidney disease that is a common cause of ESRD in children. NPHP is characterized by normal or reduced kidney size, cysts concentrated at the cortical medullary junction, and tubulointerstitial fibrosis. Mutations in one of several NPHP genes, such as NPHP1, have been identified in NPHP patients. Provided herein is a method for treating NPHP, comprising administering to a subject suffering from or suspected of having NPHP a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0194] In certain embodiments, the subject with polycystic kidney disease suffers from Joubert syndrome and related conditions (JSRD).JSRD includes extensive hallmark features, including brain, retinal and skeletal abnormalities.In addition to the hallmark features of JSRD, some subjects with JSRD suffer from polycystic kidney disease.Therefore, provided herein is a method for treating polycystic kidney disease in a subject with JSRD, comprising administering a compound provided herein to a subject with JSRD, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.In certain embodiments, the subject is suspected of suffering from JSRD.

[0195] In certain embodiments, the subject with polycystic kidney disease suffers from Meckel's syndrome (MKS). MKS is a disease with severe signs and symptoms in many parts of the body including the central nervous system, skeletal system, liver, kidneys and heart. The common feature of MKS is the presence of many fluid-filled cysts in the kidneys, as well as enlarged kidneys. Therefore, provided herein is a method for treating MKS, comprising administering a compound provided herein to a subject with MKS, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In certain embodiments, the subject is suspected of suffering from MKS.

[0196] In certain embodiments, the subject with polycystic kidney disease suffers from Bardet-Bieder syndrome (BBS). BBS is a disease affecting many parts of the body including the eyes, heart, kidneys, liver, and digestive system. The hallmark feature of BBS is the presence of renal cysts. Therefore, provided herein is a method for treating polycystic kidney disease in a subject with BBS, comprising administering a compound provided herein to a subject with BBS, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In certain embodiments, the subject is suspected of suffering from BBS.

[0197] In certain embodiments, before using the compound comprising the oligonucleotide of modification, the subject has been diagnosed as suffering from PKD.The diagnosis of PKD can be realized by evaluating parameters, including but not limited to the subject's family history, clinical features (including but not limited to hypertension, albuminuria, hematuria and GFR impaired), renal imaging studies (including but not limited to MRI, ultrasound and CT scanning) and / or histological analysis.

[0198] In certain embodiments, the diagnosis of PKD includes screening for mutations in one or more PKD1 or PKD2 genes. In certain embodiments, the diagnosis of ARPKD includes screening for mutations in PKHP1 genes. In certain embodiments, the diagnosis of NPHP includes screening for one or more mutations in one or more of NPHP1, NPHP2, NPHP3, NPHP4, NPHP5, NPHP6, NPHP7, NPHP8 or NPHP9 genes. In certain embodiments, the diagnosis of JSRD includes screening for mutations in NPHP1, NPHP6, AHI1, MKS3 or RPGRIP1L genes. In certain embodiments, the diagnosis of MKS includes screening for mutations in NPHP6, MKS3, RPGRIP1L, NPHP3, CC2D2A, BBS2, BBS4, BBS6 or MKS1 genes. In certain embodiments, diagnosis of BBS comprises screening for mutations in the BBS2, BBS4, BBS6, MKS1, BBS1, BBS3, BBS5, BBS7, BBS7, BBS8, BBS9, BBS10, BBS11, or BBS12 genes.

[0199] In certain embodiments, the subject has an increased total kidney volume. In certain embodiments, the total kidney volume is height-adjusted total kidney volume (HtTKV). In certain embodiments, the subject suffers from hypertension. In certain embodiments, the subject suffers from impaired renal function. In certain embodiments, the subject needs improved renal function. In certain embodiments, the subject is identified as suffering from impaired renal function.

[0200] In certain embodiments, the level of one or more miR-17 family members increases in the kidney of a subject suffering from PKD. In certain embodiments, prior to administration, it is determined that the subject has one or more miR-17 family members with increased levels in the kidney. The level of miR-17 family members can be measured from renal biopsy material. In certain embodiments, prior to administration, it is determined that the subject has one or more miR-17 family members with increased levels in the urine or blood of the subject.

[0201] In any of the embodiments provided herein, the subject can undergo certain tests to diagnose polycystic kidney disease in the subject, for example, to determine the cause of polycystic kidney disease, to evaluate the extent of polycystic kidney disease in the subject, and / or to determine the subject's response to treatment. Such tests can assess the markers of polycystic kidney disease. Some of these tests, such as glomerular filtration rate and blood urea nitrogen levels, are also indicators of renal function. Markers of polycystic disease include, but are not limited to: measuring the total renal volume of the subject; measuring hypertension in the subject; assessing renal pain in the subject; measuring fibrosis in the subject; measuring the blood urea nitrogen level in the subject; measuring the serum creatinine level in the subject; measuring the creatinine clearance rate in the subject; measuring albuminuria in the subject; measuring the albumin: creatinine ratio in the subject; measuring the glomerular filtration rate in the subject; measuring hematuria in the subject; measuring NGAL protein in the urine of the subject; and / or measuring KIM-1 protein in the urine of the subject. Unless otherwise indicated herein, blood urea nitrogen level, serum creatinine level, creatinine clearance, albuminuria, albumin to creatinine ratio, glomerular filtration rate, and hematuria refer to measurements in the blood (such as whole blood or serum) of a subject.

[0202] The markers of polycystic kidney disease are determined by laboratory tests. The reference ranges of individual markers may vary from laboratory to laboratory. The variation may be due to, for example, the differences in the specific assays used. Therefore, the upper and lower limits of the normal distribution of markers within a population, also referred to as the upper limit of normal (ULN) and the lower limit of normal (LLN), may vary from laboratory to laboratory. For any particular marker, a health professional may determine which levels outside the normal distribution are clinically relevant and / or indicate a disease. For example, a health professional may determine a glomerular filtration rate, which may indicate the rate of decline in renal function in a subject with polycystic kidney disease.

[0203] In certain embodiments, the administration of the compounds provided herein results in one or more clinically beneficial results. In certain embodiments, administration improves the renal function of the subject. In certain embodiments, administration slows down the rate of decline of renal function in the subject. In certain embodiments, administration reduces the total renal volume of the subject. In certain embodiments, administration slows down the rate of increase of the total renal volume in the subject. In certain embodiments, administration reduces the total renal volume (HtTKV) adjusted for height. In certain embodiments, administration slows down the rate of increase of HtTKV.

[0204] In certain embodiments, administration inhibits cyst growth in a subject. In certain embodiments, administration slows the increased rate of cyst growth in a subject. In some embodiments, the cyst is present in the kidney of the subject. In some embodiments, the cyst is present in an organ other than the kidney, such as the liver.

[0205] In certain embodiments, administration relieves renal pain in a subject. In certain embodiments, administration slows down an increase in renal pain in a subject. In certain embodiments, administration delays the onset of renal pain in a subject.

[0206] In certain embodiments, administration reduces hypertension in a subject. In certain embodiments, administration slows the worsening of hypertension in a subject. In certain embodiments, administration delays the onset of hypertension in a subject.

[0207] In certain embodiments, administering reduces fibrosis in the kidney of a subject. In certain embodiments, administering slows the progression of fibrosis in the kidney of a subject.

[0208] In certain embodiments, administration delays the onset of end-stage renal disease in a subject. In certain embodiments, administration delays the time a subject undergoes dialysis. In certain embodiments, administration delays the time a subject undergoes a kidney transplant. In certain embodiments, administration improves the life expectancy of a subject.

[0209] In certain embodiments, administration reduces albuminuria in a subject. In certain embodiments, administration slows the worsening of albuminuria in a subject. In certain embodiments, administration delays the onset of albuminuria in a subject. In certain embodiments, administration reduces hematuria in a subject. In certain embodiments, administration slows the worsening of hematuria in a subject. In certain embodiments, administration delays the onset of hematuria in a subject. In certain embodiments, administration reduces blood urea nitrogen levels in a subject. In certain embodiments, administration reduces serum creatinine levels in a subject. In certain embodiments, administration improves creatinine clearance in a subject. In certain embodiments, administration reduces the albumin: creatinine ratio in a subject.

[0210] In certain embodiments, administration improves the glomerular filtration rate of the subject. In certain embodiments, administration slows down the rate of decline of the glomerular filtration rate in the subject. In certain embodiments, the glomerular filtration rate is an estimated glomerular filtration rate (eGFR). In certain embodiments, the glomerular filtration rate is a measured glomerular filtration rate (mGFR).

[0211] In certain embodiments, administration reduces neutrophil gelatinase associated lipocalin (NGAL) protein in the urine of a subject. In certain embodiments, administration reduces kidney injury molecule-1 (KIM-1) protein in the urine of a subject.

