Methods and compositions for treating polycystic kidney disease

Abstract

The present invention relates to methods and compositions for treating polycystic kidney disease, providing methods of treating polycystic kidney disease, including autosomal dominant polycystic kidney disease, using modified oligonucleotides targeting miR-17.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202280066728.7, filed on October 7, 2022, entitled "Method and Composition for Treating Polycystic Kidney Disease".

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 63 / 253,933, filed October 8, 2021, which is incorporated herein by reference in its entirety for any purpose. Technical Field

[0004] This article provides compositions and methods for treating polycystic kidney disease. Background Technology

[0005] Polycystic kidney disease is characterized by the accumulation of numerous fluid-filled cysts in the kidneys. These cysts are lined with a single layer of epithelial cells called cystic epithelium. Over time, the cysts increase in size due to increased cell proliferation and the active secretion of fluid by the cystic epithelium. The enlarged cysts compress surrounding normal tissue, leading to a decline in kidney function. The disease eventually progresses to end-stage renal disease, requiring dialysis or kidney transplantation. At this stage, the cysts may be surrounded by fibrotic areas containing atrophic tubules. Polycystic kidney disease can also cause cysts to develop in the liver and other parts of the body.

[0006] Polycystic kidney disease (PKD) can be caused by a variety of genetic disorders. Different forms of PKD are distinguished by their mode of inheritance, such as autosomal dominant or autosomal recessive inheritance; extrarenal organ involvement and phenotype presentation; age of onset of end-stage renal disease, such as at birth, in childhood, or in adulthood; and underlying genetic mutations associated with the disease. See, for example, Kurschat et al., 2014, Nature Reviews Nephrology, 10: 687-699. Summary of the Invention

[0007] Implementation Scheme 1. A compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0008] (N'') p -(N) r -(N') q

[0009] Each N'' is independently a modified or unmodified nucleoside;

[0010] p is between 0 and 14; where if p is not 0, then (N'') pThe nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0011] (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3';

[0012] N' is a nucleoside containing a modified sugar moiety;

[0013] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; and

[0014] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof.

[0015] Implementation Scheme 2. The compound as described in Implementation Scheme 1, wherein (N) r The structure is as follows:

[0016] A S G S C M A F C F U F U M U S

[0017] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside;

[0018] The nucleoside followed by the subscript "F" is a 2'-fluoronucleoside; and

[0019] Nucleosides followed by the subscript "S" are S-cEt nucleosides.

[0020] Implementation Scheme 3. The compound as described in Implementation Scheme 1 or Implementation Scheme 2, wherein at least one nucleoside link is a thiophosphate nucleoside link.

[0021] Implementation Scheme 4. The compound as described in any one of Implementation Schemes 1 to 3, wherein the internucleotide linkage is a thiophosphate internucleotide linkage.

[0022] Implementation Scheme 5. The compound as described in any one of Implementation Schemes 1 to 4, wherein q is 1.

[0023] Implementation Scheme 6. The compound as described in any one of Implementation Schemes 1 to 4, wherein q is 0.

[0024] Implementation Scheme 7. The compound as described in any one of Implementation Schemes 1 to 6, wherein p is 0.

[0025] Implementation Scheme 8. The compound of any one of Implementation Schemes 1 to 6, wherein p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0026] Implementation Scheme 9. The compound as described in Implementation Scheme 8, wherein (N'') p The nucleobase sequence of the nucleotide has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0027] Implementation Scheme 10. The compound as described in Implementation Scheme 8, wherein (N'') p The nucleobase sequence of the sample does not mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0028] Implementation Scheme 11. The compound as described in any one of Implementation Schemes 8, 9, or 10, wherein (N'') p The nucleobase sequence is selected from CUACCUGCACUGUA (SEQ ID NO: 7), CUACCUGCACUGU (SEQ ID NO: 8), CUACCUGCACUG (SEQ ID NO: 9), CUACCUGCACU (SEQ ID NO: 10), CUACCUGCAC (SEQ ID NO: 11), CUACCUGCA, CUACCUGC, CUACCUG, CUACCU, CUACC, CUAC, CUA, CU and C.

[0029] Implementation Scheme 12. The compound of any one of Implementation Schemes 1 to 5 or 7 to 11, wherein the nucleobase of N' is a purine nucleobase that does not have a hydrogen bond acceptor at position 6.

[0030] Implementation Scheme 13. The compound as described in Implementation Scheme 12, wherein the nucleobase of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0031] Implementation Scheme 14. The compound of any one of Implementation Schemes 1 to 13, wherein the sugar moiety of N' is not a 2'-O-methyl sugar.

[0032] Implementation Scheme 15. The compound of any one of Implementation Schemes 1 to 14, wherein the sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar.

[0033] Implementation Scheme 16. The compound as described in Implementation Scheme 2, wherein the modified oligonucleotide has a 5'-A structure. S G S CM A F C F U F U M U S A S -3', where each cytosine is a nonmethylated cytosine.

[0034] Implementation Scheme 17. The compound as described in Implementation Scheme 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S U S -3', where each cytosine is a nonmethylated cytosine.

[0035] Implementation Scheme 18. The compound as described in Implementation Scheme 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S C S -3', where each cytosine is a nonmethylated cytosine.

[0036] Implementation Scheme 19. The compound as described in Implementation Scheme 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S -3', where each cytosine is a nonmethylated cytosine.

[0037] Implementation Scheme 20. The compound of any one of Implementation Schemes 1 to 19, wherein the compound comprises the modified oligonucleotide.

[0038] Implementation Scheme 21. The compound of any one of Implementation Schemes 1 to 20, wherein the pharmaceutically acceptable salt is a sodium salt.

[0039] Implementation Scheme 22. A modified oligonucleotide having the following structure:

[0040]

[0041] Wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; or a pharmaceutically acceptable salt thereof.

[0042] Implementation Scheme 23. The modified oligonucleotide as described in Implementation Scheme 22, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine and isoguanosine.

[0043] Implementation Scheme 24. The modified oligonucleotide as described in Implementation Scheme 22 or Implementation Scheme 23, wherein the pharmaceutically acceptable salt is a sodium salt.

[0044] Implementation Scheme 25. A modified oligonucleotide having the following structure:

[0045]

[0046] Where B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6.

[0047] Implementation Scheme 26. The modified oligonucleotide as described in Implementation Scheme 25, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine and isoguanosine.

[0048] Implementation Scheme 27. A modified oligonucleotide having the following structure:

[0049]

[0050] Or its pharmaceutically acceptable salt.

[0051] Implementation Scheme 28. The modified oligonucleotide as described in Implementation Scheme 27, wherein the pharmaceutically acceptable salt is a sodium salt.

[0052] Implementation Scheme 29. A modified oligonucleotide having the following structure:

[0053] .

[0054] Implementation Scheme 30. A pharmaceutical composition comprising a compound as described in any one of Implementation Schemes 1 to 21 or a modified oligonucleotide as described in any one of Implementation Schemes 22 to 29, and a pharmaceutically acceptable diluent.

[0055] Implementation Scheme 31. The pharmaceutical composition of Implementation Scheme 30, wherein the pharmaceutically acceptable diluent is an aqueous solution.

[0056] Implementation Scheme 32. The pharmaceutical composition as described in Implementation Scheme 31, wherein the aqueous solution is a saline solution.

[0057] Implementation Scheme 33. A pharmaceutical composition comprising any one of the compounds of embodiments 1 to 21 or any one of the modified oligonucleotides of embodiments 22 to 29, wherein the composition is a lyophilized composition.

[0058] Implementation Scheme 34. A pharmaceutical composition comprising substantially a compound of any one of Implementation Schemes 1 to 21 or a modified oligonucleotide of any one of Implementation Schemes 22 to 29 in a saline solution.

[0059] Implementation Scheme 35. A method for inhibiting the activity of one or more members of the miR-17 family in cells, the method comprising contacting the cells with a compound of any one of Implementation Schemes 1 to 21 or a modified oligonucleotide of any one of Implementation Schemes 22 to 29.

[0060] Implementation Scheme 36. A method for inhibiting the activity of one or more members of the miR-17 family in a subject, the method comprising administering to the subject a compound of any one of Implementation Schemes 1 to 21, a modified oligonucleotide of any one of Implementation Schemes 22 to 29, or a pharmaceutical composition of any one of Implementation Schemes 30 to 34.

[0061] Implementation Scheme 37. The method as described in Implementation Scheme 36, wherein the subject suffers from a miR-17-related disease.

[0062] Implementation Scheme 38. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0063] (N'') p -(N) r -(N') q

[0064] Each N'' is independently a modified or unmodified nucleoside;

[0065] p is between 0 and 14; where if p is not 0, then (N'') p The nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0066] (N) r Each N is independently a modified or unmodified nucleotide, and (N) rThe nucleobase sequence is 5'-AGCACUUU-3';

[0067] N' is a nucleoside containing a modified sugar moiety;

[0068] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have an H bond acceptor at position 6; and

[0069] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof.

[0070] Implementation Scheme 39. The method as described in Implementation Scheme 38, wherein (N) r The structure is as follows:

[0071] A S G S C M A F C F U F U M U S

[0072] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside;

[0073] The nucleoside followed by the subscript "F" is 2'-fluoronucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside.

[0074] Implementation Scheme 40. The method as described in Implementation Scheme 38 or Implementation Scheme 39, wherein at least one nucleoside link is a phosphate thioside link.

[0075] Implementation Scheme 41. The method of any one of Implementation Schemes 38 to 40, wherein the inter-nucleoside linkage is a phosphate thioside linkage.

[0076] Implementation Scheme 42. The method as described in any one of Implementation Schemes 38 to 41, wherein q is 1.

[0077] Implementation Scheme 43. The method as described in any one of Implementation Schemes 38 to 41, wherein q is 0.

[0078] Implementation Scheme 44. The method as described in any one of Implementation Schemes 38 to 43, wherein p is 0.

[0079] Implementation Scheme 45. The method of any one of Implementation Schemes 38 to 43, wherein p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0080] Implementation Scheme 46. The method as described in Implementation Scheme 45, wherein (N'') p The nucleobase sequence of the nucleotide has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0081] Implementation Scheme 47. The compound as described in Implementation Scheme 45, wherein (N'') p The nucleobase sequence of the sample does not mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0082] Implementation Scheme 48. The compound as described in Implementation Scheme 47, wherein (N'') p The nucleobase sequences are selected from CUACCUGCACUGUA (SEQ ID NO: 7), CUACCUGCACUGU (SEQ ID NO: 8), CUACCUGCACUG (SEQ ID NO: 9), CUACCUGCACU (SEQ ID NO: 10), CUACCUGCAC (SEQ ID NO: 11), CUACCUGCA, CUACCUGC, CUACCUG, CUACCU, CUACC, CUAC, CUA, CU, and C.

[0083] Implementation Scheme 49. The method of any one of Implementation Schemes 38 to 42 or 44 to 48, wherein the nucleobase of N' is a purine nucleobase that does not have a hydrogen bond acceptor at position 6.

[0084] Implementation Scheme 50. The method as described in Implementation Scheme 49, wherein the nucleobase of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0085] Implementation Scheme 51. The method of any one of Implementation Schemes 38 to 50, wherein the sugar portion of N' is not a 2'-O-methyl sugar.

[0086] Implementation Scheme 52. The compound of any one of Implementation Schemes 38 to 51, wherein the sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar.

[0087] Implementation Scheme 53. The method of Implementation Scheme 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3', and each cytosine is a nonmethylated cytosine.

[0088] Implementation Scheme 54. The method of Implementation Scheme 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S U S -3', where each cytosine is a nonmethylated cytosine.

[0089] Implementation Scheme 55. The method of Implementation Scheme 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S C S -3', where each cytosine is a nonmethylated cytosine.

[0090] Implementation Scheme 56. The method of Implementation Scheme 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S -3', where each cytosine is a nonmethylated cytosine.

[0091] Implementation Scheme 57. The method of any one of Implementation Schemes 38 to 56, wherein the compound comprises the modified oligonucleotide.

[0092] Implementation Scheme 58. The method of any one of Implementation Schemes 38 to 57, wherein the pharmaceutically acceptable salt is a sodium salt.

[0093] Implementation Scheme 59. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a modified oligonucleotide having the following structure:

[0094]

[0095] Or its pharmaceutically acceptable salt.

[0096] Implementation Scheme 60. The method as described in Implementation Scheme 59, wherein the pharmaceutically acceptable salt is a sodium salt.

[0097] Implementation Scheme 61. The method of Implementation Scheme 60, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0098] Implementation Scheme 62. The method of Implementation Scheme 61, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0099] Implementation Scheme 63. The method as described in Implementation Scheme 62, wherein the sterile aqueous solution is a saline solution.

[0100] Implementation Scheme 64. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a modified oligonucleotide having the following structure:

[0101] .

[0102] Implementation Scheme 65. The method of Implementation Scheme 64, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0103] Implementation Scheme 66. The method as described in Implementation Scheme 65, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0104] Implementation Scheme 67. The method as described in Implementation Scheme 66, wherein the sterile aqueous solution is a saline solution.

[0105] Implementation Scheme 68. The method of any one of Implementation Schemes 38 to 67, wherein the subject suffers from polycystic kidney disease.

[0106] Implementation Scheme 69. The method of any one of Implementation Schemes 38 to 67, wherein the subject is suspected of having polycystic kidney disease.

[0107] Implementation Scheme 70. The method of any one of Implementation Schemes 38 to 68, wherein the subject has been diagnosed with polycystic kidney disease using clinical, histopathological and / or genetic criteria.

[0108] Implementation Scheme 71. The method of any one of Implementation Schemes 38 to 70, wherein prior to administration of the compound, the modified oligonucleotide, or the pharmaceutical composition, the subject is determined to have reduced polycystin-1 (PC1) and / or polycystin-2 (PC2) levels in the subject's kidneys, urine, or blood.

[0109] Implementation Scheme 72. The method of any one of Implementation Schemes 38 to 71, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease.

[0110] Implementation Scheme 73. The method of any one of Implementation Schemes 38 to 71, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease.

[0111] Implementation Scheme 74. The method of any one of Implementation Schemes 38 to 73, wherein the subject has a mutation selected from PKD1 gene mutation or PKD2 gene mutation.

[0112] Implementation Scheme 75. The method of any one of Implementation Schemes 38 to 74, wherein the subject has an increased total kidney volume.

[0113] Implementation Scheme 76. The method of any one of Implementation Schemes 38 to 75, wherein the subject suffers from hypertension.

[0114] Implementation Scheme 77. The method of any one of Implementation Schemes 38 to 76, wherein the subject has impaired renal function.

[0115] Implementation Scheme 78. The method of any one of Implementation Schemes 38 to 77, wherein the administration reduces the total kidney volume of the subject.