[0212] In any of the embodiments provided herein, certain tests may be performed on a subject to evaluate the extent of the disease in the subject. These tests include, but are not limited to, measuring the total renal volume of the subject; measuring hypertension in the subject; measuring renal pain in the subject; measuring fibrosis in the kidneys of the subject; measuring blood urea nitrogen levels in the subject; measuring serum creatinine levels in the subject; measuring creatinine clearance in the blood of the subject; measuring albuminuria in the subject; measuring albumin: creatinine ratio in the subject; measuring glomerular filtration rate in the subject, wherein the glomerular filtration rate is estimated or measured; measuring neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject; and / or measuring kidney injury molecule-1 (KIM-1) protein in the urine of the subject.

[0213] In certain embodiments, the quality of life of a subject with polycystic kidney disease is reduced. For example, a subject with polycystic kidney disease may experience kidney pain, which may reduce the quality of life of the subject. In certain embodiments, administration improves the quality of life of the subject.

[0214] In any of the embodiments provided herein, the subject is a human subject. In certain embodiments, the human subject is an adult. In certain embodiments, an adult is at least 21 years old. In certain embodiments, the human subject is a pediatric subject, i.e., the subject is less than 21 years old. The pediatric population can be determined by a regulatory agency. In certain embodiments, the human subject is a teenager. In certain embodiments, the teenager is at least 12 years old and less than 21 years old. In certain embodiments, the human subject is a child. In certain embodiments, the child is at least two years old and less than 12 years old. In certain embodiments, the human subject is an infant. In certain embodiments, the infant is at least one month old and less than two years old. In certain embodiments, the subject is a neonate. In certain embodiments, the neonate is less than one month.

[0215] Any compound described herein can be used in therapy. Any compound provided herein can be used to treat polycystic kidney disease. In certain embodiments, polycystic kidney disease is autosomal dominant polycystic kidney disease. In certain embodiments, polycystic kidney disease is autosomal recessive polycystic kidney disease. In certain embodiments, polycystic kidney disease is nephronophthisis. In certain embodiments, the subject suffers from Joubert syndrome and related conditions (JSRD), Meckel syndrome (MKS) or Budd-Bieder syndrome (BBS).

[0216] Any modified oligonucleotide described herein can be used in therapy.Any modified oligonucleotide provided herein can be used to treat polycystic kidney disease.

[0217] Any compound provided herein can be used to prepare a medicament. Any compound provided herein can be used to prepare a medicament for treating polycystic kidney disease.

[0218] Any modified oligonucleotide provided herein can be used to prepare a medicament. Any modified oligonucleotide provided herein can be used to prepare a medicament for treating polycystic kidney disease.

[0219] Any of the pharmaceutical compositions provided herein can be used to treat polycystic kidney disease.

[0220] Some additional treatments

[0221] Treatment of polycystic kidney disease or any condition listed herein may include more than one therapy. Thus, in certain embodiments, provided herein are methods for treating a subject suffering from or suspected of suffering from polycystic kidney disease, comprising administering at least one therapy in addition to administering a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0222] In certain embodiments, the at least one additional therapy comprises a pharmaceutical agent.

[0223] In certain embodiments, the pharmaceutical agent is an antihypertensive agent.Antihypertensive agents are used to control a subject's blood pressure.

[0224] In certain embodiments, the pharmaceutical agent is a vasopressin receptor 2 antagonist. In certain embodiments, the vasopressin receptor 2 antagonist is tolvaptan.

[0225] In certain embodiments, the pharmaceutical agent comprises an angiotensin II receptor blocker (ARB). In certain embodiments, the angiotensin II receptor blocker is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan or eprosartan.

[0226] In certain embodiments, the pharmaceutical agent comprises angiotensin II converting enzyme (ACE) inhibitor. In certain embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril or ramipril.

[0227] In certain embodiments, the pharmaceutical agent is a diuretic. In certain embodiments, the pharmaceutical agent is a calcium channel blocker.

[0228] In certain embodiments, the pharmaceutical agent is a kinase inhibitor. In certain embodiments, the kinase inhibitor is bosutinib or KD019.

[0229] In certain embodiments, the pharmaceutical agent is an adrenergic receptor antagonist.

[0230] In certain embodiments, the medicament is an aldosterone receptor antagonist. In certain embodiments, the aldosterone receptor antagonist is spironolactone. In certain embodiments, spironolactone is administered at a dosage of 10 mg to 35 mg per day. In certain embodiments, spironolactone is administered at a dosage of 25 mg per day.

[0231] In certain embodiments, the agent is a mammalian target of rapamycin (mTOR) inhibitor. In certain embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

[0232] In certain embodiments, the pharmaceutical agent is a hormone analog. In certain embodiments, the hormone analog is somatostatin or adrenocorticotropic hormone.

[0233] In certain embodiments, the agent is an anti-fibrotic agent. In certain embodiments, the anti-fibrotic agent is a modified oligonucleotide complementary to miR-21.

[0234] In certain embodiments, the additional therapy is dialysis. In certain embodiments, the additional therapy is kidney transplantation.

[0235] In certain embodiments, the medicament comprises an anti-inflammatory agent. In certain embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In certain embodiments, the steroidal anti-inflammatory agent is a corticosteroid. In certain embodiments, the corticosteroid is prednisone. In certain embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug. In certain embodiments, the non-steroidal anti-inflammatory agent is ibuprofen, a COX-1 inhibitor, or a COX-2 inhibitor.

[0236] In certain embodiments, the agent is one that blocks one or more responses to a fibrotic signal.

[0237] In certain embodiments, additional therapies may be agents that enhance the body's immune system, including low-dose cyclophosphamide, thymus-stimulating hormone, vitamins and nutritional supplements (e.g., antioxidants, including vitamin A, vitamin C, vitamin E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea), and vaccines, such as immunostimulatory complexes (ISCOMs) that comprise vaccine formulations combining antigens in multimeric form and adjuvants.

[0238] In certain embodiments, additional therapies are selected to treat or mitigate side effects of one or more pharmaceutical compositions of the invention. Such side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal renal function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, increased transaminase levels in serum can indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin can indicate hepatotoxicity or abnormal liver function.

[0239] Certain microRNA nucleobase sequences

[0240] The miR-17 family includes miR-17, miR-20a, miR-20b, miR-93, miR-106a and miR-106b. Each member of the miR-17 family has a core base sequence or a miR-17 seed sequence comprising a core base sequence 5'-AAAGUG-3', which is a core base sequence of positions 2 to 7 of SEQ ID NO:1. In addition, each member of the miR-17 family shares some core base sequence identities outside the seed region. Therefore, in addition to miR-17, the modified oligonucleotides comprising a core base sequence complementary to the miR-17 seed sequence can target other microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotides target two or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotides target three or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotides target four or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotide targets five or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotide targets six microRNAs of the miR-17 family. For example, the modified oligonucleotide with the core base sequence 5'-AGCACUUUG-3' targets all members of the miR-17 family.

[0241] In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence of 5'-CACUUU-3'. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence of 5'-GCACUUUG-3'. In certain embodiments, the modified oligonucleotide comprises a nucleobase sequence of 5'-AGCACUUU-3'. In certain embodiments, the nucleobase sequence of the modified oligonucleotide is 5'-AGCACUUUG-3'.

[0242] In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTUT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTU-3'.

[0243] In certain embodiments, each cytosine is independently selected from non-methylated cytosine and 5-methylcytosine. In certain embodiments, at least one cytosine is non-methylated cytosine. In certain embodiments, each cytosine is non-methylated cytosine. In certain embodiments, at least one cytosine is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0244] In certain embodiments, the number of nucleosides connected to the modified oligonucleotide is less than the length of its target microRNA. The modified oligonucleotide with multiple connected nucleosides is considered to be a modified oligonucleotide with a nucleobase sequence that is completely complementary (also referred to as 100% complementary) to the region of the target microRNA sequence, and the length of the modified oligonucleotide is less than the length of the target microRNA, wherein each nucleobase of the modified oligonucleotide is complementary to the nucleobase at the corresponding position of the target microRNA. For example, the modified oligonucleotide consisting of 9 connected nucleosides is completely complementary to miR-17, wherein each nucleobase is complementary to the corresponding position of miR-17.

[0245] In certain embodiments, the oligonucleotide of modification has a core base sequence, and the core base sequence has a mispairing relative to the core base sequence of the target micro RNA. In certain embodiments, the oligonucleotide of modification has a core base sequence, and the core base sequence has two mispairings relative to the core base sequence of the target micro RNA. In certain such embodiments, the oligonucleotide of modification has a core base sequence, and the core base sequence has no more than two mispairings relative to the core base sequence of the target micro RNA. In certain such embodiments, the core base of mispairing is continuous. In certain such embodiments, the core base of mispairing is not continuous.

[0246] Although the sequence listing accompanying this document identifies each nucleobase sequence as a desired "RNA" or "DNA", in fact, these sequences may be modified with combinations of chemical modifications specified herein. Those skilled in the art will readily appreciate that in the sequence listing, names such as "RNA" or "DNA" describing modified oligonucleotides are arbitrary to some extent. For example, a modified oligonucleotide comprising a nucleoside containing a 2'-O-methoxyethyl sugar moiety and a thymine base provided herein may be described as a DNA residue in the sequence listing, even though the nucleoside is modified and is not a natural DNA nucleoside.

[0247] Therefore, the nucleic acid sequences provided in the sequence listing are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids with modified nucleobases. As another example and not limitation, the modified oligonucleotides with the nucleobase sequence "ATCGATCG" in the sequence listing encompass any oligonucleotides with such nucleobase sequences, whether modified or unmodified, including but not limited to such compounds containing RNA bases, such as those with the sequence "AUCGAUCG" and those with some DNA bases and some RNA bases such as "AUCGATCG" and bases with other modifications such as "ATCGATCG". me CGAUCG" oligonucleotide, me C indicates 5-methylcytosine.

[0248] Some modifications

[0249] In certain embodiments, the oligonucleotide provided herein can comprise one or more modifications to a core base, sugar and / or internucleoside linkage, and is therefore a modified oligonucleotide. The modified core base, sugar and / or internucleoside linkage can be selected in preference to unmodified forms due to desirable properties such as enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases.

[0250] In certain embodiments, a modified oligonucleotide comprises one or more modified nucleobases.

[0251] In certain embodiments, the nucleoside of modification is a sugar-modified nucleoside. In certain such embodiments, the sugar-modified nucleoside can also include a natural or modified heterocyclic base moiety and / or can be connected to another nucleoside by a natural or modified internucleoside linkage and / or can include additional modifications that are independent of sugar modification. In certain embodiments, the 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.

[0252] In certain embodiments, the nucleoside of 2'-modification has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar of α configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar of β configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar of α configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar of β configuration.

[0253] Nucleosides containing 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′-CH 2-O-2')BNA; (B)β-D-methyleneoxy (4'-CH 2 -O-2')BNA; (C)ethyleneoxy (4'-(CH 2 ) 2 -O-2')BNA; (D)aminooxy (4'-CH 2 -ON(R)-2')BNA; (E)oxyamino(4'-CH 2 -N(R)-O-2')BNA; (F)methyl(methyleneoxy)(4'-CH(CH 3 )-O-2')BNA (also called constrained ethyl or cEt); (G) methylene-thio (4'-CH 2 -S-2')BNA; (H) Methylene-amino (4'-CH2-N(R)-2')BNA; (I) Methyl carbocyclic (4'-CH 2 -CH(CH 3 )-2')BNA; (J)c-MOE(4'-CH(CH 2 -OMe)-O-2')BNA; and (K) propylene carbocyclic ring (4'-(CH 2 ) 3 -2')BNA.

[0254]

[0255] wherein Bx is a nucleobase moiety and R is independently H, a protecting group or C 1 -C 12 alkyl.

[0256] In certain embodiments, the 2'-modified nucleoside comprises a moiety selected from the group consisting of F, OCF 3 、O-CH 3 (also known as "2'-OMe"), OCH 2 CH 2 OCH 3 (also known as "2'-O-methoxyethyl" or "2'-MOE"), 2'-O(CH 2 ) 2 SCH 3 、O-(CH 2 ) 2 -ON(CH 3 ) 2 、-O(CH 2 ) 2 O(CH 2 ) 2 N(CH 3 ) 2 and O-CH 2 -C(=O)-N(H)CH 3The 2'-substituted group.

[0257] In certain embodiments, the 2'-modified nucleoside comprises a moiety selected from the group consisting of F, O-CH 3 and OCH 2 CH 2 OCH 3 The 2'-substituted group.

[0258] In certain embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In certain embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. The 4'-thio-modified nucleoside has a β-D-ribonucleoside in which the 4'-O is replaced by a 4'-S. The 4'-thio-2'-modified nucleoside is a 4'-thio-modified nucleoside in which the 2'-OH is replaced by a 2'-substituted group. Suitable 2'-substituted groups include 2'-OCH 3 , 2'-OCH 2 CH 2 OCH 3 and 2′-F.

[0259] In certain embodiments, the modified oligonucleotide comprises one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage comprises a phosphorus atom.

[0260] In certain embodiments, the modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0261] In certain embodiments, the oligonucleotide of modification comprises one or more modified nucleobases. In certain embodiments, the nucleobase of modification is selected from 5-hydroxymethylcytosine, 7-deazaguanine and 7-deazaadenine. In certain embodiments, the nucleobase of modification is selected from 7-deaza-adenine, 7-deazaguanine, 2-aminopyridine and 2-pyridone. In certain embodiments, the nucleobase of modification is selected from 5-substituted pyrimidine, 6-azapyrimidine and N-2, N-6 and O-6 substituted purine, including 2 aminopropyl adenine, 5-propynyl uracil and 5-propynyl cytosine.

[0262] In certain embodiments, the modified nucleobase comprises a polycyclic heterocycle. In certain embodiments, the modified nucleobase comprises a tricyclic heterocycle. In certain embodiments, the modified nucleobase comprises a phenoxazine derivative. In certain embodiments, the phenoxazine can be further modified to form a G-clamped nucleobase known in the art.

[0263] In certain embodiments, the oligonucleotide of modification is conjugated to one or more antisense oligonucleotides that enhance the activity, cellular distribution or cellular uptake of the antisense oligonucleotides produced. In certain such embodiments, the part is a cholesterol part. In certain embodiments, the part is a lipid part. The additional part for conjugation includes carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and dye. In certain embodiments, the carbohydrate part is N-acetyl-D-galactosamine (GalNac). In certain embodiments, the conjugated group is directly connected to the oligonucleotide. In certain embodiments, the conjugate group is linked to the modified oligonucleotide via a linking moiety selected from the group consisting of amino, azido, hydroxyl, carboxylic acid, sulfhydryl, unsaturated moieties (e.g., double or triple bonds), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (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, the substituent group is selected from the group consisting of hydroxyl, amino, alkoxy, azido, carboxyl, benzyl, phenyl, nitro, sulfhydryl, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0264] In some such embodiments, the compound comprises a modified oligonucleotide with one or more stabilizing groups, and the one or more stabilizing groups are connected to one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. Cap structures are included in the stabilizing group. These end modifications protect the modified oligonucleotide from exonuclease degradation, and can help to deliver and / or locate in the cell. The cap can be present in the 5'-end (5'-cap) or the 3'-end (3'-cap), or can be present on both ends. Cap structures include, for example, reverse deoxy abasic caps.

[0265] Certain pharmaceutical compositions

[0266] Provided herein is a pharmaceutical composition comprising a compound or modified oligonucleotide provided herein and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In certain embodiments, the aqueous solution is a saline solution. As used herein, a pharmaceutically acceptable diluent is understood to be a sterile diluent. Suitable routes of administration include, but are not limited to, intravenous and subcutaneous administration.

[0267] In certain embodiments, the pharmaceutical composition is administered in the form of a dosage unit. For example, in certain embodiments, the dosage unit is in the form of a tablet, capsule, or bolus injection.

[0268] In certain embodiments, the medicament is a modified oligonucleotide, which is prepared in a suitable diluent, adjusted to pH 7.0-9.0 with acid or alkali during preparation, and then lyophilized under aseptic conditions. The lyophilized modified oligonucleotide is then reconstructed with a suitable diluent such as an aqueous solution, such as water or physiologically compatible buffer such as saline solution, Hanks's solution or Ringer's solution. The reconstructed product is administered in a subcutaneous injection or intravenous infusion form. The lyophilized drug product can be packaged in a 2mL I type (ammonium sulfate treated) transparent glass vial, plugged with a bromobutyl rubber stopper and sealed with an aluminum top seal.

[0269] In certain embodiments, the pharmaceutical compositions provided herein may additionally contain other auxiliary components that are typically present in pharmaceutical compositions, and these auxiliary components have usage amounts determined in the art. Thus, for example, the composition may contain additional compatible pharmaceutically active materials, such as antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents.

[0270] In some embodiments, the pharmaceutical compositions provided herein may contain additional substances that can be used to physically prepare various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners and stabilizers; Such additional substances also include, but are not limited to, excipients, such as alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone. In various embodiments, when such substances are added, they should not excessively interfere with the biological activity of the components of the compositions of the present invention. The preparations can be sterilized and mixed with adjuvants such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, coloring substances, aromatic substances and / or aromatic substances, etc., if necessary, and the adjuvants do not interact harmfully with one or more oligonucleotides of the preparation. Some pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain preparatons, such as suspending agents, stabilizers and / or dispersants. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil; synthetic fatty acid esters, such as ethyl oleate or triglycerides; and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the agent to allow the preparation of a high concentration solution.