[0116] Implementation Scheme 79. The method of any one of Implementation Schemes 38 to 78, wherein the administration slows the rate of increase in total kidney volume in the subject.

[0117] Implementation Scheme 80. The method as described in Implementation Scheme 78 or Implementation Scheme 79, wherein the total kidney volume is a height-adjusted total kidney volume.

[0118] Implementation Scheme 81. The method of any one of Implementation Schemes 38 to 80, wherein the administration slows the rate of decline of the glomerular filtration rate in the subject.

[0119] Implementation Scheme 82. The method of any one of Implementation Schemes 38 to 81, wherein the administration increases the glomerular filtration rate in the subject.

[0120] Implementation Scheme 83. The method as described in Implementation Scheme 81 or Implementation Scheme 82, wherein the glomerular filtration rate is an estimated glomerular filtration rate.

[0121] Implementation Scheme 84. The method of any one of Implementation Schemes 38 to 83, wherein the administration slows the increase in cyst growth in the kidneys and / or liver of the subject.

[0122] Implementation Scheme 85. The method as described in any one of Implementation Schemes 38 to 84, wherein the application:

[0123] a) Improved renal function in the subjects;

[0124] b) Delay the deterioration of the subject's renal function;

[0125] c) Reduce kidney pain in the subjects;

[0126] d) Reduced the increase in kidney pain in the subjects;

[0127] e) Delay the onset of kidney pain in the subjects;

[0128] f) Lower the high blood pressure of the subjects;

[0129] g) To slow the worsening of hypertension in the subjects;

[0130] h) Delay the onset of hypertension in the subjects;

[0131] i) Reduce fibrosis in the kidneys of the subjects;

[0132] j) Slowing down the progression of fibrosis in the kidneys of the subjects;

[0133] k) Delay the onset of end-stage renal disease in the subjects;

[0134] l) Delay the dialysis time of the subjects;

[0135] m) Delaying the time for the subject to undergo kidney transplantation; and / or

[0136] n) Improve the life expectancy of the subjects.

[0137] Implementation Scheme 86. The method as described in any one of Implementation Schemes 38 to 85, wherein the application:

[0138] a) Reduce the albuminuria of the subjects;

[0139] b) Slowing the progression of albuminuria in the subjects;

[0140] c) Delay the onset of albuminuria in the subjects;

[0141] d) Reduce the hematuria of the subjects;

[0142] e) To slow the worsening of hematuria in the subjects;

[0143] f) Delay the onset of hematuria in the subject;

[0144] g) Reduce the blood urea nitrogen level of the subject;

[0145] h) Reduce the serum creatinine level of the subjects;

[0146] i) Improve the creatinine clearance rate of the subjects;

[0147] j) Reduce the albumin:creatinine ratio of the subjects;

[0148] k) Increase polycystic protein-1 (PC1) in the urine of the subjects;

[0149] l) Increase polycystic protein-2 (PC2) in the urine of the subjects;

[0150] m) Reduce neutrophil gelatinase-associated lipocalin (NGAL) in the urine of the subjects; and / or

[0151] n) Reduce the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

[0152] Implementation Scheme 87. The method of any one of Implementation Schemes 38 to 86, said method comprising:

[0153] a) Measure the total kidney volume of the subject;

[0154] b) Measure the subject's blood pressure;

[0155] c) Measure the kidney pain of the subject;

[0156] d) Measure polycystic protein-1 (PC1) in the urine of the subjects;

[0157] e) Measure polycystic protein-2 (PC2) in the urine of the subjects;

[0158] f) Measure the fibrosis of the subject's kidneys;

[0159] g) Measure the blood urea nitrogen level of the subject;

[0160] h) Measure the serum creatinine level of the subjects;

[0161] i) Measure the creatinine clearance rate of the subjects;

[0162] j) Measure the albuminuria of the subjects;

[0163] k) Measure the albumin:creatinine ratio of the subject;

[0164] l) Measure the glomerular filtration rate of the subject;

[0165] m) Measure neutrophil gelatinase-associated lipocalin (NGAL) in the urine of the subjects; and / or

[0166] n) Measure the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

[0167] Implementation Scheme 88. The method of any one of Implementation Schemes 38 to 87, wherein the method includes administering at least one additional therapy, wherein the at least one additional therapy is an antihypertensive agent.

[0168] Implementation Scheme 89. The method of any one of Implementation Schemes 38 to 87, wherein the method comprises administering at least one additional therapy selected from: angiotensin II converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), diuretics, calcium channel blockers, kinase inhibitors, adrenergic receptor antagonists, vasodilators, benzodiazepines, renin inhibitors, aldosterone receptor antagonists, endothelin receptor blockers, mammalian target of rapamycin (mTOR) inhibitors, hormone analogs, vasopressin receptor 2 antagonists, aldosterone receptor antagonists, glucose-ceramide synthase inhibitors, antihyperglycemic agents, dialysis, and kidney transplantation.

[0169] Implementation Scheme 90. The method of Implementation Scheme 89, wherein the angiotensin II converting enzyme (ACE) inhibitor is selected from captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril.

[0170] Implementation Scheme 91. The method of Implementation Scheme 89, wherein the angiotensin II receptor blocker (ARB) is selected from candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, and eprosartan.

[0171] Implementation Scheme 92. The method of Implementation Scheme 89, wherein the vasopressin receptor 2 antagonist is tolvaptan.

[0172] Implementation Scheme 93. The method of Implementation Scheme 89, wherein the aldosterone receptor antagonist is spironolactone.

[0173] Implementation Scheme 94. The method of Implementation Scheme 89, wherein the kinase inhibitor is selected from bosutinib and KD019.

[0174] Implementation Scheme 95. The method of Implementation Scheme 89, wherein the mTOR inhibitor is selected from everolimus, rapamycin, and sirolimus.

[0175] Implementation Scheme 96. The method of Implementation Scheme 89, wherein the hormone analogue is selected from somatostatin and adrenocorticotropic hormone.

[0176] Implementation Scheme 97. The method of Implementation Scheme 89, wherein the glucose ceramide synthase inhibitor is venglustat.

[0177] Implementation Scheme 98. The method as described in Implementation Scheme 89, wherein the antihyperglycemic agent is metformin.

[0178] Implementation Scheme 99. The method of any one of Implementation Schemes 38 to 96, wherein the method comprises administering a therapeutically effective amount of the compound.

[0179] Implementation scheme 100. The method of any one of implementation schemes 38 to 99, wherein the subject is a human subject.

[0180] Implementation Scheme 101. A compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0181] (N'') p -(N) r -(N') q

[0182] Each N'' is independently a modified or unmodified nucleoside;

[0183] p is between 0 and 14; where if p is not 0, then (N'') p The nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0184] (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3';

[0185] N' is a nucleoside containing a modified sugar moiety;

[0186] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have an H bond acceptor at position 6; and

[0187] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof, which are used in therapy.

[0188] Implementation Scheme 102. The compound as described in Implementation Scheme 101, wherein the therapy is a treatment for polycystic kidney disease.

[0189] Implementation Scheme 103. The compound as described in Implementation Scheme 102, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

[0190] Implementation Scheme 104. The compound as described in Implementation Scheme 102, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD).

[0191] Implementation Scheme 105. The compound of any one of Implementation Schemes 1 to 22, the modified oligonucleotide of any one of Implementation Schemes 23 to 29, or the pharmaceutical composition of any one of Implementation Schemes 30 to 33, used in a therapy. Attached Figure Description

[0192] Figure 1 Purine nucleobase structure.

[0193] Figures 2A to 2C Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Therapeutic effects: (2A) kidney / body weight ratio, (2B) blood urea nitrogen (BUN) level and (2C) blood creatinine level.

[0194] Figure 3 Maximum tolerated dose (MTD) studies and comparative dose assessments of RG-NG-1001, RGLS4326, and RG-NG-1017. Six- to seven-week-old male C57BL / 6J mice were administered a single intraventricular (ICV) injection of RG-NG-1001 and RGLS4326 (an anti-miR-17 oligomer that inhibits AMPA-R), and RG-NG-1017 (an anti-miR-17 oligomer that does not inhibit AMPA-R; RG-NG-1017), at different doses, and monitored for seven days. Mice mortality was indicated by the different doses of these three compounds.

[0195] Figures 4A to 4FThe activity of RG-NG-1015 and RGLS4326 against miR-17 (4A), miR-20a (4B), miR-93 (4C), and miR106(a) (4D) luciferase sensors in HeLa cells in vitro was evaluated. The activity of RG-NG-1015 and RGLS4326 against the full-length 3' untranslated region (UTR) containing the direct target genes of miR-17, PKD1 (4E) and PKD2 (4F), was also evaluated.

[0196] Figures 5A to 5D The pharmacokinetics and target binding of RGLS4326 and RG-NG-1015 following a single subcutaneous administration in C57BL6 mice were measured (as measured by miPSA). Plasma concentrations (5A), tissue concentrations (5B), renal target binding (5C), and hepatic target binding (5D) are shown.

[0197] Figures 6A to 6E The effects of different doses and regimens of RG-NG-1015, as well as its combination with tolvaptan, on a PKDPcy / DBA mouse model were measured. Dosing time is expressed as... Figure 6A In the middle, and Figures 6C to 6E Legends for each chart are shown in Figure 6B The results show kidney weight / body weight (6C), cystic area (%) (6D), and urine Ngal / Cr (6E). Error bars represent standard deviations. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, (ns)p>0.05, compared with the Pcy mediator treatment group; univariate ANOVA Bonferroni multiple comparison test. #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001, (ns)p>0.05, compared with the tolvaptan alone group; univariate ANOVA Sadik multiple comparison test. $p<0.05, $$p<0.01, $$p<0.001, $$$p<0.001, (ns)p>0.05, compared with dose-matched RG-NG-1015 alone; univariate ANOVA Sadik multiple comparison test. Detailed Implementation

[0198] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless specifically defined, the nomenclature used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, as well as the procedures and techniques of analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, are those well-known and commonly used in the art. Where multiple definitions exist for terms herein, those defined in this section shall prevail. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of subjects. Some such techniques and procedures can be found, for example, in “Carbohydrate Modifications in Antisense Research,” edited by Sanghvi and Cook, American Chemical Society, Washington DC, 1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and are incorporated herein by reference for any purpose. Where permitted, unless otherwise noted, all patents, patent applications, publications and filings, GENBANK sequences, websites and other public materials mentioned throughout this disclosure are incorporated herein by reference in their entirety. Where a URL or other such identifier or address is referenced, it should be understood that such identifiers are subject to change, and specific information on the Internet may change, but equivalent information can be found by searching the Internet. Such citations attest to the availability and public dissemination of this information.

[0199] Before disclosing and illustrating the compositions and methods of the present invention, it should be understood that the terminology used herein is for the purpose of illustrating particular embodiments only and is not intended to be limiting. It must be noted that, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” as used in this specification and the appended claims include a plural of indicators.

[0200] definition

[0201] Polycystic kidney disease (PKD) is a cystic kidney disease characterized by the accumulation of numerous fluid-filled cysts in the kidneys. Multiple cysts form in at least one kidney, often leading to enlargement of the affected kidney and progressive loss of kidney function.

[0202] "Markers of polycystic kidney disease" refers to medical parameters used to assess the severity of polycystic kidney disease, renal function, and / or the response of a subject with polycystic kidney disease to treatment. Non-limiting examples of markers for polycystic kidney disease include total kidney volume, hypertension, glomerular filtration rate, and renal pain.

[0203] "Markers of renal function" refers to medical parameters used to assess the renal function of a subject. Non-limiting examples of markers of renal function include glomerular filtration rate, blood urea nitrogen level, and serum creatinine level.

[0204] Autosomal dominant polycystic kidney disease (ADPKD) is a polycystic kidney disease caused by one or more genetic mutations in the PKD1 and / or PKD2 genes. 85% of ADPKD cases are caused by PKD1 mutations located on chromosome 16, while the vast majority of other ADPKD cases are caused by PKD2 mutations located on chromosome 4.

[0205] Autosomal recessive polycystic kidney disease (ARPKD) is a 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 intrauterine kidney disease, and about one-third of those who survive develop end-stage renal disease (ESRD) within 10 years.

[0206] "Nen wasting disease" or "NPHP" refers to autosomal recessive cystic kidney disease characterized by cortico-medullary cysts, rupture of the tubular basement membrane, and tubulointerstitial nephropathy.

[0207] "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) scans, or ultrasound (US) imaging, and the volume is calculated using standard methods such as the elliptic volume equation (for ultrasound), or by quantitative stereometry or boundary tracking (for CT / MRI).

[0208] "Height-adjusted total kidney volume" or "HtTKV" is a measure of the total kidney volume per unit height. Patients with an HtTKV value ≥ 600 ml / m are expected to develop stage 3 chronic kidney disease within 8 years.

[0209] "Kidney pain" refers to clinically significant kidney pain that requires sick leave, medication (anesthetics or as a last resort) or invasive intervention.

[0210] "Worsening hypertension" refers to changes in blood pressure that require initiation or increased treatment for hypertension.

[0211] "Fibrosis" refers to the formation or development of an excessive amount of fibrous connective tissue in an organ or tissue. In some embodiments, fibrosis occurs as a reparative or reactive process. In some embodiments, fibrosis occurs in response to damage or injury. The term "fibrosis" should be understood as the formation or development of an excessive amount of fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to the formation of fibrous tissue as a normal component of an organ or tissue.

[0212] "Hematuria" means the presence of red blood cells in the urine.

[0213] Albuminuria refers to the presence of excessive albumin in urine, and includes (but is not limited to) normoalbuminuria, hypernoralbuminuria, microalbuminuria, and macroalbuminuria. Normally, the glomerular filtration permeability barrier, composed of podocytes, the glomerular basement membrane, and endothelial cells, prevents serum proteins from leaking into the urine. Albuminuria reflects damage to this barrier. Albuminuria can be calculated from 24-hour urine samples, overnight urine samples, or spot urine samples.

[0214] "Hypernormal albuminuria" refers to elevated albuminuria characterized by: (i) excretion of 15 to <30 mg of albumin in the urine every 24 hours and / or (ii) an albumin / creatinine ratio of 1.25 to <2.5 mg / mmol (or 10 to <20 mg / g) in men or 1.75 to <3.5 mg / mmol (or 15 to <30 mg / g) in women.

[0215] “Microalbuminuria” refers to elevated albuminuria characterized by: (i) excretion of 30 to 300 mg of albumin in the urine every 24 hours and / or (ii) an albumin / creatinine ratio of 2.5 to <25 mg / mmol (or 20 to <200 mg / g) in men and 3.5 to <35 mg / mmol (or 30 to <300 mg / g) in women.