[0271] Lipid moieties have been used in nucleic acid therapy in various methods. In one method, the nucleic acid is introduced into a preformed liposome or a lipid complex made of a mixture of cationic lipids and neutral lipids. In another method, a DNA complex with a monocationic lipid or a polycationic lipid is formed in the absence of a neutral lipid. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to a specific cell or tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to adipose tissue. In certain embodiments, the lipid moiety is selected to increase the distribution of the agent to muscle tissue.

[0272] In certain embodiments, the pharmaceutical compositions provided herein comprise a polyamine compound or a lipid portion complexed with a nucleic acid. In certain embodiments, such formulations comprise one or more compounds each individually having a structure defined by formula (Z) or a pharmaceutically acceptable salt thereof,

[0273]

[0274] Each X a and X b C independently at each occurrence 1-6 alkylene; n is 0, 1, 2, 3, 4 or 5; each R is independently H, wherein at least n+2 R moieties in at least about 80% of the molecules of the compound of formula (Z) in the formulation are not H; m is 1, 2, 3 or 4; Y is O, NR 2 or S; R 1 is alkyl, alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; and R 2 R is H, alkyl, alkenyl or alkynyl; each of which is optionally substituted with one or more substituents; provided that, if n=0, at least n+3 R moieties are not H. Such formulations are described in PCT publication WO / 2008 / 042973, which is incorporated herein by reference in its entirety for the purpose of disclosing lipid formulations. Certain additional formulations are described in Akinc et al., Nature Biotechnology 26, 561-569 (May 1, 2008), which is incorporated herein by reference in its entirety for the purpose of disclosing lipid formulations.

[0275] In certain embodiments, the pharmaceutical compositions provided herein are prepared using known techniques, including, but not limited to, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.

[0276] In certain embodiments, the pharmaceutical compositions provided herein are solid (e.g., powders, tablets, and / or capsules). In certain such embodiments, solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using ingredients known in the art, including but not limited to starch, sugar, diluents, granulating agents, lubricants, binders, and disintegrants.

[0277] In certain embodiments, the pharmaceutical compositions provided herein are prepared as depot preparations. Some such depot preparations typically act longer than non-depot preparations. In certain embodiments, such preparations are administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymeric materials or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins or as slightly soluble derivatives such as as slightly soluble salts.

[0278] In certain embodiments, the pharmaceutical compositions provided herein include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are suitable for preparing certain pharmaceutical compositions, including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents are used, such as dimethyl sulfoxide.

[0279] In certain embodiments, the pharmaceutical compositions provided herein include one or more tissue-specific delivery molecules designed to deliver one or more agents of the invention to specific tissues or cell types. For example, in certain embodiments, the pharmaceutical compositions include liposomes coated with tissue-specific antibodies.

[0280] In certain embodiments, the pharmaceutical composition provided herein comprises a sustained release system. A non-limiting example of such sustained release system is a semi-permeable matrix of a solid hydrophobic polymer. In certain embodiments, sustained release systems can release medicaments over a period of hours, days, weeks, or months depending on their chemical properties.

[0281] Certain pharmaceutical compositions for injection are presented in unit dosage form, for example, in ampoules or in multi-dose containers.

[0282] In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a modified oligonucleotide. In certain embodiments, a therapeutically effective amount is sufficient to prevent, alleviate or relieve the symptoms of a disease or prolong the survival of the subject being treated.

[0283] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, when administered in vivo, prodrugs are chemically converted into more active forms biologically, pharmaceutically or therapeutically of oligonucleotides. In certain embodiments, prodrugs are useful because they are easier to administer than corresponding active forms. For example, in some cases, prodrugs can be more bioavailable (for example, by oral administration) than corresponding active forms. In some cases, compared with corresponding active forms, prodrugs can have improved solubility. In certain embodiments, prodrugs are less water-soluble than corresponding active forms. In some cases, such prodrugs have excellent ability to transmit across cell membranes, wherein water solubility is not conducive to mobility. In certain embodiments, prodrugs are esters. In certain such embodiments, the esters are metabolically hydrolyzed into carboxylic acids when administered. In certain cases, compounds containing carboxylic acids are corresponding active forms. In certain embodiments, prodrugs include short peptides (polyamino acids) bonded to acid groups. In certain such embodiments, the peptides are cracked to form corresponding active forms when administered.

[0284] In certain embodiments, prodrugs are produced by modifying pharmaceutically active compounds so that the active compounds will be regenerated when administered in vivo. The prodrugs can be designed to change the metabolic stability or transport characteristics of the drug, mask side effects or toxicity, improve the fragrance of the drug or change other characteristics or properties of the drug. With the knowledge of pharmacodynamic processes and drug metabolism in vivo, those skilled in the art, once understanding pharmaceutically active compounds, can design prodrugs of compounds (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392).

[0285] Additional routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, by inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In certain embodiments, pharmaceutical intrathecal drugs are administered to achieve local exposure rather than systemic exposure. For example, the pharmaceutical composition can be injected directly in the area of ​​desired effect (e.g., into the kidney).

[0286] Certain kits

[0287] The present invention also provides a kit. In some embodiments, the kit comprises one or more compounds comprising a modified oligonucleotide disclosed herein. In some embodiments, the kit can be used to administer the compound to a subject.

[0288] In certain embodiments, the kit comprises a pharmaceutical composition ready for administration. In certain embodiments, the pharmaceutical composition is present in a vial. A plurality of vials, e.g., 10, may be present in, e.g., a dispensing package. In some embodiments, the vials are made so as to be accessible with a syringe. The kit may also contain instructions for use of the compound.

[0289] In some embodiments, the kit comprises the pharmaceutical composition in a pre-filled syringe (such as a single-dose syringe having, for example, a 27-gauge, 1 / 2-inch needle with a needle shield), rather than in a vial. A plurality of pre-filled syringes, for example, 10, may be present in, for example, a dispensing package. The kit may also contain instructions for administering a compound comprising a modified oligonucleotide disclosed herein.

[0290] In some embodiments, the kit comprises a modified oligonucleotide provided herein as a lyophilized drug product and a pharmaceutically acceptable diluent. When ready for administration to a subject, the lyophilized drug product is reconstituted in a pharmaceutically acceptable diluent.

[0291] In some embodiments, in addition to a compound comprising a modified oligonucleotide disclosed herein, the kit may further comprise one or more of the following: a syringe, an alcohol swab, a cotton ball, and / or a gauze pad.

[0292] Some experimental models

[0293] In certain embodiments, the present invention provides methods of using and / or testing the modified oligonucleotides of the present invention in experimental models. One skilled in the art is able to select and modify protocols for such experimental models to evaluate the agents of the present invention.

[0294] In general, the oligonucleotide of modification is first tested in cultured cells. Suitable cell types include cell types related to the cell types to which the oligonucleotide of modification needs to be delivered in vivo. For example, suitable cell types for studying the methods described herein include primary cells or cultured cells.

[0295] In certain embodiments, the modified oligonucleotides are assessed in cultured cells to interfere with the activity of one or more miR-17 family members. In certain embodiments, the inhibition of microRNA activity can be assessed by measuring one or more levels in the transcripts of the microRNAs regulated by the prediction or verification. Inhibition of microRNA activity may cause the transcripts regulated by miR-17 family members and / or the proteins encoded by the transcripts regulated by miR-17 family members to increase (that is, the transcripts regulated by miR-17 family members are de-repressed). In addition, in certain embodiments, certain phenotypic results can be measured.

[0296] Those skilled in the art can use several animal models to study one or more miR-17 family members in human disease models. Models of polycystic kidney disease include, but are not limited to, models with mutations and / or deletions in Pkd1 and / or Pkd2; and models comprising mutations in other genes. Non-limiting exemplary models of PKD comprising mutations and / or deletions in Pkd1 and / or Pkd2 include hypoallele models, such as models comprising missense mutations in Pkd1 and models with reduced or unstable expression of Pkd2; inducible conditional knockout models; and conditional knockout models. Non-limiting exemplary PKD models comprising mutations in genes other than Pkd1 and Pkd2 include models with mutations in Pkhd1, Nek8, Kif3a, and / or Nphp3. The PKD model is reviewed in, for example, Shibazaki et al., Human Mol. Genet., 2008; 17(11): 1505-1516; Happe and Peters, Nat Rev Nephrol., 2014; 10(10): 587-601; and Patel et al., PNAS, 2013; 110(26): 10765-10770.

[0297] Some quantitative analysis

[0298] In certain embodiments, the microRNA level is quantitatively measured in cells or tissues in vitro or in vivo. In certain embodiments, the change in microRNA level is measured by microarray analysis. In certain embodiments, the change in microRNA level is measured by one of several commercially available PCR assays such as MicroRNA assay (Applied Biosystems) was used to measure.

[0299] The regulation of microRNA activity with anti-miR or microRNA analogs can be assessed by the microarray spectrum analysis of mRNA.Search for the sequence of the mRNA regulated (increase or decrease) by anti-miR or microRNA analogs for microRNA seed sequence, with the regulation of the mRNA as the target of microRNA and the regulation of the mRNA not as the target of microRNA compared.In this way, the interaction of anti-miR and its target microRNA or microRNA analogs and its target can be evaluated.In the case of anti-miR, the mRNA sequence of the mRNA whose expression level increases is screened, and the mRNA sequence comprises the seed matching of the microRNA complementary to anti-miR.

[0300] Regulating microRNA activity with anti-miR compounds can be assessed by measuring the level of the messenger RNA itself or the level of the protein transcribed therefrom by measuring the level of the messenger RNA target of the microRNA. Antisense inhibition of microRNA generally results in an increase in the level of the messenger RNA of the microRNA and / or the protein of the messenger RNA target, i.e., anti-miR treatment results in the derepression of one or more target messenger RNAs.

[0301] Example

[0302] The following examples are proposed to more fully illustrate some embodiments of the present invention. However, the examples should not be construed as limiting the broad scope of the present invention. Those of ordinary skill in the art will be easy to design various compounds using the basic principles of this discovery without departing from the spirit of the present invention.

[0303] Example 1: Role of miR-17 in PKD

[0304] The miR-17 family members of the miR-17-92 cluster of microRNAs are upregulated in the mouse PKD model. Genetic deletion of the miR-17-92 cluster in the mouse PKD model reduces renal cyst growth, improves renal function and prolongs survival (Patel et al., PNAS, 2013; 110(26): 10765-10770). The miR-17-92 cluster contains 6 different microRNAs, each with a different sequence: miR-17, miR-18a, miR-19a, miR-19-b-1 and miR-92a-1.

[0305] The miR-17-92 cluster includes two microRNAs, miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Each member of the family shares a seed sequence identity, as well as varying degrees of sequence identity outside the seed region. Other members of the miR-17 family are miR-20b, miR-93, miR-106a, and miR-106b. MiR-20b and miR-106a are located in the miR-106a-363 ​​cluster on human chromosome X, and miR-93 and miR-106b are located in the miR-106b-25 cluster on human chromosome 7. The sequences of the miR-17 family members are shown in Table 1.

[0306] Table 1: miR-17 family of microRNAs

[0307]

[0308] Previous studies using anti-miR-17 compounds have been conducted in two different PKD models: the Pkd2-KO model (also known as Pkhd1 / cre; Pkd2 F / FModel) and PCy model to identify the role of miR-17 in PKD. Oligonucleotides modified by research tools complementary to miR-17 were tested in the mouse PKD model. Anti-miR-17 compounds are fully phosphorothioated oligonucleotides with a length of 19 linked nucleosides (5'-CTGCACTGTAAGCACTTTG-3'; SEQ ID NO:7) with DNA, 2'-MOE and S-cEt sugar moieties. Although the compound has mismatches relative to other members of the miR-17 family, tests in in vitro assays reveal that it hybridizes with all members of the miR-17 family and inhibits these members.

[0309] Pkd2-KO mice spontaneously develop polycystic kidney disease. Mice were treated with 20 mg / kg tool anti-miR-17 compound or control oligonucleotide or with PBS. The results showed that anti-miR-17 treatment of Pkd2-KO mice reduced the primary treatment endpoint, kidney weight-to-body weight ratio, by 17% (p=0.017) relative to control treatment. Anti-miR-17 treatment also significantly reduced the expression of BUN and renal injury mRNA biomarkers Kim1 and Ngal in Pkd2-KO mice. Finally, anti-miR-17 treatment resulted in a trend toward decreased serum creatinine levels and decreased cyst index in Pkd2-KO mice. These results were not observed with anti-miR controls, indicating that they were specifically attributed to miR-17 inhibition.

[0310] Pcy mice carrying mutations in Nphp3 spontaneously develop polycystic kidney disease, with slower disease progression than observed in Pkd2-KO mice. Mice were treated with 50 mg / kg of the tool anti-miR-17 compound or PBS once a week for a total of 26 weeks. The average ratio of kidney weight to body weight in Pcy mice treated with anti-miR-17 was 19% lower (p=0.0003) than the average ratio of kidney weight to body weight in Pcy mice administered PBS alone. Pcy mice treated with anti-miR-17 showed an average reduction of 28% in cyst index compared to Pcy mice administered PBS alone (p=0.008).

[0311] These data suggest that miR-17 is a valid target for the treatment of PKD in two different experimental models of PKD.

[0312] Example 2: Compound design and screening

[0313] Although research tool compounds have shown efficacy in PKD models, they have been observed to be mildly pro-inflammatory in in vivo studies. Additionally, research tool compounds are not effective enough to be developed as agents for the treatment of PKD.

[0314] Therefore, a screen was performed to identify inhibitors of one or more miR-17 family members that were sufficiently potent, convenient to administer, and safe to administer to subjects with PKD.Another criterion was that the kidney-liver delivery ratio was high enough to enhance the proportion of anti-miR-17 compound delivered to the target organ.

[0315] Designed about 200 kinds of oligonucleotides containing modifications of the nucleobase sequence complementary to the miR-17 seed sequence, which have different lengths and chemical compositions. The length range of the compound is 9 to 20 connected nucleosides, and the number, type and position of the chemical modification of the compound are different. Because it is not possible to simply predict efficacy and safety based on the nucleobase chemical structure of the compound, in a series of tests designed to eliminate compounds with unfavorable properties, the characteristics of the compound are evaluated in vitro and in vivo, including efficacy, efficacy, pharmacokinetic behavior, viscosity, safety and metabolic stability. In some assays, tool anti-miR-17 compounds are used as the benchmark for comparing library compounds. As described below, each of nearly 200 compounds is first tested in several in vitro assays (e.g., efficacy, toxicology, metabolic stability) to identify compounds suitable for further testing in more complex in vivo assays (e.g., pharmacokinetic profiles, efficacy, toxicology). The design screening process is to identify candidate agents based on the aggregated data from all assays, with emphasis on efficacy, pharmacokinetic profiles (e.g., delivered to the kidney) and safety features.

[0316] In vitro and in vivo potency and efficacy

[0317] In vitro efficacy was evaluated using a luciferase reporter assay. The luciferase reporter plasmid for miR-17 has two fully complementary miR-17 binding sites in tandem in the 3'-UTR of the luciferase gene. If the maximum inhibition of the longer length compound is greater than the maximum inhibition of the tool anti-miR-17 compound, the longer length compound is selected. Since shorter compounds such as 9-mers do not usually have maximum activity under the same assay conditions for longer compounds, shorter compounds are selected based on maximum inhibition relative to appropriate control compounds. In this way, compounds of different lengths and chemical compositions are included in additional tests.

[0318] In vivo efficacy is evaluated using microRNA polysome shift assay (miPSA). The assay is used to determine the degree to which the compound directly engages with the miR-17 target in the kidney in normal mice and PKD mice. MiPSA relies on the following principle: active miRNA binds to its mRNA target in high molecular weight (HMW) polysomes of translational activity, while inhibited miRNA is located in low MW (LMW) polysomes. Treatment with anti-miR causes microRNA to shift from HMW polysomes to LMW polysomes. Therefore, miPSA provides direct measurement of microRNA target engagement by complementary anti-miR (Androsavich et al., Nucleic Acids Research, 2015, 44: e13).

[0319] Selected compounds that have passed various screening criteria are evaluated for efficacy in experimental models of PKD such as the Pkd2-KO mouse model and the Pcy mouse model. Mice are treated with anti-miR-17 compounds and clinically relevant endpoints are evaluated, including the ratio of kidney weight to body weight, blood urea nitrogen levels, serum creatinine levels, and renal cyst index.

[0320] Pharmacokinetic properties

[0321] Metabolic stability was evaluated by incubating each anti-miR-17 compound in mouse liver lysate. After 24 hours, the percentage of intact compound remaining was calculated. Compounds that were unstable after 24 hours of incubation are likely unstable in vivo.