[0216] "Major albuminuria" refers to elevated albuminuria characterized by: excretion of more than 300 mg of albumin in the urine every 24 hours and / or (ii) an albumin / creatinine ratio >25 mg / mmol (or >200 mg / g) in men or >35 mg / mmol (or >300 mg / g) in women.

[0217] The "albumin / creatinine ratio" refers to the ratio of urinary albumin (mg / dL) to urinary creatinine (g / dL), and is expressed in mg / g. In some 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.

[0218] "Glomerular filtration rate" or "GFR" refers to the rate at which filtrate flows through the kidneys and is used as an indicator of a subject's renal function. In some embodiments, a subject's GFR is determined by calculating an estimated glomerular filtration rate. In some embodiments, the subject's GFR is measured directly in the subject using the inulin method.

[0219] "Estimated glomerular filtration rate" or "eGFR" refers to a measurement of the extent to which the kidneys filter creatinine and is used to estimate glomerular filtration rate. Because direct measurement of GFR is complex, eGFR is more commonly used in clinical practice. Normal results are typically 90-120 mL / min / 1.73 mL. 2 Within the range. Levels below 60 mL / min / 1.73 m 2 A level lasting 3 months or longer may be an indicator of chronic kidney disease. Levels below 15 mL / min / 1.73 m 2 It could be an indicator of kidney failure.

[0220] “Proteinuria” refers to the presence of excessive serum proteins in the urine. Proteinuria is characterized by the excretion of >250 mg of protein in the urine every 24 hours and / or a urinary protein-to-creatinine ratio ≥ 0.20 mg / mg. Elevated serum proteins accompanying proteinuria include (but are not limited to) albumin.

[0221] "Blood urea nitrogen level," or "BUN level," refers to a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea as a waste product of protein digestion during the urea cycle, and urea is removed from the blood by the kidneys. Normal adult blood contains 7 to 21 mg of urea nitrogen per 100 ml of blood (7-21 mg / dL). Measurements of blood urea nitrogen levels are used as an indicator of kidney health. If the kidneys are unable to properly remove urea from the blood, the subject's BUN level will be elevated.

[0222] "Elevated" means an increase in a medical parameter that is considered clinically relevant. Healthcare professionals can determine whether the increase is clinically significant.

[0223] "End-stage renal disease (ESRD)" refers to complete or near-complete failure of kidney function.

[0224] "Quality of life" refers to the degree to which a subject's physical, psychological, and social functioning is impaired due to the disease and / or treatment of the disease. Subjects with polycystic kidney disease may experience a reduced quality of life.

[0225] "Impaired kidney function" means that kidney function is reduced compared to normal kidney function.

[0226] "Slowing down the progression of..." and "slowing down the progression of..." both refer to reducing the rate at which a medical condition progresses to a late stage.

[0227] "Delayed dialysis time" means maintaining sufficient kidney function so that the need for dialysis treatment can be delayed.

[0228] "Delaying kidney transplantation" means maintaining sufficient kidney function so that the need for a kidney transplant can be postponed.

[0229] "Improving life expectancy" means extending the lifespan of a subject by treating one or more symptoms of the subject's disease.

[0230] "Subject" refers to a human or non-human animal that is selected to undergo treatment or therapy.

[0231] "Subjects in need" refers to subjects identified as requiring therapy or treatment.

[0232] "Subjects suspected of having..." refers to subjects who exhibit one or more clinical indicators of a disease.

[0233] "MiR-17-related diseases" refers to diseases or disorders that are regulated by the activity of one or more members of the miR-17 family.

[0234] "Administration" means providing a medicine or composition to a subject, and includes (but is not limited to) administration by a medical professional and self-administration.

[0235] "Parenteral administration" means administration via injection or infusion. Parenteral administration includes (but is not limited to) subcutaneous, intravenous, and intramuscular administration.

[0236] "Subcutaneous application" means application slightly below the skin.

[0237] "Intravenous administration" means administration via intravenous infusion.

[0238] "Concomitant administration" refers to the simultaneous administration of two or more agents in any manner, where the pharmacological effects of both agents are simultaneously observed in the patient. Concomitant administration does not require the two agents to be administered as a single drug combination, in the same dosage form, or via the same route of administration. The effects of the two agents do not need to be simultaneous. The effects only need to overlap over a period of time, not necessarily be concurrent.

[0239] "Duration" refers to the period during which the activity or event continues. In some embodiments, the duration of treatment is the period during which a certain dose of a pharmaceutical agent or drug composition is administered.

[0240] "Therapy" refers to a treatment for a disease. In some implementations, a therapy includes (but is not limited to) administering one or more pharmaceutical agents to a subject suffering from a disease.

[0241] "Treatment" means the application of one or more specific procedures to improve at least one indicator of a disease. In some embodiments, the specific procedure is the administration of one or more pharmaceutical agents. In some embodiments, treatment of PKD includes (but is not limited to) reducing total kidney volume, improving renal function, lowering blood pressure, and / or relieving kidney pain.

[0242] "Improvement" means reducing the severity of at least one indicator of a disease or ailment. In some embodiments, improvement includes delaying or slowing the progression of one or more indicators of a disease or ailment. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art.

[0243] "At risk of developing..." means a state in which the subject is susceptible to an illness or disease. In some embodiments, a subject at risk of developing an illness or disease exhibits one or more symptoms of the illness or disease, but not a sufficient number of symptoms to be diagnosed with the illness or disease. In some embodiments, a subject at risk of developing an illness or disease exhibits one or more symptoms of the illness or disease, but to a degree less than that required to be diagnosed with the illness or disease.

[0244] "Prevention of the onset of..." means preventing a subject at risk of developing a disease or ailment from developing the disease or ailment. In some embodiments, a subject at risk of developing a disease or ailment receives treatment similar to that received by a subject who already has the disease or ailment.

[0245] "Delayed onset of..." means delaying the onset of a disease or ailment in a subject who is at risk of developing such a disease or ailment. In some embodiments, the subject at risk of developing a disease or ailment receives treatment similar to that received by a subject who already has the disease or ailment.

[0246] "Dosage" refers to the specified amount of pharmaceutical agent provided in a single administration. In some embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in some embodiments, where subcutaneous administration is desired, the volume required for the desired dose is not readily provided by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In some embodiments, a dose may be administered in two or more injections to minimize individual injection site reactions. In some embodiments, a dose is administered as a slow infusion.

[0247] "Dosage unit" refers to the form of delivery of a pharmaceutical preparation. In some embodiments, the dosage unit is a vial containing lyophilized oligonucleotides. In some embodiments, the dosage unit is a vial containing reconstituted oligonucleotides.

[0248] "Therapeutic effective dose" refers to the amount of a medicine that provides therapeutic benefit to an animal.

[0249] "Pharmaceutical composition" means a mixture of substances suitable for individual administration, including pharmaceutical preparations. For example, a pharmaceutical composition may contain a sterile aqueous solution.

[0250] "Medicinal agent" refers to a substance that provides a therapeutic effect when administered to a subject.

[0251] "Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that provides the desired effect.

[0252] “Pharmaceutical-acceptable salt” means a physiologically and pharmaceutically acceptable salt of the compounds provided herein, i.e., a salt that retains the desired biological activity of the compound when administered to a subject and does not have undesirable toxicological effects. Non-limiting exemplary pharmaceutically acceptable salts of the compounds provided herein include sodium and potassium salt forms. Unless otherwise expressly indicated, the terms “compound,” “oligonucleotide,” and “modified oligonucleotide” as used herein include their pharmaceutically acceptable salts.

[0253] "Salt solution" refers to a solution of sodium chloride in water.

[0254] "Improved organ function" refers to a change in organ function toward normal limits. In some implementations, organ function is assessed by measuring molecules found in the subject's blood or urine. For example, in some implementations, improved kidney function is measured by a decrease in blood urea nitrogen levels, a reduction in proteinuria, a reduction in albuminuria, etc.

[0255] "Acceptable safety profile" refers to the pattern of side effects within clinically acceptable limits.

[0256] "Side effects" refers to physiological responses attributable to treatment other than the desired effects. In some implementations, side effects include (but are not limited to) injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. Such side effects can be detected directly or indirectly. For example, an increase in serum transaminase levels may indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin may indicate hepatotoxicity or abnormal liver function.

[0257] As used in this article, the term "blood" encompasses whole blood and blood components such as serum and plasma.

[0258] "Anti-miR" refers to an oligonucleotide having a nucleobase sequence complementary to microRNA. In some implementations, anti-miR is a modified oligonucleotide.

[0259] "Anti-miR-17" refers to a modified oligonucleotide having a nucleobase sequence complementary to one or more miR-17 family members. In some embodiments, anti-miR-17 is fully complementary (i.e., 100% complementary) to one or more miR-17 family members. In some 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.

[0260] “miR-17” refers to a mature miRNA with the nucleobase sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO:1).

[0261] “miR-20a” refers to a mature miRNA with the nucleobase sequence 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' (SEQ ID NO:2).

[0262] “miR-20b” refers to a mature miRNA with the nucleobase sequence 5'- CAAAGUGCUCAUAGUGCAGGUAG-3' (SEQ ID NO: 3).

[0263] “miR-93” refers to a mature miRNA with the nucleobase sequence 5'- CAAAGUGCUGUUCGUGCAGGUAG-3' (SEQ ID NO:4).

[0264] “miR-106a” refers to a mature miRNA with the nucleobase sequence 5'-AAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO: 5).

[0265] “miR-106b” refers to a mature miRNA with the nucleobase sequence 5'-UAAAAGUGCUGACAGUGCAGAU-3' (SEQ ID NO:6).

[0266] The “miR-17 seed sequence” refers to the nucleobase sequence 5'-AAAGUG-3', which is present in every member of the miR-17 family.

[0267] "miR-17 family member" refers to a mature miRNA that has a nucleobase sequence containing the miR-17 seed sequence, and is selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0268] The “miR-17 family” refers to the following miRNA groups: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b, each of which has a nucleobase sequence containing the miR-17 seed sequence.

[0269] "Target nucleic acid" refers to nucleic acid that has been designed to hybridize with oligomeric compounds.

[0270] "Targeting" refers to the process of designing and selecting the nucleobase sequence that will hybridize with the target nucleic acid.

[0271] "Targeted" means having a nucleobase sequence that allows hybridization with the target nucleic acid.

[0272] "Regulation" refers to a disturbance of function, quantity, or activity. In some embodiments, regulation means an increase in function, quantity, or activity. In other embodiments, regulation means a decrease in function, quantity, or activity.

[0273] "Expression" refers to any function and step that translates the encoded information of a gene into a structure that exists and functions within the cell.

[0274] "Nucleobase sequence" refers to the sequence of adjacent nucleobases in an oligomer or nucleic acid, usually arranged in a 5' to 3' orientation, and is independent of any sugars, linkages, and / or nucleobase modifications.

[0275] "Neighboring nucleobases" refers to the nucleobases that are adjacent to each other in nucleic acids.

[0276] "Nucleobase complementarity" refers to the ability of two nucleobases to pair non-covalently via hydrogen bonds.

[0277] "Complementarity" means that one nucleic acid can hybridize with another nucleic acid or oligonucleotide. In some implementations, complementarity refers to the ability of an oligonucleotide to hybridize with a target nucleic acid.

[0278] "Complete complementarity" means that each nucleobase of the oligonucleotide is able to pair with a nucleobase at a corresponding position in the target nucleic acid. In some embodiments, the oligonucleotide is completely complementary to the microRNA (also known as 100% complementarity), meaning that each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. The modified oligonucleotide can be completely complementary to the microRNA and has a linker number less than the length of the microRNA. For example, an oligonucleotide having 16 linker nucleotides is completely complementary to the microRNA, wherein each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. In some embodiments, an oligonucleotide whose each nucleobase is complementary to a nucleobase within the stem-loop sequence region of the microRNA is completely complementary to the stem-loop sequence of the microRNA.

[0279] "Complementarity percentage" refers to the percentage of nucleobases in an oligonucleotide that are complementary to the same length portion of the target nucleic acid. It is calculated by dividing the number of nucleobases in the oligonucleotide that are complementary to the corresponding position in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

[0280] "Identity percentage" refers to the number of nucleotides in the first nucleic acid that are identical to those in the corresponding position in the second nucleic acid, divided by the total number of nucleotides in the first nucleic acid. In some embodiments, both the first and second nucleic acids are microRNAs. In some embodiments, both the first and second nucleic acids are oligonucleotides.

[0281] "Hybridization" refers to the annealing of complementary nucleic acids that occur through nucleobase complementarity.

[0282] "Mismatch" means that the nucleobases of the first nucleic acid cannot be paired with the corresponding nucleobases of the second nucleic acid in a Watson-Crick pairing.

[0283] In the context of nucleobase sequence, "identical" means having the same nucleobase sequence, regardless of sugar, bonding, and / or nucleobase modification, and regardless of the methylation state of any pyrimidine present.

[0284] “MicroRNA” refers to an endogenous non-coding RNA of 18 to 25 nucleotides in length, which is the product of precursor microRNA being cleaved by the enzyme Dicer. Examples of mature microRNAs can be found in a microRNA database called miRBase (microrna.sanger.ac.uk / ). In some implementations, microRNA is abbreviated as “miR”.

[0285] "MicroRNA-regulated transcripts" refers to transcripts regulated by microRNAs.

[0286] "Seed matching sequence" refers to a nucleobase sequence that is complementary to the seed sequence and has the same length as the seed sequence.

[0287] "Oligomers" refer to compounds that contain multiple linked monomeric subunits. Oligomeric compounds include oligonucleotides.

[0288] "Oligonucleotide" refers to a compound containing multiple linked nucleosides, each of which may be modified or unmodified independently of each other.

[0289] "Naturally occurring nucleoside linkages" refers to 3' to 5' phosphodiester linkages between nucleosides.

[0290] "Natural sugars" refers to sugars found in DNA (2'-H) or RNA (2'-OH).

[0291] "Nucleoside linkage" refers to the covalent linkage between adjacent nucleosides.

[0292] "Linked nucleosides" refers to nucleosides that are linked together by covalent bonds.

[0293] "Nucleobase" refers to a heterocyclic moiety that can non-covalently pair with another nucleobase.

[0294] "Nucleoside" refers to a nucleobase that is attached to a sugar.

[0295] "Nucleotide" refers to a nucleoside that has a phosphate ester group covalently linked to the sugar moiety of the nucleoside.

[0296] "A compound comprising a modified oligonucleotide consisting of a specified number of linked nucleosides" means a compound comprising a modified oligonucleotide having a specified number of linked nucleosides. Therefore, the compound may include additional substituents or conjugates. Unless otherwise indicated, the modified oligonucleotide does not hybridize with the complementary strand, and the compound does not contain any additional nucleosides other than those of the modified oligonucleotide.