[0322] The pharmacokinetic properties and tissue distribution of the selected compound are evaluated in wild-type C57BL6 mice and JCK mice (experimental models of PKD). The compound is administered to wild-type mice at a dosage of 0.3mg / kg, 3mg / kg or 30mg / kg, or to JCK mice at a dosage of 3mg / kg, 30mg / kg or 100mg / kg. Seven days later, mice are killed. Kidney and liver tissues are collected. The concentration of anti-miR-17 compounds in liver and kidney is measured. Preferably, the compound accumulated to a higher level in kidney relative to liver (i.e., with a higher kidney-liver ratio).

[0323] A complete pharmacokinetic profile of selected compounds that passed multiple screening criteria was obtained in C57BL6 mice. In one study, mice were injected with a single subcutaneous injection of anti-miR-17 compound at 30 mg / kg. In another study, mice were injected with anti-miR-17 compound three times subcutaneously at 39 mg / kg over a two-month period. In each study, liver and kidney samples were collected at 1 hour, 4 hours, 8 hours, 1 day, 4 days, 7 days, 14 days, 28 days, and 56 days after injection.

[0324] toxicology

[0325] In the in vitro assay, the possibility of toxicity was assessed using a biochemical fluorescence binding assay (FBA) and liver or kidney slice assay. FBA was performed by incubating a fluorescent dye with each compound and measuring fluorescence immediately. Highly fluorescent compounds have the possibility of producing toxicity in vivo. Liver or kidney slice assays were performed by incubating a piece of tissue from a core liver sample isolated from a rat. After incubation for 24 hours, RNA was extracted from the tissue slices, and the expression levels of 18 pro-inflammatory genes were measured. The induction of pro-inflammatory gene expression indicates the possibility of pro-inflammatory effects in vivo.

[0326] Additional in vivo toxicology evaluations were performed by subcutaneous injection of 300 mg / kg anti-miR-17 compounds into normal mice (Sv129 mice). Four days later, mice were killed, blood was collected for serum chemistry analysis, liver and spleen were weighed, and RNA was isolated from kidney and liver tissue. The expression level of proinflammatory genes, interferon-induced proteins with triangular tetrapeptide repeats (IFIT) was measured. Since the induction in IFIT expression may indicate toxicity, compounds that do not induce IFIT expression are preferred.

[0327] Throughout the screening process, certain anti-miR-17 compounds performed well in multiple assays. Although no compound was the top performer in each assay, after multiple screening stages, certain compounds showed particularly favorable characteristics, such as high potency and a relatively high kidney-liver ratio. From nearly 200 compounds tested in in vitro assays, about 20 met the criteria for further testing in vivo. These 20 compounds were ultimately narrowed down to 5 compounds, and finally to one compound, RG4326, which had the best overall profile and was selected as a candidate agent. After identifying the compound, additional studies were performed to evaluate efficacy, pharmacokinetic profile, and efficacy.

[0328] RG4326 has the following sequence and chemical modification pattern A S G S C M A F C F U F U M U S G S, wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, each cytosine is a non-methylated cytosine, and all linkages are phosphorothioate linkages. As shown in the following examples, the compound exhibits strong targeted engagement of miR-17 in vivo, efficacy in a mouse PKD model, and a pharmacokinetic profile that is favorable for distribution to the kidneys. In addition, the viscosity of RG4326 was determined to be 6 cP at a concentration of about 150 mg / mL (in water at 20°C), so RG4326 in solution is suitable for administration by subcutaneous injection.

[0329] Example 3: Additional short anti-miR-17 compounds

[0330] An additional 9-nucleotide compound (RG4047), in which each nucleoside is an S-cEt nucleoside, was tested in the selected assays to compare activity, safety, and pharmacokinetic profiles with RG4326.

[0331] One assay used is a luciferase assay. As described above, short (e.g., 9 nucleotides) anti-miR-17 compounds, although they may have advantages in in vivo studies, may not perform well in in vitro transfection assays. Therefore, luciferase assay transfection conditions are optimized for short anti-miR-17 compounds so that the inhibitory activity of the compounds can be measured.

[0332] RG5124 was used as a control compound. RG5124 is 9 linked nucleosides in length and has the same sugar modification pattern as RG4326, but has a nucleobase sequence that is not complementary to miR-17.

[0333] The luciferase reporter plasmid of miR-17 contains a fully complementary miR-17 binding site in the 3'-UTR of the luciferase gene. HeLa cells were transfected with microRNA mimics and their cognate luciferase reporter genes, and then transfected with anti-miR-17 at doses of 0.001nM, 3nM, 10nM, 30nM, 100nM, and 300nM. At the end of the 24-hour transfection period, luciferase activity was measured. As shown in Table 2-1, RG4047, although not as effective as RG4326, inhibited miR-17 activity in a dose-dependent manner. SD indicates standard deviation.

[0334] Table 2-1: Luciferase reporter gene assay

[0335]

[0336] The in vivo efficacy, safety, and distribution to the kidney and liver of RG4047 were evaluated. As with larger library screening, in vitro efficacy cannot predict in vivo behavior. RG4047 produces mild pro-inflammatory signals in both the kidney and liver, and is a less potent miR-17 inhibitor in wild-type mice and PKD mice compared to RG4326, and has a much lower kidney-liver ratio (see Table 2-2). These studies reveal that the activity and properties of RG4047 are not improved relative to RG4326.

[0337] To further explore the effects of the location, type, and number of chemical modifications on the activity and kidney-liver ratio of the 9-mer compounds, additional anti-miR-17 compounds were evaluated in wild-type mice and JCK mice. The JCK model is a mouse model of chronic progressive cystic renal disease associated with the same gene that causes human nephronophthisis type 9. Renal cysts in this mouse develop in multiple regions of the nephron.

[0338] miPSA was used to assess the potency of each compound measured by displacement fraction in wild-type and JCK mice. Tissue accumulation of anti-miR-17 compounds was measured by extracting the compounds using liquid-liquid extraction (LLE) and / or solid phase extraction (SPE), followed by analysis of compound identity and concentration using ion-paired reversed-phase high-performance liquid chromatography coupled to time-of-flight mass spectrometry (IP-RP-HPLC-TOF).

[0339] The results for these additional compounds, as well as RG4326 and RG4047, are shown in Table 2-2.

[0340] For miPSA analysis, a single dose of 3 mg / kg was administered to wild-type mice, and for tissue accumulation analysis, a single dose of 30 mg / kg was administered to wild-type mice. For miPSA and tissue accumulation analysis, a single dose of 30 mg / kg was administered to JCK mice. Kidney tissue was collected 7 days after administration of the anti-miR-17 compound. As shown in Table 2-2, changes in the type and position of the modified nucleosides showed significant effects on the miR-17 inhibitory activity and / or kidney-liver ratio of the anti-miR-17 compound. For example, although RG4324 showed efficacy as measured by miPSA, the kidney: liver ratio was lower than the efficacy observed for other compounds. For diseases where the primary site of action is the kidney, a higher kidney-liver ratio is generally preferred. In contrast, RG4327 showed a high kidney: liver ratio, but low efficacy in PKD mice. As described above, RG4326 showed the most suitable efficacy and pharmacokinetic profile for PKD treatment.

[0341] Table 2-2: Comparison of activity and tissue accumulation of anti-miR-17 compounds

[0342]

[0343] Example 4: Activity of RG4326 in additional in vitro assays

[0344] Additional in vitro assays were performed to further explore the efficacy of RG4326. Luciferase reporter gene assays were used to test the ability of RG4326 to inhibit miR-17 family members miR-17, miR-20a, miR-93, and miR-106b. Luciferase reporter plasmids were constructed for each of miR-20a, miR-93, and miR-106b, with fully complementary microRNA binding sites in the 3'-UTR of the luciferase gene. HeLa cells were transfected with microRNA mimics and their cognate luciferase reporter genes, and then transfected with anti-miR-17 at a dose of 100nm. As shown in Table 3, miR-17, miR-20a, miR-93, and miR-106b were each inhibited by RG4326, indicating that anti-miR-17 compounds inhibit multiple members of the miR-17 family. Since RG4326 is 100% complementary to other miR-17 family members, miR-20b and miR-106b, which were not tested, it is expected to inhibit these microRNAs as well. The data in Table 3 are also shown in Figure 1A middle.

[0345] Table 3: In vitro inhibition of miR-17 family

[0346]

[0347] To test the ability of RG4326 to inhibit miR-17 regulation of endogenous targets, miR-17 target gene derepression was evaluated in vitro in several renal cell types from normal and PKD mouse kidneys. Mouse renal collecting duct cells (IMCD3) were treated with 0.3nM, 1.2nM, 4.7nM, 18.8nM, 75nM and 300nM of RG4326 or control oligonucleotide RG5124. Additional control groups included untreated cells and mock-transfected cells (cells treated with transfection reagent only). After a 24-hour transfection period, cells were collected and RNA was extracted. The mRNA levels of 18 genes targeted by miR-17 were measured and averaged to provide a pharmacodynamic marker score (PD marker score), which was expressed as a Log2 fold change (Log2FC) relative to the mock transfection. As shown in Table 4, RG4326, but not the control treatment, derepressed the miR-17 target in a dose-dependent manner. The data are also shown in Figure 2 In B.