[0297] "Modified oligonucleotides" refer to single-stranded oligonucleotides that have one or more modifications relative to their naturally occurring ends, sugars, nucleobases, and / or nucleoside linkages. Modified oligonucleotides may contain unmodified nucleosides.

[0298] "Modified nucleoside" means a nucleoside that has any variation compared to naturally occurring nucleosides. Modified nucleosides may have modified sugars and unmodified nucleobases. Modified nucleosides may have modified sugars and modified nucleobases. Modified nucleosides may have natural sugars and modified nucleobases. In some embodiments, the modified nucleoside is a bicyclic nucleoside. In some embodiments, the modified nucleoside is a non-bicyclic nucleoside.

[0299] "Modified nucleoside linkages" refers to any alteration compared to natural nucleoside linkages.

[0300] "Thiophosphate nucleoside linkage" refers to the linkage between nucleosides, in which one of the non-bridging atoms is a sulfur atom.

[0301] "Modified sugar portion" means the substitution and / or any change compared to natural sugar.

[0302] "Unmodified nucleobases" refers to naturally occurring heterocyclic bases in RNA or DNA: purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

[0303] "5-methylcytosine" refers to cytosine containing a methyl group attached to the 5-position.

[0304] "Unmethylated cytosine" refers to cytosine that does not have a methyl group attached to the 5-position.

[0305] "Modified nucleobase" means any nucleobase that is not an unmodified nucleobase.

[0306] "Sugar fraction" refers to naturally occurring furanyl groups or modified sugar fractions.

[0307] "Modified sugar portion" refers to the sugar portion that has been replaced or a sugar substitute.

[0308] "2'-O-methyl sugar" or "2'-OMe sugar" refers to a sugar with an O-methyl modification at the 2' position.

[0309] "2'-O-methoxyethyl sugar" or "2'-MOE sugar" refers to a sugar with an O-methoxyethyl modification at the 2' position.

[0310] "2'-F" or "2'-F" refers to sugars that have fluorine modification at the 2' position.

[0311] "Bicyclic sugar moiety" refers to a modified sugar moiety comprising a 4- to 7-membered ring (including, but not limited to, a furanyl group), wherein the 4- to 7-membered ring includes a bridge connecting two atoms of the 4- to 7-membered ring to form a second ring, thereby producing a bicyclic structure. In some embodiments, the 4- to 7-membered ring is a sugar ring. In some embodiments, the 4- to 7-membered ring is a furanyl group. In some such embodiments, the bridge connects the 2'-carbon and 4'-carbon of the furanyl group. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

[0312] "Locked nucleoside (LNA) sugar moiety" refers to a substituted sugar moiety containing a (CH2)-O bridge between the 4' and 2' furanose ring atoms.

[0313] "ENA sugar moiety" refers to a substituted sugar moiety containing a (CH2)2-O bridge between the 4' and 2' furanose ring atoms.

[0314] "Constrained ethyl (cEt) sugar moiety" refers to a substituted sugar moiety containing a CH(CH3)-O bridge between the 4' and 2' furanose ring atoms. In some embodiments, the CH(CH3)-O bridge is constrained in the S direction. In some embodiments, the CH(CH3)-O is constrained in the R direction.

[0315] "S-cEt sugar moiety" refers to a substituted sugar moiety containing an S-bound CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0316] "R-cEt sugar moiety" refers to a substituted sugar moiety containing an R-bound CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0317] "2'-O-methyl nucleoside" refers to a 2'-modified nucleoside with 2'-O-methyl sugar modification.

[0318] "2'-O-methoxyethyl nucleoside" refers to a 2'-modified nucleoside with a 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleoside may contain modified or unmodified nucleobases.

[0319] "2'-Fluoronucleotide" refers to a 2'-modified nucleoside with 2'-fluoro sugar modification. 2'-Fluoronucleotides may contain modified or unmodified nucleobases.

[0320] "Bicyclic nucleoside" refers to a nucleoside with a 2'-modified bicyclic sugar moiety. Bicyclic nucleosides may have modified or unmodified nucleobases.

[0321] "cEt nucleoside" refers to a nucleoside that contains a cEt sugar moiety. cEt nucleosides may contain modified or unmodified nucleobases.

[0322] "S-cEt nucleoside" refers to a nucleoside that contains the S-cEt sugar moiety.

[0323] "R-cEt nucleoside" refers to a nucleoside that contains the R-cEt sugar moiety.

[0324] “β-D-deoxyribonucleoside” refers to naturally occurring DNA nucleosides.

[0325] “β-D-ribonucleoside” refers to naturally occurring RNA nucleosides.

[0326] "LNA nucleoside" refers to a nucleoside that contains the LNA sugar moiety.

[0327] "ENA nucleoside" refers to nucleosides that contain the ENA sugar moiety.

[0328] "Hydrogen bond acceptor" refers to a hydrogen bond component that does not provide a shared hydrogen atom.

[0329] "Hydrogen bond donor" refers to a bond or molecule that provides a hydrogen atom for a hydrogen bond.

[0330] Overview

[0331] Polycystic kidney disease (PKD) is a form of inherited kidney disease in which fluid-filled cysts develop in the kidneys, leading to renal insufficiency and often end-stage renal disease. Some cases of PKD are also characterized by enlarged kidneys. Excessive cyst proliferation is a hallmark pathological feature of PKD. In the management of PKD, the primary goals of treatment are to manage symptoms (such as hypertension and infections), maintain kidney function, and prevent the onset of end-stage renal disease (ESRD), which in turn improves the life expectancy of individuals with PKD.

[0332] miR-17 has been identified as a target for the treatment of PKD. The anti-miR-17 compound RGLS4326 was discovered through screening chemically diverse and rationally designed anti-miR-17 oligonucleotide libraries to obtain optimal pharmaceutical properties. RGLS4326 preferentially distributes to renal and urinary duct-derived cysts, releases miR-17 from translationally active polyribosomes, and derepresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, after subcutaneous administration, RGLS4326 attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models. A Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) was initiated in October 2020.

[0333] Following the initiation of the Phase 1b clinical trial, nonclinical toxicology studies revealed CNS-related findings in mice at high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or exhaustion. RGLS4326 was found to be an antagonist of AMPA receptors (AMPA-R), which are glutamate receptors and ion channels at excitatory synapses in the central nervous system (CNS) that mediate rapid excitatory neurotransmission and are therefore a key component of all neuronal networks. Antagonism of AMPA receptors explains the CNS-mediated findings observed in nonclinical toxicology models at high doses of RGLS4326. Although such CNS-related findings have not been observed in human subjects, antagonism of AMPA receptors is still preferred. Therefore, a library of antimiR-17 compounds was screened to identify compounds with comparable physicochemical and pharmacological properties to RGLS4326 and also with a more favorable safety profile. One such compound, RG-NG-1015, was identified and selected as a candidate therapeutic agent for the treatment of ADPKD.

[0334] compound

[0335] This document provides compounds comprising modified oligonucleotides, wherein the modified oligonucleotides have the following structure in the 5' to 3' direction:

[0336] (N'')p -(N) r -(N') q

[0337] Each N'' is independently a modified or unmodified nucleoside; p ranges from 0 to 14; where if p is not 0, then (N'') p The nucleobase sequence of [the component] is complementary to the same length portion of the nucleobase sequence of miR-17; (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3'; N' is a nucleoside containing a modified sugar moiety; q is 0 or 1; wherein if q is 1, the nucleobase of N' is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; and each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof.

[0338] According to the standard numbering convention for nucleobases, purine nucleobases are numbered from 1 to 9, as shown in the following structure:

[0339] .

[0340] The number of atoms or groups bonded to the ring atoms of the nucleobase is the same as the number of ring atoms to which they are bonded.

[0341] Some nucleobases (such as guanosine and inosine) contain hydrogen bond acceptors at the 6-position. The hydrogen bond acceptor at the 6-position of guanosine is an oxygen atom bonded to the 6-carbon. The hydrogen bond acceptor at the 6-position of inosine is an oxygen atom bonded to the 6-carbon.

[0342] Purine nucleobases that do not have hydrogen bond acceptors at the 6-position include (but are not limited to) 2-aminopurine, 2,6-diaminopurine, isoguanosine, and adenosine. The NH2 at the 6-position in each of 2,6-diaminopurine, isoguanosine, and adenosine acts as a hydrogen bond donor. 2-aminopurine lacks a substituent at the 6-position and therefore lacks either a hydrogen bond acceptor or donor.

[0343] In some implementations, (N) r The structure is: A S G S C M A F C F U F U M U S The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside.

[0344] In some embodiments, at least one nucleoside link is a phosphate thioside link. In some embodiments, each nucleoside link is a phosphate thioside link.

[0345] In some implementations, q is 1. In some implementations, q is 0. In some implementations, p is 0. In some implementations, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0346] In some implementations, (N'') p The nucleobase sequence of [the component] has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1). In some embodiments, (N'') p The nucleobase sequence of [the component] does not mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1). In some embodiments, (N'') p The nucleobase sequences are selected from CUACCUGCACUGUA (SEQ ID NO: 7), CUACCUGCACUGU (SEQ ID NO: 8), CUACCUGCACUG (SEQ ID NO: 9), CUACCUGCACU (SEQ ID NO: 10), CUACCUGCAC (SEQ ID NO: 11), CUACCUGCA, CUACCUGC, CUACCUG, CUACCU, CUACC, CUAC, CUA, CU, and C.

[0347] In some embodiments, the nucleotide of N' is a purine nucleotide that does not have a hydrogen bond acceptor at position 6. In some embodiments, the nucleotide of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0348] In some embodiments, the sugar moiety of N' is not a 2'-O-methyl sugar. In some embodiments, the sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar.

[0349] In some implementations, the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3', where each cytosine is an unmethylated cytosine. In some embodiments, the modified oligonucleotide has a 5'-A structure. S GS C M A F C F U F U M U S U S -3', where each cytosine is an unmethylated cytosine. In some embodiments, the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S C S -3', wherein each cytosine is a nonmethylated cytosine. The compound of claim 2, wherein the modified oligonucleotide has a structure of 5'-A. S G S C M A F C F U F U M U S -3', where each cytosine is a nonmethylated cytosine.

[0350] In some implementations, the compound consists of modified oligonucleotides.

[0351] In some implementations, the pharmaceutically acceptable salt is a sodium salt.

[0352] This article provides modified oligonucleotides with the following structures:

[0353]

[0354] Wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; or a pharmaceutically acceptable salt thereof. In some embodiments, B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0355] This article provides modified oligonucleotides with the following structures:

[0356]

[0357] Wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In some embodiments, B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0358] This article provides a modified oligonucleotide named RG-NG-1015, wherein the structure of the modified oligonucleotide is as follows:

[0359] .

[0360] This document also provides pharmaceutically acceptable salts of the modified oligonucleotide RG-NG-1015. Therefore, in some embodiments, the modified oligonucleotide has the following structure:

[0361]

[0362] Or a pharmaceutically acceptable salt thereof. A non-limiting exemplary pharmaceutically acceptable salt of RG-NG-1015 has the following structure:

[0363] .

[0364] In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide contains a cationic counterion (e.g., Na+). + Less than those present in the thiophosphate and / or phosphodiester bonds per molecule (i.e., some thiophosphate and / or phosphodiester bonds are protonated). In some embodiments, pharmaceutically acceptable salts of RG-NG-1015 contain fewer than 8 cationic counterions (e.g., Na+) per molecule of RG-NG-1015. + In other words, in some embodiments, each molecule of pharmaceutically acceptable salt of RG-NG-1015 may contain an average of 1, 2, 3, 4, 5, 6 or 7 cationic counterions, with the remaining thiophosphate groups being protonated.

[0365] certain uses

[0366] This article provides a method for inhibiting the activity of one or more members of the miR-17 family in cells, the method comprising contacting cells with a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0367] This document provides a method for inhibiting the activity of one or more members of the miR-17 family in a subject, the method comprising administering to the subject a pharmaceutical composition provided herein. In some embodiments, the subject suffers from a disease associated with one or more members of the miR-17 family.

[0368] This document provides a method for treating polycystic kidney disease (PKD), the method comprising administering a compound provided herein to a subject in need, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject has polycystic kidney disease. In some embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and non-autosomal wasting disease (NPHP). In some embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD).

[0369] In some implementations, the subject suffers from a condition characterized by multiple non-renal markers and polycystic kidney disease. Such conditions include, for example, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). Therefore, this document provides a method for treating polycystic kidney disease (PKD) comprising administering to a subject a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject suffers from Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). This document provides a method for treating polycystic kidney disease (PKD) comprising administering to a subject a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject is suspected of having Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0370] In some implementations, polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the PKD1 or PKD2 genes. ADPKD is a progressive disease in which cyst formation and kidney enlargement lead to renal insufficiency and ultimately result in end-stage renal disease in 50% of patients by age 60. Patients with ADPKD may require lifelong dialysis and / or kidney transplantation. ADPKD is the most common genetic cause of kidney failure. Excessive cyst proliferation is a hallmark pathological feature of ADPKD. In the management of PKD, the primary goal of treatment is to maintain renal function and prevent the onset of end-stage renal disease (ESRD), which in turn improves the life expectancy of subjects with PKD. In patients with ADPKD, the total kidney volume typically increases steadily, with this increase being associated with a decline in renal function. This article provides a method for treating ADPKD, which comprises administering a compound provided herein to a subject who has or is suspected of having ADPKD, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0371] In some implementations, polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). ARPKD is caused by mutations in the PKHD1 gene and is a cause of chronic kidney disease in children. The typical renal phenotype of ARPKD is enlarged kidneys; however, ARPKD has significant effects on other organs, particularly the liver. Patients with ARPKD progress to end-stage renal disease and require kidney transplantation as young as 15 years of age. This article provides a method for treating ARPKD, comprising administering a compound provided herein to a subject who has or is suspected of having ARPKD, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0372] In some implementations, polycystic kidney disease is referred to as renal wasting disease (NPHP). Renal wasting disease is an autosomal recessive cystic kidney disease and a common cause of ESRD in children. NPHP is characterized by normal or reduced kidney size, cysts concentrated at the corticomedullary junction, and tubulointerstitial fibrosis. Mutations in one of several NPHP genes (e.g., NPHP1) have been identified in patients with NPHP. This document provides a method for treating NPHP, comprising administering a compound provided herein to a subject who has or is suspected of having NPHP, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0373] In some embodiments, the subject with polycystic kidney disease also has Jupiter syndrome and related conditions (JSRD). JSRD includes a wide range of hallmark features, including brain, retinal, and skeletal abnormalities. In addition to the hallmark features of JSRD, some subjects with JSRD also have polycystic kidney disease. Therefore, this document provides a method for treating polycystic kidney disease in subjects with JSRD, the method comprising administering to the subject with JSRD a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having JSRD.