[0348] Table 4: miR-17PD signature scores in IMCD3 cells

[0349]

[0350] The ability of RG4326 to de-repress miR-17 targets was also evaluated in additional renal cell types, which were derived from the kidneys of normal mice and PKD mice. Cells were treated with 30nM of RG4326 or control oligonucleotide RG5124. After a 24-hour transfection period, cells were collected and RNA was extracted. The mRNA levels of 18 genes targeted by miR-17 were measured, and the average was taken to provide a pharmacodynamic marker score (PD marker score), which was expressed as a Log2 fold change (Log2FC) relative to a simulated transfection. As shown in Table 5, RG4326, but not control oligonucleotides, de-repressed miR-17 targets in several different healthy and diseased kidney-derived cell types. "P < 0.05" indicates that the p value calculated by one-way ANOVA is less than 0.05. "NS" indicates no statistically significant changes.

[0351] Table 5: Derepression of miR-17 targets in renal cell types

[0352]

[0353] Example 5: In vivo efficacy of RG4326

[0354] The microRNA polysome shift assay (miPSA) was used to identify compounds that directly bind miR-17 in the kidneys of normal mice and PKD mice. miPSA relies on the following principle: active miRNAs bind to their mRNA targets in translationally active high molecular weight (HMW) polysomes, while inhibited miRNAs are located in low MW (LMW) polysomes. Treatment with anti-miRs causes microRNAs to shift from HMW polysomes to LMW polysomes. Therefore, miPSA provides a direct measurement of microRNA target engagement by complementary anti-miRs (Androsavich et al., Nucleic Acids Research, 2015, 44: e13).

[0355] For this experiment, the PKD model of choice was the JCK model, a mouse model of a slowly progressive cystic renal disease associated with the same gene that causes human nephronophthisis type 9. Renal cysts in this mouse develop in multiple zones of the nephron.

[0356] C57BL6 mice were treated with a single subcutaneous dose of 0.3 mg / kg, 3 mg / kg and 30 mg / kg of RG4326 or tool anti-miR-17 (described in Example 1). JCK mice were treated with a single subcutaneous dose of 3 mg / kg, 30 mg / kg and 100 mg / kg of RG4326 or tool anti-miR-17. PBS treatment was used as an additional control.

[0357] Seven days after treatment, mice were sacrificed and kidney tissue was isolated for miPSA. The calculated shift scores shown in Table 6 demonstrate robust target engagement of RG4326 in normal and PKD kidneys. The shift scores after treatment with RG4326 were greater than those after treatment with the tool anti-miR-17 compound. Data for wild-type mice and JCK mice are also shown in Table 6, respectively. Figure 3A and Figure 3B middle.

[0358] Table 6: RG4326 in vivo target engagement

[0359]

[0360] Example 6: In vivo efficacy of RG4326 in PKD experimental models

[0361] Two experimental models of PKD were used to evaluate efficacy. Pkd2-KO mice spontaneously develop polycystic kidney disease and are used as a model of ADPKD. See Patel et al., PNAS, 2013; 110(26): 10765-10770. Pcy mice carrying mutations in Nphp3 spontaneously develop polycystic kidney disease with slower disease progression than observed in Pkd2-KO mice. The Pcy model was used as a model of nephronophthisis. See Happe and Peters, Nat. Rev. Nephrol., 2014; 10: 587-601.

[0362] Pkd2-KO model

[0363] RG4326 was tested in the Pkd2-KO mouse model of ADPKD. This model is also called the PKD2-KO model. Wild-type mice were used as control mice. Oligonucleotides complementary to miRNAs unrelated to miR-17 were used as specific treatment controls (RG5124).

[0364] Gender-matched littermates were injected subcutaneously with RG4326 at a dose of 20 mg / kg (n=12), RG5124 at a dose of 20 mg / kg (n=12), tool anti-miR-17 at a dose of 20 mg / kg (n=12), or PBS (n=12) every day at 10 days, 11 days, 12 days, and 19 days of age. Mice were sacrificed at 28 days of age, and kidney weight, body weight, cyst index, serum creatinine level, and blood urea nitrogen (BUN) level were measured. BUN level is a marker of renal function. Higher BUN levels are associated with poorer renal function, so a decrease in BUN levels is an indicator of reduced renal injury and damage and improved function. Statistical significance was calculated by one-way ANOVA with Dunnett's multiple correction.

[0365] The cyst index is a histological measurement of the cystic area relative to the total renal area. For this analysis, one kidney was perfused with cold PBS and 4% (weight / volume) paraformaldehyde and then harvested. The kidney was fixed with 4% paraformaldehyde for 2 hours and then embedded in paraffin for sectioning. The sagittal sections of the kidney were stained with hematoxylin and eosin (H&E). All image processing steps were automated and performed in free and open source software: R1 script, which uses functions from the EBImage Bioconductor package 2 and ImageMagick3 suite of image processing tools. The kidney H&E images in Aperio SVS format were converted to TIFF images, and the first frame was retained for image analysis. First, the total renal cross-sectional area was calculated using image segmentation. Image segmentation was similarly used to find all internal structures including renal cysts. A filter was applied to remove all objects with an average radius less than three pixels. The cystic index is the image area associated with the cyst divided by the total renal area. The cystic index was calculated for the longitudinal renal sections and transverse renal sections of each individual animal, respectively. For each treatment group, the combined cystic index of individual animals was compared.

[0366] The results are shown in Table 7. The mean ratio of kidney weight to body weight (KW / BW ratio) in Pkd2-KO mice treated with RG4326 was 29% lower than the mean KW / BW ratio in Pkd2-KO mice administered PBS (p=0.0099). Pkd2-KO mice treated with RG4326 showed an average 12% decrease in cyst index compared to Pkd2-KO mice administered PBS, although the difference was not statistically significant. The mean BUN levels of Pkd2-KO mice treated with PBS were reduced by 13%, although the difference was not statistically significant. The mean serum creatinine levels of Pkd2-KO mice treated with RG4326 were 18% lower than those of Pkd2-KO mice administered PBS, although the difference was not statistically significant. These results were not observed with control oligonucleotides, indicating that they were specifically attributed to miR-17 inhibition. Although previous studies have shown a decrease in the KW / BW ratio, BUN, and cystic index in Pkd2-KO after treatment with tool anti-miR-17 compounds, no statistically significant changes were observed in this study. Treatment with the control oligonucleotide RG5124 did not reduce the ratio of kidney weight to body weight, cystic index, or BUN. The KW / BW ratio, BUN, and cystic index are also shown in Figure 4A , Figure 4B and Figure 4C middle.

[0367] Table 7: Efficacy of RG4326 in the Pkd2-KO model of PKD

[0368]

[0369] These results suggest that RG4326 treatment produces positive results in Pkd2-KO mice for a biological endpoint relevant to PKD treatment, kidney volume relative to body weight. RG4326 was more effective than the tool anti-miR-17 compound with regard to this specific endpoint. RG4326 treatment resulted in a trend toward decreased BUN and decreased cystic index in Pkd2-KO mice.

[0370] PCy Model

[0371] RG4326 was tested in the Pcy mouse model. Wild-type mice were used as a control group. Starting from 4 weeks of age, Pcy mice were treated with subcutaneous injection of RG4326 at a dose of 25 mg / kg, tool anti-miR-17 at a dose of 25 mg / kg, control oligonucleotide RG5124 at a dose of 25 mg / kg, or PBS once a week. Each treatment group contained 15 male mice. Three treatments were administered at 55 days, 56 days, and 57 days of age, and then at 6 weeks of age, 7 weeks of age, 8 weeks of age, 9 weeks of age, 10 weeks of age, 11 weeks of age, 12 weeks of age, 13 weeks of age, and 14 weeks of age. Tolvaptan, a vasopressin V2 receptor antagonist (VRA), was also tested for the treatment of some patients with polycystic kidney disease. Mice were killed at 15 weeks of age. Body weight was recorded. One kidney was extracted and weighed, and the other kidney was processed for histological analysis to calculate the cyst index as in Pkhd1 / cre;Pkd2 F / F Blood urea nitrogen (BUN) levels and serum creatinine levels were measured. Statistical significance was calculated by one-way ANOVA with Dunnett's multiple correction.