[0374] In some embodiments, the subject with polycystic kidney disease is diagnosed with Meckel syndrome (MKS). MKS is a condition characterized by severe signs and symptoms in many parts of the body, including the central nervous system, skeletal system, liver, kidneys, and heart. A common feature of MKS is the presence of multiple fluid-filled cysts in the kidneys and kidney enlargement. Therefore, this document provides a method for treating MKS, comprising administering to a subject with MKS a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having MKS.

[0375] In some embodiments, the subject with polycystic kidney disease has Budd-Bied syndrome (BBS). BBS is a condition that affects many parts of the body, including the eyes, heart, kidneys, liver, and digestive system. A hallmark feature of BBS is the presence of kidney cysts. Therefore, this document provides a method for treating polycystic kidney disease in a subject with BBS, the method comprising administering to the subject a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having BBS.

[0376] In some implementations, the subject has been diagnosed with PKD prior to administration of a compound containing a modified oligonucleotide. The diagnosis of PKD can be achieved by evaluating parameters including (but not limited to) the following: the subject's family history, clinical characteristics (including but not limited to hypertension, albuminuria, hematuria, and impaired GFR), renal imaging studies (including but not limited to MRI, ultrasound, and CT scans), and / or histological analysis.

[0377] In some embodiments, PKD diagnosis includes screening for mutations in one or more of the PKD1 or PKD2 genes. In some embodiments, ARPKD diagnosis includes screening for mutations in the PKHP1 gene. In some embodiments, NPHP diagnosis includes screening for mutations in one or more of the NPHP1, NPHP2, NPHP3, NPHP4, NPHP5, NPHP6, NPHP7, NPHP8, or NPHP9 genes. In some embodiments, JSRD diagnosis includes screening for mutations in the NPHP1, NPHP6, AHI1, MKS3, or RPGRIP1L genes. In some embodiments, MKS diagnosis includes screening for mutations in the NPHP6, MKS3, RPGRIP1L, NPHP3, CC2D2A, BBS2, BBS4, BBS6, or MKS1 genes. In some implementations, BBS diagnosis includes screening for mutations in the BBS2, BBS4, BBS6, MKS1, BBS1, BBS3, BBS5, BBS7, BBS8, BBS9, BBS10, BBS11, or BBS12 genes.

[0378] In some implementations, the subject has an increased total kidney volume. In some implementations, the total kidney volume is a highly adjusted total kidney volume (HtTKV). In some implementations, the subject has hypertension. In some implementations, the subject has impaired kidney function. In some implementations, the subject requires improved kidney function. In some implementations, the subject is identified as having impaired kidney function.

[0379] In some embodiments, the levels of one or more miR-17 family members are increased in the kidneys of a subject with PKD. In some embodiments, the increased levels of one or more miR-17 family members in the kidneys of the subject are determined prior to administration. The levels of miR-17 family members can be measured from kidney biopsy material. In some embodiments, the increased levels of one or more miR-17 family members in the urine or blood of the subject are determined prior to administration. In some embodiments, the decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine of the subject are determined prior to administration. In some embodiments, the decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine of the subject are determined prior to administration. In some embodiments, the decreased levels of polycystic protein-1 (PC1) and / or polycystic protein-2 (PC2) in the urine of the subject are determined prior to administration.

[0380] In any of the embodiments provided herein, a subject may undergo certain tests to diagnose the subject's polycystic kidney disease, such as determining the cause of the polycystic kidney disease, evaluating the severity of the subject's polycystic kidney disease, and / or measuring the subject's response to treatment. Such tests may assess biomarkers of polycystic kidney disease. Some of these tests (e.g., glomerular filtration rate and blood urea nitrogen levels) are also indicators of kidney function. Biomarkers for polycystic disease include (but are not limited to): measurements of total kidney volume; measurements of hypertension; assessment of renal pain; measurements of fibrosis; measurements of polycystic protein-1 (PC1) in urine; measurements of polycystic protein-2 (PC2) in urine; measurements of blood urea nitrogen levels; measurements of serum creatinine levels; measurements of creatinine clearance; measurements of albuminuria; measurements of albumin:creatinine ratio; measurements of glomerular filtration rate; measurements of hematuria; measurements of NGAL protein in urine; and / or measurements of KIM-1 protein in urine. Unless otherwise indicated herein, blood urea nitrogen levels, serum creatinine levels, creatinine clearance, albuminuria, albumin:creatinine ratio, glomerular filtration rate, and hematuria refer to measurements of the subject's blood (e.g., whole blood or serum).

[0381] Biomarkers for polycystic kidney disease are determined through laboratory tests. Reference ranges for individual biomarkers may vary between laboratories. Such deviations may be due to, for example, differences in the specific assays used. Therefore, the upper and lower limits of the normal distribution of a biomarker within a population (also referred to as the upper limit of normal (ULN) and lower limit of normal (LLN) respectively) may vary from laboratory to laboratory. For any given biomarker, a healthcare professional can determine which levels outside the normal distribution are clinically relevant and / or indicative of disease. For example, a healthcare professional can determine glomerular filtration rate (GFR), which may indicate the rate of decline in renal function in a subject with polycystic kidney disease.

[0382] In some embodiments, administration of the compounds provided herein produces one or more clinically beneficial results. In some embodiments, the administration improves renal function in a subject. In some embodiments, the administration slows the rate of decline in renal function in a subject. In some embodiments, the administration reduces total kidney volume in a subject. In some embodiments, the administration slows the rate of increase in total kidney volume in a subject. In some embodiments, the administration reduces highly adjusted total kidney volume (HtTKV). In some embodiments, the administration slows the rate of increase in HtTKV.

[0383] In some embodiments, the administration increases polycystic protein-1 (PC1) in the subject's urine. In some embodiments, the administration increases polycystic protein-2 (PC2) in the subject's urine. In some embodiments, the administration increases both polycystic protein-1 (PC1) and polycystic protein-2 (PC2) in the subject's urine.

[0384] In some embodiments, the application inhibits cyst growth in the subject. In some embodiments, the application slows the rate of increase in cyst growth in the subject. In some embodiments, the cyst is located in the subject's kidney. In some embodiments, the cyst is located in an organ other than the kidney (e.g., the liver).

[0385] In some embodiments, the administration alleviates the subject's kidney pain. In some embodiments, the administration slows the increase in the subject's kidney pain. In some embodiments, the administration delays the onset of the subject's kidney pain.

[0386] In some embodiments, the administration lowers the subject's blood pressure. In some embodiments, the administration slows the worsening of the subject's blood pressure. In some embodiments, the administration delays the onset of the subject's blood pressure.

[0387] In some embodiments, the administration reduces fibrosis in the subject's kidneys. In some embodiments, the administration slows the progression of fibrosis in the subject's kidneys.

[0388] In some embodiments, the administration delays the onset of end-stage renal disease in the subject. In some embodiments, the administration delays the duration of dialysis in the subject. In some embodiments, the administration delays the time to kidney transplantation in the subject. In some embodiments, the administration increases the life expectancy of the subject.

[0389] In some embodiments, the administration reduces albuminuria in the subject. In some embodiments, the administration slows the worsening of albuminuria in the subject. In some embodiments, the administration delays the onset of albuminuria in the subject. In some embodiments, the administration reduces hematuria in the subject. In some embodiments, the administration slows the worsening of hematuria in the subject. In some embodiments, the administration delays the onset of hematuria in the subject. In some embodiments, the administration lowers the blood urea nitrogen level in the subject. In some embodiments, the administration lowers the serum creatinine level in the subject. In some embodiments, the administration improves the creatinine clearance rate in the subject. In some embodiments, the administration lowers the albumin:creatinine ratio in the subject.

[0390] In some embodiments, the administration improves the subject's glomerular filtration rate (GFR). In some embodiments, the administration slows the rate of decline in the subject's GFR. In some embodiments, the GFR is an estimated GFR (eGFR). In some embodiments, the GFR is a measured GFR (mGFR).

[0391] In some embodiments, the administration reduces neutrophil gelatinase-associated lipocalin (NGAL) in the subject's urine. In some embodiments, the administration reduces kidney injury molecule-1 (KIM-1) protein in the subject's urine.

[0392] In any of the embodiments provided herein, a subject may be subjected to certain tests to assess the severity of the subject's disease. Such tests include, but are not limited to, measuring the subject's total kidney volume; measuring the subject's hypertension; measuring the subject's renal pain; measuring the subject's renal fibrosis; measuring the subject's blood urea nitrogen level; measuring the subject's serum creatinine level; measuring the subject's creatinine clearance in the subject's blood; measuring the subject's albuminuria; measuring the subject's albumin:creatinine ratio; measuring the subject's glomerular filtration rate, wherein the glomerular filtration rate is estimated or measured; measuring neutrophil gelatinase-associated lipocalin (NGAL) in the subject's urine; and / or measuring kidney injury molecule-1 (KIM-1) protein in the subject's urine.

[0393] In some implementations, the quality of life of subjects with polycystic kidney disease is reduced. For example, subjects with polycystic kidney disease may experience kidney pain, which can reduce their quality of life. In some implementations, the administration improves the subject's quality of life.

[0394] In any of the embodiments provided herein, the subjects are human subjects. In some embodiments, the human subjects are adults. In some embodiments, the adults are at least 21 years old. In some embodiments, the human subjects are pediatric subjects, i.e., subjects under 21 years of age. The pediatric population may be defined by the regulatory authority. In some embodiments, the human subjects are adolescents. In some embodiments, the adolescents are at least 12 years old and under 21 years old. In some embodiments, the human subjects are children. In some embodiments, the children are at least two years old and under 12 years old. In some embodiments, the human subjects are infants. In some embodiments, the infants are at least one month old and under two years old. In some embodiments, the subjects are newborns. In some embodiments, the newborns are under one month old.

[0395] Any compound described herein may be used in a therapeutic context. Any compound provided herein may be used to treat polycystic kidney disease. In some embodiments, the polycystic kidney disease is autosomal dominant polycystic kidney disease. In some embodiments, the polycystic kidney disease is autosomal recessive polycystic kidney disease. In some embodiments, the polycystic kidney disease is renal wasting disease. In some embodiments, the subject has Jupiter syndrome and related disorders (JSRD), Merkel syndrome (MKS), or Budd-Bead syndrome (BBS).

[0396] Any modified oligonucleotides described in this article can be used in therapy. Any modified oligonucleotides provided in this article can be used to treat polycystic kidney disease.

[0397] Any of the compounds described herein can be used to prepare pharmaceutical preparations. Any of the compounds described herein can be used to prepare pharmaceutical preparations for the treatment of polycystic kidney disease.

[0398] Any modified oligonucleotides provided in this article can be used to prepare pharmaceutical agents. Any modified oligonucleotides provided in this article can be used to prepare pharmaceutical agents for the treatment of polycystic kidney disease.

[0399] Any of the pharmaceutical compositions described herein can be used to treat polycystic kidney disease.

[0400] Some additional treatments

[0401] Treatment for polycystic kidney disease or any of the disorders listed herein may include more than one therapy. Therefore, in some embodiments, this document provides a method for treating a subject who has or is suspected of having polycystic kidney disease, the method 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.

[0402] In some embodiments, the at least one additional therapy comprises a pharmaceutical agent. In some embodiments, the pharmaceutical agent is an antihypertensive agent. The antihypertensive agent is used to control the subject's blood pressure.

[0403] In some embodiments, the pharmaceutical agent is an angiotensin receptor 2 antagonist. In some embodiments, the angiotensin receptor 2 antagonist is tolvaptan.

[0404] In some embodiments, the pharmaceutical agent includes an angiotensin II receptor blocker (ARB). In some embodiments, the angiotensin II receptor blocker is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, or eprosartan.

[0405] In some embodiments, the pharmaceutical agent includes an angiotensin II converting enzyme (ACE) inhibitor. In some embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, or ramipril.

[0406] In some embodiments, the pharmaceutical agent is a diuretic. In some embodiments, the pharmaceutical agent is a calcium channel blocker.

[0407] In some embodiments, the pharmaceutical agent is a glucose-ceramide synthase inhibitor. In some embodiments, the glucose-ceramide synthase inhibitor is venulstat.

[0408] In some embodiments, the pharmaceutical agent is an antihyperglycemic agent. In some embodiments, the antihyperglycemic agent is biguanide. In some embodiments, the biguanide is metformin.

[0409] In some embodiments, the pharmaceutical agent is a kinase inhibitor. In some embodiments, the kinase inhibitor is besutinib or KD019.

[0410] In some implementations, the pharmaceutical agent is an adrenergic receptor antagonist.

[0411] In some embodiments, the pharmaceutical agent is an aldosterone receptor antagonist. In some embodiments, the aldosterone receptor antagonist is spironolactone. In some embodiments, spironolactone is administered at a dose ranging from 10 mg to 35 mg daily. In some embodiments, spironolactone is administered at a dose of 25 mg daily.

[0412] In some embodiments, the pharmaceutical agent is a mammalian target of rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

[0413] In some embodiments, the pharmaceutical agent is a hormone analogue. In some embodiments, the hormone analogue is somatostatin or adrenocorticotropic hormone.

[0414] In some embodiments, the pharmaceutical agent is an antifibrotic agent. In some embodiments, the antifibrotic agent is a modified oligonucleotide complementary to miR-21.

[0415] In some implementations, the additional treatment is dialysis. In some implementations, the additional treatment is a kidney transplant.

[0416] In some embodiments, the pharmaceutical agent includes an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In some embodiments, the steroidal anti-inflammatory agent is a corticosteroid. In some embodiments, the corticosteroid is prednisone. In some embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug. In some embodiments, the non-steroidal anti-inflammatory agent is ibuprofen, a COX-1 inhibitor, or a COX-2 inhibitor.

[0417] In some implementations, the pharmaceutical agent is a pharmaceutical agent that blocks one or more responses to the fiber generation signal.

[0418] In some implementations, additional therapies may be pharmaceutical agents that enhance the body’s immune system, including low-dose cyclophosphamide, thymosin, vitamins and nutritional supplements (such as antioxidants, including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea), and vaccines, such as immunostimulatory complexes (ISCOM), which are multipolymer-presented vaccine formulations containing a combination of antigens and adjuvants.

[0419] In some embodiments, additional therapies are selected to treat or improve side effects of one or more of the pharmaceutical compositions provided herein. Such side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and muscle lesions. For example, an increase in serum transaminase levels may indicate hepatotoxicity or abnormal liver function. For example, an increase in bilirubin may indicate hepatotoxicity or abnormal liver function.