[0372] The results are shown in Table 8. The mean KW / BW ratio of the treated Pcy mice in the group treated with 25 mg / kg RG4326 was 19% lower (p=0.0055) relative to the mean KW / BW ratio in PBS-treated mice. In addition, the cyst index of Pcy mice treated with RG4326 was reduced by 34% (p=0.016) compared to Pcy mice administered PBS. Treatment with RG4326 reduced BUN in Pcy mice by 16% (p=0.0070) relative to BUN in PBS-treated Pcy mice. Treatment with control oligonucleotides or tool anti-miR-17 compounds did not result in a statistically significant reduction in KW / BW ratio, BUN, or cyst index. Tolvaptan was ineffective in this study. The data in Table 8 are also shown in Figure 5.

[0373] Table 8: Efficacy of RG4326 in the PCy model

[0374]

[0375] These data demonstrate that treatment with RG4326 results in a decrease in kidney weight, BUN, and cystic index in another PKD model.

[0376] Example 7: RG4326 Pharmacokinetic Evaluation

[0377] Due to their reduced serum protein binding ability, which is a property that drives the distribution of oligonucleotides in the body, it is expected that short oligonucleotides may not have pharmacokinetic properties that make them suitable for use as drugs. RG4326 was incubated in mouse, monkey or human liver homogenates. The properties and concentrations of RG4326 and metabolites were determined after 24 hours of incubation. RG4326 and metabolites were extracted using liquid-liquid extraction (LLE) and / or solid phase extraction (SPE), and then the properties and concentrations were analyzed using ion pairing reversed phase high performance liquid chromatography (IP-RP-HPLC-TOF) coupled to time-of-flight mass spectrometry. As shown in Table 9, despite its short length, RG4326 was found to have a particularly favorable pharmacokinetic profile, with more than 95% of the parent compound RG4326 remaining intact after 24 hours of incubation.

[0378] Table 9: In vitro metabolic stability in mouse, monkey and human liver lysates

[0379]

[0380] Pharmacokinetic behavior was evaluated by administering a single subcutaneous 30 mg / kg dose of RG4326 or tool anti-miR-17 compounds to wild-type mice. At 1 hour, 4 hours, 8 hours, 1 day, 7 days, 14 days, 28 days and 56 days after a single injection, mice were killed and the average concentration (ug / g) of anti-miR compounds in kidney and liver tissues was measured as described above. The area under the curve (AUC) of kidney and liver tissues was calculated using the formula ug*h / g, where ug is the amount of oligonucleotides in the tissue, h is the time point of tissue collection in hours, and g is the weight of the tissue. The ratio of kidney AUC to liver AUC was determined. Kidney tissue was also processed for miPSA to determine the target engagement of each compound in this study. PSA AUC was calculated using the formula Log2FC*h, where Log2FC is the displacement value and h is the time point of tissue collection in hours. The efficacy of the kidney at day 7 was calculated using the formula Log2FC+g / ug, where Log2FC is the displacement value as determined by miPSA, g is the weight of the kidney tissue, and ug is the amount of anti-miR in the kidney tissue at day 7.

[0381] As shown in Table 10, the ratio of kidney AUC to liver AUC for RG4326 is greater than the ratio of kidney AUC to liver AUC for tool anti-miR-17 compounds. Interestingly, although the kidney AUC for RG4326 is lower than the kidney AUC for tool anti-miR-17 compounds, the potency as determined by miPSA is significantly higher. Thus, RG4326 exhibits higher potency at lower concentrations in the kidney, a major target tissue for PKD.

[0382] Table 10: Pharmacokinetic profile of RG4326

[0383]

[0384] The pharmacokinetic behavior of RG4326 was further characterized in wild-type (C57B16) mice and PKD (JCK) mice. Five mice in each group received three 10 mg / kg subcutaneous injections on each of three consecutive days. On the 1st, 4th, 7th, 14th and 21st day after the third and last injections, the mice were killed and plasma, kidney and liver samples were collected. In order to measure RG4326, RG4326 was extracted using liquid-liquid extraction (LLE) and / or solid phase extraction (SPE), and then the properties and concentrations were analyzed using ion pairing reversed phase high performance liquid chromatography (IP-RP-HPLC-TOF) coupled to time-of-flight mass spectrometry.

[0385] The data are summarized in Table 11. RG4326 was observed to be stable in both plasma and tissues, with more than 90% of the parent compound remaining after 21 days. The anti-miR was rapidly distributed to tissues within hours of injection, and primarily to the kidneys. The half-life in the liver and kidneys of wild-type mice was approximately 8 days, the half-life in the liver of JCK mice was approximately 6 days, and the half-life in the kidneys of JCK mice was approximately 8 days. In wild-type mice, the ratio of kidney AUC to liver AUC was 17. In PKD mice, the ratio of kidney AUC to liver AUC was 13. These data indicate that the pharmacokinetic profile of RG4326 is comparable in normal mice and PKD mice.

[0386] Table 11: Pharmacokinetic profile of RG4326 in normal mice and PKD mice

[0387]

[0388] Example 8: RG4326 safety assessment

[0389] The potential for renal and liver toxicity was evaluated in in vitro, ex vivo, and in vivo assays.

[0390] The potential for toxicity was assessed using a biochemical fluorescence binding assay (FBA). FBA was performed by incubating a fluorescent dye with each compound and measuring fluorescence immediately. Results were expressed as a fold change (linear FC) relative to control treated samples. Highly fluorescent compounds have the potential to produce toxicity in vivo.

[0391] In vitro assay was performed with liver or kidney tissue slices. Liver or kidney slice assay was performed by incubating a piece of tissue from the core liver or kidney sample isolated from rats. After incubation for 24 hours, RNA was extracted from tissue slices, and the expression levels of 18 proinflammatory genes including IFIT were measured. The log2 conversion (Log2-FC) of the multiple change relative to PBS treatment was performed. The induction of proinflammatory gene expression indicates the possibility of proinflammatory effect in vivo.

[0392] In vivo assays were performed in normal Sv129 mice. A single subcutaneous dose of RG4326 at 300 mg / kg was administered. As control treatments, PBS and two anti-miRs unrelated to miR-17 were included, one known to be pro-inflammatory (positive control) and the other non-pro-inflammatory (negative control). Mice were killed four days later. Kidney and liver tissues were isolated for RNA extraction. The levels of the gene IFIT, known to be induced during inflammatory responses, were measured and normalized to mouse GAPDH. Log2 transformation (Log2-FC) of the fold change relative to PBS treatment was performed.

[0393] Table 11: Safety profile of RG4326

[0394]

[0395] These data suggest that RG4326 displays a favorable safety profile and minimal risk of pro-inflammatory liability based on multiple assays. Sequence Listing <110> REGULUS THERAPEUTICS INC. <120> Composition for treating polycystic kidney disease <130> 01138-0029-00PCT <150> US 62 / 430,139 <151> 2016-12-05 <160> 7 <170> PatentIn Version 3.5 <210> 1 <211> twenty three <212> RNA <213> Homo sapiens <400> 1 caaagugcuu acagugcagg uag 23 <210> 2 <211> twenty three <212> RNA <213> Homo sapiens <400> 2 uaaagugcuu auagugcagg uag 23 <210> 3 <211> twenty three <212> RNA <213> Homo sapiens <400> 3 caaagugcuc auagugcagg uag 23 <210> 4 <211> twenty three <212> RNA <213> Homo sapiens <400> 4 caaagugcug uucgugcagg uag 23 <210> 5 <211> twenty three <212> RNA <213> Homo sapiens <400> 5 aaaagugcuu acagugcagg uag 23 <210> 6 <211> twenty one <212> RNA <213> Homo sapiens <400> 6 uaaagugcug acagugcaga u 21 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Modified oligonucleotides <400> 7 ctgcactgta agcactttg 19

Claims

1. A modified oligonucleotide having the structure: or a pharmaceutically acceptable salt thereof.

2. The modified oligonucleotide according to claim 1, which is a pharmaceutically acceptable salt of said structure.

3. The modified oligonucleotide of claim 1, which is a sodium salt of the structure.

4. A modified oligonucleotide having the structure:

5. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 4 and a pharmaceutically acceptable diluent.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutically acceptable diluent is an aqueous solution.

7. The pharmaceutical composition of claim 6, wherein the aqueous solution is a saline solution.

8. A pharmaceutical composition comprising the modified oligonucleotide according to any one of claims 1 to 4, which is a lyophilized composition.

9. A pharmaceutical composition consisting of the modified oligonucleotide according to any one of claims 1 to 4 in saline solution.

10. Use of the modified oligonucleotide according to any one of claims 1 to 4 in the preparation of a medicament for inhibiting the activity of one or more members of the miR-17 family in a cell.

11. Use of the pharmaceutical composition according to any one of claims 5 to 9 in the preparation of a medicament for inhibiting the activity of one or more members of the miR-17 family in a subject.

12. The use of claim 11, wherein the subject suffers from a disease associated with miR-17.

Images