[0420] Certain microRNA nucleobase sequences

[0421] 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 nucleotide sequence comprising the 5'-AAAGUG-3' nucleotide sequence or a miR-17 seed sequence, wherein the seed sequence is the nucleotide sequence from position 2 to position 7 of SEQ ID NO: 1. Furthermore, each member of the miR-17 family shares a certain nucleotide sequence identity outside the seed region. Therefore, modified oligonucleotides containing a nucleotide sequence complementary to the miR-17 seed sequence can target other microRNAs in the miR-17 family besides miR-17. In some embodiments, the modified oligonucleotide targets two or more microRNAs of the miR-17 family. In some embodiments, the modified oligonucleotide targets three or more microRNAs of the miR-17 family. In some embodiments, the modified oligonucleotide targets four or more microRNAs of the miR-17 family. In some embodiments, the modified oligonucleotide targets five or more microRNAs of the miR-17 family. In some embodiments, the modified oligonucleotide targets six microRNAs of the miR-17 family. For example, a modified oligonucleotide having the nucleotide sequence 5'-AGCACUUU-3' targets all members of the miR-17 family.

[0422] In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUU-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUU-3'.

[0423] In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-GCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In some embodiments, the modified oligonucleotide is the nucleobase sequence 5'-AGCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6.

[0424] In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUUA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUU-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUU-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACU-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCAC-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-GCACUUUA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUUA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-ACUUUA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CUUUA-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AAGCACUUUA-3'.

[0425] In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTT-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUT-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTU-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In some embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTU-3'.

[0426] In some embodiments, each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. In some embodiments, at least one cytosine is unmethylated cytosine. In some embodiments, each cytosine is unmethylated cytosine. In some embodiments, at least one cytosine is 5-methylcytosine. In some embodiments, each cytosine is 5-methylcytosine.

[0427] In some embodiments, the number of linking nucleotides in the modified oligonucleotide is less than the length of its target microRNA. A modified oligonucleotide having a number of linking nucleotides less than the length of the target microRNA (where each nucleobase of the modified oligonucleotide is complementary to a nucleobase at a corresponding position in the target microRNA) is considered a modified oligonucleotide having a nucleobase sequence that is completely complementary (also referred to as 100% complementary) to a region of the target microRNA sequence. For example, a modified oligonucleotide consisting of nine linking nucleotides (where each nucleobase is complementary to a corresponding position in miR-17) is completely complementary to miR-17.

[0428] In some embodiments, the modified oligonucleotide has one mismatched nucleotide sequence relative to the target microRNA's nucleotide sequence. In some embodiments, the modified oligonucleotide has two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some such embodiments, the modified oligonucleotide has no more than two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some such embodiments, the mismatched nucleotides are adjacent. In some such embodiments, the mismatched nucleotides are not adjacent.

[0429] Although the sequence listing accompanying this application identifies each nucleobase sequence as “RNA” or “DNA” as required, in practice, those sequences may be modified with the chemical modification combinations specified herein. Those skilled in the art will readily appreciate that the names used in the sequence listing, such as “RNA” or “DNA,” to describe modified oligonucleotides are somewhat arbitrary. For example, a modified oligonucleotide comprising a nucleoside containing a 2'-O-methoxyethyl sugar moiety and a thymine base may be described in the sequence listing as a DNA residue, even if the nucleoside is modified and not a native DNA nucleoside.

[0430] Therefore, the nucleic acid sequences provided in the sequence listing are intended to cover nucleic acids containing any combination of natural or modified RNA and / or DNA, including (but not limited to) such nucleic acids having modified nucleotides. As another example, and without limitation, the modified oligonucleotides in the sequence listing having the nucleotide sequence “ATCGATCG” cover any oligonucleotide having this nucleotide sequence, whether modified or unmodified, including (but not limited to) compounds containing RNA bases, such as those having the sequence “AUCGAUCG”; and those having some DNA bases and some RNA bases such as “AUCGATCG”; and those having other modified bases such as “AT”. me The oligonucleotide “CGAUCG”, in which me C indicates 5-methylcytosine.

[0431] certain modifications

[0432] In some embodiments, the oligonucleotides provided herein may comprise one or more modifications to the inter-nucleobase, sugar, and / or nucleoside linkages, and are therefore modified oligonucleotides. The modified inter-nucleobase, sugar, and / or nucleoside linkages may be selected relative to the unmodified form for desired properties, such as enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases.

[0433] In some implementations, the modified oligonucleotide comprises one or more modified nucleosides.

[0434] In some embodiments, the modified nucleoside is a sugar-modified nucleoside. In some such embodiments, the sugar-modified nucleoside may also comprise a natural or modified heterocyclic base moiety, and / or may be linked to another nucleoside via a natural or modified internucleotide bond, and / or may include other modifications independent of the sugar modification. In some embodiments, the sugar-modified nucleoside is a 2'-modified nucleoside, wherein the sugar ring is modified at the 2' carbon of the natural ribose or 2'-deoxy-ribose.

[0435] In some embodiments, the 2'-modified nucleoside has a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety is a D sugar with an α-configuration. In some such embodiments, the bicyclic sugar moiety is a D sugar with a β-configuration. In some such embodiments, the bicyclic sugar moiety is an L sugar with an α-configuration. In some such embodiments, the bicyclic sugar moiety is an L sugar with a β-configuration.

[0436] Nucleosides containing such bicyclic sugar moieties are called bicyclic nucleosides or BNAs. In some embodiments, bicyclic nucleosides include (but are not limited to) the following illustrated below: (A) α-L-methyleneoxy(4'-CH2-O-2') BNA; (B) β-D-methyleneoxy(4'-CH2-O-2') BNA; (C) ethyloxy(4'-(CH2)2-O-2') BNA; (D) aminooxy(4'-CH2-ON(R)-2') BNA; (E) oxyamino(4'-CH2-N(R)-O-2') BNA; (F) methyl(methyleneoxy)(4'-CH(CH3)-O-2') BNA (also known as bound ethyl or cEt); (G) methylene-thio(4'-CH2-S-2') BNA; (H) methylene-amino(4'-CH2-N(R)-2') BNA; (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNA; (J) c-MOE (4'-CH(CH2-OMe)-O-2') BNA and (K) propylene carbocyclic (4'-(CH2)3-2') BNA.

[0437]

[0438]

[0439] Where Bx is the nucleobase moiety, and R is independently H, a protecting group, or C1-C. 12 alkyl.

[0440] In some embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from the following: F, OCF3, O-CH3 (also known as "2'-OMe"), OCH2CH2OCH3 (also known as "2'-O-methoxyethyl" or "2'-MOE"), 2'-O(CH2)2SCH3, O-(CH2)2-ON(CH3)2, -O(CH2)2O(CH2)2N(CH3)2 and O-CH2-C(=O)-N(H)CH3.

[0441] In some embodiments, the 2'-modified nucleoside contains a 2'-substituent selected from the following: F, O-CH3, and OCH2CH2OCH3.

[0442] In some embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In some embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. The 4'-thio-modified nucleoside has β-D-ribonucleoside, wherein 4'-O is replaced by 4'-S. The 4'-thio-2'-modified nucleoside is a 4'-thio-modified nucleoside in which 2'-OH is replaced by a 2'-substituent. Suitable 2'-substituents include 2'-OCH3, 2'-OCH2CH2OCH3, and 2'-F.

[0443] In some embodiments, the modified oligonucleotide comprises one or more inter-nucleoside modifications. In some such embodiments, each inter-nucleoside link of the modified oligonucleotide is a modified inter-nucleoside link. In some embodiments, the modified inter-nucleoside link comprises a phosphorus atom.

[0444] In some embodiments, the modified oligonucleotide comprises at least one phosphate thioester nucleoside link. In some embodiments, each nucleoside link of the modified oligonucleotide is a phosphate thioester nucleoside link.

[0445] In some embodiments, the modified oligonucleotide comprises one or more modified nucleobases. In some embodiments, the modified nucleobase is selected from 5-hydroxymethylcytosine, 7-deazoguanine, and 7-deazoadenine. In some embodiments, the modified nucleobase is selected from 7-deazoadenine, 7-deazoguanosine, 2-aminopyridine, and 2-pyridone. In some embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0446] In some embodiments, the modified nucleobase comprises a polycyclic heterocycle. In some embodiments, the modified nucleobase comprises a tricyclic heterocycle. In some embodiments, the modified nucleobase comprises a phenoxazine derivative. In some embodiments, the phenoxazine may be further modified to form a nucleobase known in the art as a G-clamp.

[0447] In some embodiments, the modified oligonucleotide is conjugated to one or more portions that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. In some such embodiments, the portion is a cholesterol moiety. In some embodiments, the portion is a lipid moiety. Other moieties used for conjugation include carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinone, acridine, fluorescein, rose red, coumarin, and dyes. In some embodiments, the carbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In some embodiments, the conjugating group is directly linked to the oligonucleotide. In some embodiments, the conjugating group is linked to the modified oligonucleotide via a linker selected from amino, azide, hydroxy, carboxylic acid, thiol, unsaturated moiety (e.g., double or triple bond), 8-amino-3,6-dioxanoic acid (ADO), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide (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 some such embodiments, the substituent is selected from hydroxyl, amino, alkoxy, azide, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0448] In some such embodiments, the compound comprises a modified oligonucleotide having one or more stabilizing groups attached to one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. A cap structure is included within the stabilizing group. These end modifications protect the modified oligonucleotide from exonuclease degradation and may facilitate intracellular delivery and / or localization. The cap may be present at the 5' end (5'-cap) or the 3' end (3'-cap), or may be present at both ends. The cap structure includes, for example, a reverse deoxygenated baseless cap.

[0449] Certain pharmaceutical compositions

[0450] This document provides pharmaceutical compositions comprising the compounds or modified oligonucleotides provided herein and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In some embodiments, the aqueous solution is a saline solution. As used herein, a pharmaceutically acceptable diluent should be understood as a sterile diluent. Suitable routes of administration include (but are not limited to) intravenous and subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. In some embodiments, administration is oral.

[0451] In some embodiments, the pharmaceutical composition is administered in the form of dosage units. For example, in some embodiments, the dosage units are in the form of tablets, capsules, or bolus injections.

[0452] In some embodiments, the pharmaceutical agent is a modified oligonucleotide prepared in a suitable diluent, adjusted to pH 7.0–9.0 with acid or base during preparation, and then lyophilized under aseptic conditions. The lyophilized modified oligonucleotide is subsequently reconstituted with a suitable diluent, such as an aqueous solution, like water, or a physiologically compatible buffer, such as saline solution, Hanks' solution, or Ringer's solution. The reconstituted product is administered subcutaneously or intravenously. The lyophilized pharmaceutical product can be packaged in 2 mL Type I clear glass vials (treated with ammonium sulfate), plugged with bromobutyl rubber and sealed with an aluminum top seal.

[0453] In some embodiments, the pharmaceutical compositions provided herein may additionally contain other excipients conventionally present in pharmaceutical compositions, at levels established in the industry. Thus, for example, the composition may contain additional compatible pharmaceutically active materials, such as antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents.

[0454] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials that can be used to physically formulate various dosage forms of the compositions provided herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers; such additional materials also include (but are not limited to) excipients, such as alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone. In various embodiments, such materials, when added, should not excessively interfere with the bioactivity of the components of the compositions provided herein. The formulation may be sterilized and, where desired, mixed with excipients (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, etc.) that do not adversely interact with the oligonucleotides of the formulation. Some injectable pharmaceutical compositions are suspensions, solutions, or emulsions in oily or aqueous media and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Suitable solvents for injectable pharmaceutical compositions include (but are not limited to) lipophilic solvents and fatty oils (e.g., sesame oil), synthetic fatty acid esters (e.g., ethyl oleate or triglycerides), and liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent to allow for the preparation of high-concentration solutions.

[0455] Lipid moieties have been used in nucleic acid therapy in various methods. In one method, nucleic acids are introduced into a pre-formed liposome or lipoplex prepared from a mixture of cationic and neutral lipids. In another method, a DNA complex comprising monocationic or polycationic lipids is formed in the absence of neutral lipids. In some embodiments, the lipid moieties are selected to increase the distribution of the pharmaceutical agent to specific cells or tissues. In some embodiments, the lipid moieties are selected to increase the distribution of the pharmaceutical agent to adipose tissue. In some embodiments, the lipid moieties are selected to increase the distribution of the pharmaceutical agent to muscle tissue.

[0456] In some embodiments, the pharmaceutical compositions provided herein comprise a polyamine compound or a lipid moiety complexed with a nucleic acid. In some embodiments, such formulations comprise one or more compounds, each individually having a structure defined by formula (Z) or a pharmaceutically acceptable salt thereof.

[0457]

[0458] Each X a and X b It is C independently each time it appears. 1-6Alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least about 80% of the compounds of formula (Z) in the formulation have at least n + 2 R portions that are not H; m is 1, 2, 3, or 4; Y is O, NR. 2 Or S; R 1 It is alkyl, alkenyl, or ynyl; each of which is optionally substituted by one or more substituents; and R 2 The R group is H, alkyl, alkenyl, or alkynyl; each is optionally substituted with one or more substituents; provided that if n = 0, at least n + 3 R groups 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.

[0459] In some embodiments, the pharmaceutical compositions provided herein are prepared using known techniques, including (but not limited to) mixing, dissolving, granulating, forming sugar-coated pills, grinding, emulsifying, encapsulating, embedding, or tableting processes.

[0460] In some embodiments, the pharmaceutical compositions provided herein are solids (e.g., powders, tablets, and / or capsules). In some such embodiments, the solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using ingredients known in the art, including (but not limited to) starch, sugars, diluents, granulating agents, lubricants, binders, and disintegrants.

[0461] In some embodiments, the pharmaceutical compositions provided herein are formulated as accumulation formulations. Some of these accumulation formulations typically have a longer duration of action than non-accumulation formulations. In some embodiments, such formulations are administered via implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. In some embodiments, accumulation formulations are prepared using suitable polymeric or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or in the form of slightly soluble derivatives (e.g., in the form of slightly soluble salts).

[0462] In some embodiments, the pharmaceutical compositions provided herein comprise a delivery system. Examples of delivery systems include (but are not limited to) liposomes and emulsions. Certain delivery systems can be used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0463] In some embodiments, the pharmaceutical compositions provided herein comprise one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents provided herein to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0464] In some embodiments, the pharmaceutical compositions provided herein comprise a sustained-release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of a solid hydrophobic polymer. In some embodiments, the sustained-release system, depending on its chemical properties, can release the pharmaceutical agent over periods of hours, days, weeks, or months.

[0465] Some injectable pharmaceutical compositions are presented in unit dosage form (e.g., in ampoules or in multi-dose containers).

[0466] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a modified oligonucleotide. In some embodiments, the therapeutically effective amount is sufficient to prevent, alleviate, or improve disease symptoms or prolong the survival of the treated subject.

[0467] In some embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In some embodiments, upon administration in vivo, the prodrug is chemically converted into a more biologically, pharmaceutically, or therapeutically active form of the oligonucleotide. In some embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in some cases, the prodrug may have greater bioavailability than the corresponding active form (e.g., via oral administration). In some cases, the prodrug may have improved solubility compared to the corresponding active form. In some embodiments, the prodrug is less water-soluble than the corresponding active form. In some cases, such prodrugs have excellent transmembrane transport, where water solubility is detrimental to flowability. In some embodiments, the prodrug is an ester. In some such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In some cases, the compound containing the carboxylic acid is the corresponding active form. In some embodiments, the prodrug comprises a short peptide (multi-amino acid) bound to an acid group. In some such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0468] In some embodiments, prodrugs are produced by modifying the active pharmaceutical compound so that the active compound is regenerated upon administration in vivo. Prodrugs can be designed to alter the metabolic stability or transport properties of the drug, mask side effects or toxicity, improve the taste of the drug, or change other properties or characteristics of the drug. With knowledge of in vivo pharmacodynamic processes and drug metabolism, those skilled in the art can design prodrugs of the active pharmaceutical compound once they know of it (e.g., see Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392).

[0469] Other routes of administration include (but are not limited to) oral, rectal, mucosal, intestinal, enteric, topical, suppository, inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In some embodiments, intrathecal pharmaceutical agents are administered to achieve local rather than systemic exposure. For example, the pharmaceutical composition may be injected directly into the area of ​​desired effect (e.g., into the kidney).

[0470] Some medicine boxes

[0471] A pillbox is also provided. In some embodiments, the pillbox contains one or more compounds comprising the modified oligonucleotides disclosed herein. In some embodiments, the pillbox can be used to administer the compound to a subject.

[0472] In some embodiments, the kit contains a ready-to-use pharmaceutical composition. In some embodiments, the pharmaceutical composition is contained in a vial. Multiple vials (e.g., 10) may be contained in, for example, a dispensing pack. In some embodiments, the vials are manufactured to allow syringe insertion. The kit may also contain instructions for using the compound.

[0473] In some embodiments, the kit contains a pharmaceutical composition present in a pre-filled syringe (e.g., a single-dose syringe with, for example, a 27.5-gauge needle and a needle guard), rather than in a vial. Multiple pre-filled syringes (e.g., 10) may be present, for example, in a dispensing kit. The kit may also contain instructions for administering a compound comprising the modified oligonucleotides disclosed herein.

[0474] In some embodiments, the kit contains the modified oligonucleotides provided herein as a lyophilized pharmaceutical product, and a pharmaceutically acceptable diluent. The lyophilized pharmaceutical product is reconstituted in the pharmaceutically acceptable diluent before administration to a subject.

[0475] In some embodiments, in addition to compounds containing the modified oligonucleotides disclosed herein, the kit may also contain one or more of the following: a syringe, an alcohol wipe, a cotton ball, and / or a gauze pad.

[0476] Some experimental models

[0477] In some implementations, methods are provided for using and / or testing the modified oligonucleotides provided herein in experimental models. Those skilled in the art can select and modify protocols used in such experimental models to evaluate the pharmaceutical agents provided herein.

[0478] Typically, the modified oligonucleotides are first tested in cultured cells. Suitable cell types include those associated with the cell types for which the modified oligonucleotides are to be delivered in vivo. For example, suitable cell types for studying the methods described herein include primary cells or cultured cells.

[0479] In some embodiments, the extent to which modified oligonucleotides interfere with the activity of one or more miR-17 family members is assessed in cultured cells. In some embodiments, inhibition of microRNA activity can be assessed by measuring the levels of one or more predicted or validated microRNA-regulated transcripts. Inhibition of microRNA activity can lead to an increase in transcripts regulated by miR-17 family members and / or proteins encoded by transcripts regulated by miR-17 family members (i.e., derepressing transcripts regulated by miR-17 family members). Furthermore, in some embodiments, certain phenotypic outcomes can be measured.

[0480] Several animal models are available to those skilled in the art for studying one or more miR-17 family members in human disease models. Models of polycystic kidney disease include (but are not limited to) models with Pkd1 and / or Pkd2 mutations and / or deletions; and models containing other gene mutations. Non-limiting exemplary models of PKD containing Pkd1 and / or Pkd2 mutations and / or deletions include hypomorphic models, such as models containing Pkd1 missense mutations and models with reduced or unstable Pkd2 expression; inducible conditional knockout models; and conditional knockout models. Non-limiting exemplary PKD models containing mutations in genes other than Pkd1 and Pkd2 include models with Pkhd1, Nek8, Kif3a, and / or Nphp3 mutations. PKD models have been 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.

[0481] Some quantitative determinations

[0482] In some embodiments, microRNA levels in cells or tissues are quantified in vitro or in vivo. In some embodiments, changes in microRNA levels are measured by microarray analysis. In some embodiments, changes in microRNA levels are measured by one of several commercially available PCR assays, such as the TaqMan® MicroRNA Assay (Applied Biosystems).

[0483] Microarray profiling of mRNAs can be used to assess the regulation of microRNA activity by anti-miRs or microRNA mimics. Seed microRNA sequences are searched for in mRNA sequences regulated (increased or decreased) by anti-miRs or microRNA mimics to compare the regulation by mRNAs that are microRNA targets with that by mRNAs that are not. This allows for the evaluation of interactions between anti-miRs and their target microRNAs, or between microRNA mimics and their targets. In the case of anti-miRs, mRNAs with increased expression levels are screened for mRNA sequences containing seed-matching sequences of microRNAs complementary to anti-miRs.

[0484] The regulation of microRNA activity by antimiR compounds can be assessed by measuring the levels of microRNA messenger RNA target proteins, either by measuring the levels of the messenger RNA itself or the levels of proteins transcribed from it. Antisense inhibition of microRNA typically leads to an increase in the levels of microRNA messenger RNA and / or the proteins of its target proteins; that is, antimiR treatment de-represses one or more target messenger RNAs.

[0485] Example

[0486] The following embodiments are presented to more fully illustrate some embodiments of the invention. However, these embodiments should not in any way be construed as limiting the broad scope of the invention.

[0487] Those skilled in the art will readily grasp the fundamental principles of this discovery to design various compounds without departing from the spirit of the invention.

[0488] Example 1: The role of miR-17 in PKD

[0489] In a mouse model of PKD, miR-17 family members of the miR-17-92 microRNA cluster were upregulated. Genetic deletion of the miR-17-92 cluster in the PKD mouse model reduced renal cyst growth, improved renal function, and prolonged survival (Patel et al., PNAS, 2013; 110(26): 10765-10770). The miR-17-92 cluster contains six different microRNAs, each with a distinct sequence: miR-17, miR-18a, miR-19a, miR-19-b-1, and miR-92a-1.

[0490] The miR-17~92 cluster comprises two microRNAs, miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Each member of this family shares a seed sequence identity and exhibits 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.

[0491] Table 1: miR-17 family of microRNAs

[0492]

[0493] The anti-miR-17 compound RGLS4326 was discovered through screening chemically diverse and rationally designed anti-miR-17 oligonucleotide libraries to obtain optimal pharmaceutical properties. RGLS4326 preferentially distributes to renal and urinary duct-derived cysts, releasing miR-17 from translationally active polyribosomes and de-repressing multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, after subcutaneous administration, RGLS4326 attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models. A Phase 1 single-dose escalation (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a Phase 1 multiple-dose escalation (MAD) clinical trial in healthy volunteers in May 2018. A Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) was initiated in October 2020.

[0494] Following the initiation of the Phase 1 MAD clinical trial, non-clinical toxicology studies revealed central nervous system (CNS)-related findings at high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or exhaustion. To identify potential candidates for off-target pharmacology, a panel of 174 targets, including G protein-coupled receptors, transporters, ion channels, nuclear receptors, and cytokine receptors, were evaluated in vitro to understand their potential interactions with RGLS4326. RGLS4326 was found to be an antagonist of AMPA glutamate receptors, with a 50% inhibitory concentration (IC50) based on ligand binding of 4.6 μM (14.2 μg / mL) and a functional IC50 based on patch-clamp activity of 300–600 nM (0.9–1.8 μg / mL). AMPA receptors are ion channels at excitatory synapses in the CNS that mediate rapid excitatory neurotransmission and are therefore a key component of all neuronal networks. This interaction with AMPA receptors could explain the CNS-mediated findings observed in a non-clinical toxicology model at high doses of RGLS4326.

[0495] Example 2: Screening for anti-miR-17 compounds with reduced AMPA receptor binding

[0496] RGLS4326 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 SIn this model, the nucleoside followed by the subscript "M" is 2'-O-methyl, the nucleoside followed by the subscript "F" is 2'-fluoro, and the nucleoside followed by the subscript "S" is S-cEt. Each cytosine is unmethylated, and all bonds are phosphate thioester bonds. Chemical modifications and length variants of RGLS4326 were designed and screened to identify compounds that retain the potency and pharmacokinetic characteristics of RGLS4326 and exhibit reduced binding to the AMPA receptor (AMPA-R).

[0497] A library of compounds with different chemical modifications, nucleobase sequences, and lengths relative to RGLS4326 was designed.

[0498] Table 2: Anti-miR-17 Library

[0499]

[0500] The activity of the antimiR-17 compound was evaluated in a radioligand binding assay, wherein the assay measures the activity of the antimiR-17 compound in the presence of increasing concentrations. 3 [H] AMPA ligands bind to AMPA-R present on the synaptic membrane of the rat brain. AntimiR-17 compounds with affinity for AMPA-R will bind to [ 3 H] AMPA ligands bind to and compete with it for binding.

[0501] The assay was performed according to previously published methods (Honore et al., J Neurochem., 1982, 38(1):173-178; Olsen et al., Brain Res., 1987, 402(2):243-254). A 5.0 nM ligand [ 3 [H] AMPA, 1.0 mM of the nonspecific ligand L-glutamate, and μM concentrations of antimiR compounds were incubated for 90 min with synaptic membranes prepared from the cerebral cortex of Wistar rats. The compounds shown in Table 2 were tested in three experiments. AntimiR compounds targeting microRNAs other than miR-17 were used as control compounds (RG5124 targeting miR-33a; RG5365 targeting let-7a; RG8093 targeting miR-214). RGLS4326 and RG-NG-1001 were also tested in each experiment because they have been shown to bind to AMPA-R and inhibit its activity. [By radioligand binding to [] 3 The amounts of H] AMPA ligands were quantified and are shown in Tables 3, 4, and 5. As the data illustrate, the compounds exhibit varying abilities to inhibit the binding of radiolabeled ligands to AMPA-R.

[0502] Table 3: Inhibition of ligand binding to AMPA-R, Experiment 1

[0503]

[0504] Table 4: Inhibition of AMPA-R binding by ligands, Experiment 2

[0505]

[0506] Table 5: Inhibition of AMPA-R binding by ligands, Experiment 3

[0507]

[0508] To evaluate the functional antagonistic effect of antimiR-17 oligonucleotides on AMPA-R, certain oligonucleotides were tested using a manual whole-cell patch clamping technique that records membrane current as a measure of AMPA-R activity.

[0509] Manual whole-cell patch clamping studies were conducted by Metrion Biosciences (Cambridge, UK). Whole-cell voltage clamping experiments were performed at room temperature (18°C–21°C) using an EPC10 patch clamp amplifier and Patchmaster software (HEKA Elektronik). Glass patch pipettes were made of borosilicate glass capillaries (Harvard Apparatus) with resistances between 1.4 MΩ and 2.5 MΩ. Membrane currents were recorded using whole-cell patch clamping techniques. ChanTest® GluA1 / GluA4 EZ cells were clamped at a holding potential of -80 mV, and 10 μM (S)-AMPA was delivered using the VC38 perfusion system (ALA Scientific Instruments) to induce membrane currents. The minimum current amplitude was measured each time 10 μM (S)-AMPA was applied. The fractional change in current amplitude produced by each concentration of the compound was calculated relative to the control current (before the compound) and expressed as a percentage change (inhibition %) per cell. The compounds tested are shown in Table 6. RGLS4326 was tested in a separate study from all other compounds in Table 6.

[0510] As shown in Table 6, based on a manual whole-cell patch clamping study in human ChanTest® GluA1 / GluA4 EZ cells, compounds RG-NG-1015, RG-NG-1016, and RG-NG-1017 exhibited reduced functional antagonism against AMPA-R compared to RGLS4326.

[0511] Table 6: Functional antagonistic effects of AMPA-R in whole-cell patch clamping studies

[0512]

[0513] Example 3: The relationship between nucleobase properties and AMPA-R binding

[0514] As elucidated by AMPA-R binding and whole-cell patch-clamp studies, the presence of guanosine at the 3'-terminus of the anti-miR-17 oligonucleotide, complementary to the first nucleotide of miR-17, influences the functional antagonism of AMPA-R. Like guanosine, adenosine is a purine; however, adenosine does not inhibit AMPA-R. Guanosine and adenosine are similar in several properties except for hydrogen bonding; therefore, the differences in hydrogen bonding at positions 1, 2, and 6 of the purine bases were evaluated. The tested purine nucleobases are shown in... Figure 1 And in Table 7. In the “Purine Position” column of Table 7, “A” indicates the hydrogen acceptor position of purine, and “D” indicates the hydrogen donor position of purine. In the “Purine Position” column of Table 7, “N” indicates a neutral position that is neither a hydrogen acceptor nor a hydrogen donor. Different 2'-sugar moieties on the purine nucleotide bases were also tested to evaluate the effect of 2'-sugar moieties chemistry on the ability of purine nucleotide bases to inhibit AMPA-R.

[0515] Table 7: AntimiR-17 compounds with different nucleobase and sugar moieties

[0516]

[0517] In the radioligand binding assay described in this article, compounds were tested to determine the interaction between antimiR-17 compounds and […]. 3 [H] AMPA ligands bind and compete for their binding ability. As shown in Table 8, a correlation was observed between inhibition of ligand binding to AMPA-R and the presence of a hydrogen bond acceptor at the purine 6-position of the 3'-terminal nucleotide. For example, compounds with guanosine or inosine at the 3'-terminus inhibit ligand binding to AMPA-R. Compounds with hydrogen bond acceptors at the purine 6-position of the 3'-terminal nucleotide (e.g., RG-NG-1037 and RG-NG-1039) are less likely to inhibit ligand binding to AMPA-R.

[0518] Table 8: Inhibition of ligand binding to AMPA-R

[0519]

[0520] Example 4: In high-dose studies, antimiR-17 compounds with reduced binding and inhibition of AMPA-R did not show CNS toxicity.

[0521] RG-NG-1015, RG-NG-1016, and RG-NG-1017 were tested in high-dose mouse toxicity studies. Each compound was tested at a single dose of 2000 mg / kg and at escalating doses (100 mg / kg, 450 mg / kg, and 2000 mg / kg). As shown in Table 9, while escalating doses of RG-NG-1001 and RGLS4326 resulted in ataxia and somnolence, and the highest dose of RGLS4326 resulted in unconsciousness, no CNS toxicity was observed with RG-NG-1015, RG-NG-1016, or RG-NG-1017.

[0522] Table 9: Findings related to anti-miR-17 compounds and CNS

[0523]

[0524] Example 5: Maximum Tolerable Dose (MTD) Studies and Comparative Dose Assessments of Different Compounds

[0525] Data from the study below further support that AMPA-R antagonism is the cause of CNS toxicity and death observed in previous toxicity studies of RGLS4326.

[0526] Study 1: Maximum Tolerated Dose (MTD) Study and Comparative Dose Assessment of RG-NG-1017, RGLS4326, and RG-NG-1001

[0527] Compounds (RG-NG-1017, RGLS4326, and RG-NG-1001) were evaluated in a lead maximum tolerated dose (MTD) study (discussed below). RG-NG-1017, RGLS4326, and RG-NG-1001 were initially evaluated at four dose levels each. RG-NG-1017 was included as a non-AMPA-R-binding compound, compared to AMPA-R-binding compounds RGLS4326 and RG-NG-1001. Six- to seven-week-old male C57Bl / 6J mice (Jackson Laboratories) were used in this study. Mice were randomly assigned to treatment groups without the study being known. Animals were acclimatized for at least five days and housed in 12-hour light / dark cycles (lights turned on at 7:00 AM). No more than four mice were housed in each cage in a ventilated cage system. The diet consisted of free-feeding standard rodent feed and water.

[0528] MTD pilot study

[0529] The following parameters were used in this study:

[0530] 1. Route of administration: Intraventricular (ICV) administration of RG-NG-1017, RG-NG-1001, and RGLS4326.

[0531] 2. Dosage volume: 4 µL

[0532] 3. Formulation: Carrier, Ca-free 2+ and Mg 2+ dPBS

[0533] 4. Dosage frequency: once

[0534] 5. Study duration: 8 days

[0535] 6. Number of groups: 3

[0536] 7. Number of animals per group: (2-4 animals per group)

[0537] 8. Total number of animals: 54

[0538] For ICV administration, mice were anesthetized and positioned for injection. The skin above the skull was incised, and a small hole was drilled in the skull above the target using a micro-drill. Stereoscopic coordinates were determined as follows: anteroposterior to anterior fontanelle (AP) -0.4 mm; medial (ML) + / - 1.0–1.5 mm; dorsoventral (DV) -3.0 mm, for injection into the right and left lateral ventricles (Hironaka et al., 2015). 4 μl was injected unilaterally into the right lateral ventricle of the animal. The compound was injected over 1–2 min, and the needle was left in place for 0.5–1 min before withdrawal. The incision was closed with sutures, wound clips, or VetBond.

[0539] Following ICV treatment (day 0), the animals were monitored for 7 days, during which daily health checks, weight, and mortality were recorded. On day 7, the brain and kidneys were collected and fixed (in 10% formalin) and stored for histological examination.

[0540] The results of the MTD study are shown in Table 10 and Figure 3In this study, all animal deaths occurred within the first 5–8 hours following ICV injection. Mice injected with 2.5 µg of RG4326 reported some immediate signs of respiratory distress, even after being provided with a heating pad. RG-NG-1017 (a non-AMPA-R binding compound) was well tolerated at high doses, and this compound had no established MTD (0 deaths at 600 µg, 100 µg, or 50 µg; 1 death at 300 µg). RG4326 and RG-NG-1001 showed 100% mortality at high doses (e.g., 600 µg, 300 µg, 100 µg), and both AMPA-R binding compounds showed 100% mortality at 50 µg and 25 µg. The MTD of RG-NG-1001 was not obtained in this study and is expected to be below 2.5 µg. The expected MTD of ICV RG4326 is approximately 2.5 < 5.0 µg. According to reports, all animals fully recovered by the second day of observation.

[0541] Table 10: Summary Results of the 7-Day MTD Study

[0542]

[0543] Maximum tolerated dose (MTD) study of RGLS4326

[0544] A second MTD study of ICV RGL4326 was conducted to evaluate dose selection for compound evaluation in disease models (Table 11). Different mouse strains (Swiss:Rjorl male mice, 5 weeks old, from Janvier) were evaluated in this study. Mice were anesthetized with isoflurane (5% for induction and 2% for maintenance, at 100% O2) and administered 5 mg / kg sc carprofen (Rimadyl®). The mice were then placed in a stereotactic frame. A midline sagittal incision was made in the scalp, and a hole was drilled in the skull above the left lateral ventricle. A stainless steel cannula (0.51 mm outer diameter) was stereotactically inserted into the left lateral ventricle at the following coordinates: +0.5 posterior to the anterior fontanelle, L ± 0.7 mm, V = -2.7 mm. After a 2-minute delay to allow brain tissue to slide through the cannula, 4 µL of a solution containing 0.625 mg / mL RG4326 was slowly infused over 2 minutes. After infusion, leave the cannula in place for 5 minutes to prevent backflow of the solution along the cannula path. Administer 5 mg / kg sc carbofen (Rimadyl®) to mice 24 and 48 hours post-surgery. Monitor mice for 3–7 days post-surgery (starting 24 h after ICV administration) and record their weight daily to assess their health. For mice monitored for more than 7 days, record their weight on days 1 and 7 post-surgery to assess their health.

[0545] Table 11: MTD Study Design for RGLS4326

[0546]

[0547] In Study 1, six mice were injected with 4 µL of a 0.625 mg / mL solution (total 2.5 µg per ICV injection; Table 10). At the end of anesthesia, the mice remained in a lateral recumbent position. During the first few hours post-surgery, the mice were quiet, occasionally scratching. No toxic effects were observed in any of the six mice administered the solution at 24, 48, or 72 hours. In Study 2, four mice were injected with four different doses of RGLS4326 (0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, and 1.875 mg / mL, 4 µL volume). One mouse receiving the highest dose (1.875 mg / mL, or 7.5 µg / mouse) died approximately 24 hours after the ICV injection. All other mice remained in good health until the end of the pilot study (7 days post-administration).

[0548] The combined results of Studies 1 and 2 demonstrate that RG4326 was generally well tolerated in the tested subjects, but only at significantly lower doses than RG-NG-1017 (see [link to study 1]). Figure 3 ).

[0549] Table 12 summarizes the MTD data of RGLS4326 in the mouse models of Studies 1 and 2. Based on these results from Study 2, the MTD of RGLS4326 in the Swiss:Rjorl mouse strain is expected to be approximately 4 µg.

[0550] Table 12: 7-day survival data from MTD studies 1 and 2

[0551]

[0552] In summary, the evaluation of compounds RG-NG-1017, RGLS4326, and RG-NG-1001 across two MTD studies demonstrates a significant difference in tolerability between the non-AMPA-R-binding compound (RG-NG-1017) and the AMPA-R-binding compounds (RGLS4326, RG-NG-1001) (see [link to MTD study]). Figure 3 Although one death occurred at the 300 µg ICV dose, the MTD of RG-NG-1017 was not established because no deaths occurred at the higher test dose of 600 µg. Furthermore, no effect on mortality was observed at RG-NG-1017 doses of 100 µg and 50 µg. In contrast, the significant effect of the AMPA-R combination compounds RGLS4326 and RG-NG-1001 on mortality was evident, with no animal survival observed in the test dosing range of 25 µg to 600 µg. A trend toward improved survival was observed at lower doses of RGLS4326 (10 µg), with 50% survival in animals treated with 5 µg RGLS4326 and 100% survival at 2.5 µg. Similarly, in the case of RG-NG-1001 (which showed stronger AMPA-R binding compared to RGLS4326), 100% mortality was evident at a low dose of 5 µg, with a trend toward improved survival at 2.5 µg. The results from Study 1 regarding RGLS43426 were further confirmed in a second MTD study (Study 2) using different mouse strains. This study found that there may be modest differences in the tolerability of RGLS4326 between strains, with an effect on mouse survival observed only at the highest dose of 7.5 µg, compared to 5 µg in Study 1 using C57 / Bl / 6J. However, these results still support the view that the MTD of AMPA-R-bound RGLS4326 is between approximately 2.5 µg and 5–7.5 µg (depending on the strain), in contrast to the significantly higher MTD (at least >40-fold or higher) of non-AMPA-R-bound RG-NG-1017. Figure 3 ).

[0553] Example 6: In vitro and in vivo efficacy of anti-miR-17 compounds

[0554] The in vitro efficacy of certain compounds was evaluated using a miR-17 luciferase sensor assay employing a miR-17 luciferase reporter gene vector with two tandemly complementary miR-17 binding sites in the 3'-UTR of the luciferase gene. HeLa cells were co-transfected with the luciferase reporter gene vector and an exogenous miR-17 expression vector to repress luciferase signaling. HeLa cells were then individually treated with anti-miR-17 oligonucleotides at concentrations of 0.045 nM, 0.137 nM, 0.412 nM, 1.23 nM, 3.70 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM. Luciferase activity was measured at the end of the 18-24 hour transfection period. RG5124 was included as a control compound. As shown in Table 13, these compounds inhibited miR-17 function in vitro and derepressed miR-17 luciferase reporter gene activity, with EC... 50 The value is similar to that of RGLS4326.

[0555] Table 13: Inhibition of miR-17 in luciferase assay

[0556]

[0557] like Figures 4A-4B As shown in the figure, in the luciferase assay in HeLa cells, RG-NG-1015 inhibited miR-17 as well as miR-20a, miR-106a and miR-93 in vitro, with EC50% showing the highest inhibition rate. 50 The value is similar to that of RGLS4326.

[0558] RG-NG-1015 also derepressed luciferase sensors containing the full-length 3' untranslated regions (UTRs) of miR-17 direct target genes PKD1 and PKD2 in vitro, where EC 50 The value is similar to that of RGLS4326.

[0559] The activity of certain compounds was evaluated using mouse miR-17 pharmacodynamic imprinting (miR-17 PD-Sig), which consisted of the expression of 18 unique miR-17 target genes normalized to six reference housekeeping genes to provide an unbiased and comprehensive assessment of miR-17 activity. The mouse miR-17 PD-Sig score was the calculated mean of the individual log2 fold change (normalized to six housekeeping genes) of the 18 genes compared to a simulated transfection (Lee et al., Nat. Commun., 2019, 10, 4148).

[0560] As shown in Table 13, in normal and PKD kidney cell lines (mouse and human), the tested oligonucleotides inhibited miR-17 function in vitro and derepressed the expression of multiple direct miR-17 target genes (as measured by miR-17 PD-imprinting), among which EC 50 The values ​​are similar to those of RGLS4326. No PD-Sig (77.2, indicated by "*") of RGLS4326 was generated in mIMCD3 cells in this experiment; the values ​​in Table 14 are reported values ​​by Lee et al., Nat. Commun., 2019, 10, 4148. Blank cells in the tables indicate compounds that were not tested in the specific cell lines.

[0561] Table 14: miR-17 PD-Sig in normal and PKD cell lines

[0562]

[0563] In vivo potency was evaluated using the microRNA polyribosome translocation assay (miPSA). This assay was used to determine the extent to which compounds directly bind to miR-17 targets in the kidneys of normal and PKD mice. The principle underlying miPSA is that active miRNAs bind to their mRNA targets in translationally active high molecular weight (HMW) polyribosomes, while repressed miRNAs reside in low molecular weight (LMW) polyribosomes. Treatment with antimiRs causes microRNAs to translocate from HMW polyribosomes to LMW polyribosomes. Therefore, miPSA provides a direct measurement of the binding of complementary antimiRs to microRNA targets (Androsavich et al., NucleicAcids Research, 2015, 44: e13).

[0564] Wild-type mice were administered a single dose of 0.3 mg / kg, 3 mg / kg, or 30 mg / kg. Kidney tissue was collected seven days later and subjected to miPSA. The mean displacement score for each treatment is shown in Table 15 (PBS, n = 17; RGLS432630 mg / kg, n = 10; all other treatments, n = 4–5). In normal mouse kidneys, the tested oligonucleotides caused miR-17 to exit the translationally active polyribosome (as measured by miPSA).

[0565] Table 15: miPSA Displacement Score

[0566]

[0567] In addition, as shown in Table 16 and Figures 5A to 5DAs shown, RGLS4326 and RG-NG-1015 exhibited similar pharmacokinetic and target binding profiles (as measured by miPSA) after a single subcutaneous administration in C57BL6 mice.

[0568] Table 16: Overview of Pharmacokinetics and Target Binding

[0569]

[0570] Example 7: Efficacy of RG-NG-1015 in ADPKD experimental model

[0571] Evaluating the efficacy of RG-NG-1015 in the KspCre / Pkd1F / RC (Pkd1-F / RC) mouse model. Pkd1-F / RC is an orthologous ADPKD model containing a germline submorphic Pkd1 mutation (the mouse equivalent of human PKD1-R3277C (RC mutation)) in one allele and loxP sites flanking exons 2 and 4 of Pkd1 in the other allele. KspCre-mediated recombination was used to delete fluxed Pkd1 exons and generate compound mutant mice with a renal tubule-specific somatic null mutation in one allele and a germline submorphic mutation in the other allele. This is an invasive but long-lived ADPKD model (Hajarnis et al., Nat. Commun., 2017, 8, 14395).

[0572] At 8, 10, 12, and 15 days of age, sex-paired Pkd1-F / RC mice were administered subcutaneous injections of: RGLS4326 at a dose of 20 mg / kg (n = 8; 4 males and 4 females per treatment group), RG5124 at a dose of 20 mg / kg (n = 8), RG-NG-1015 at a dose of 20 mg / kg (n = 8), or PBS (n = 8). Mice were sacrificed at 18 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; therefore, a decrease in BUN level is an indicator of reduced renal injury and functional improvement. Statistical significance was calculated using one-way ANOVA and Dunnett's multiple correction.

[0573] The results are shown in Table 17 and Figures 2A-2C(**** = p<0.0001; *** = p<0.001; ** = p<0.01; ns = not significant). The efficacy of RG-NG-1015 was similar to that of RGLS4326. The mean kidney weight to body weight ratio (KW / BW ratio) of Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015 was significantly lower than that of Pkd1-F / RC mice treated with PBS. Figure 2A Compared with mice treated with PBS, Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively, showed significantly lower mean BUN levels. Figure 2B Compared to mice treated with PBS, Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively, showed a decrease in mean serum creatinine levels; however, this decrease was not statistically significant. Figure 2C Treatment with the control oligonucleotide RG5124 did not reduce the kidney weight / body weight ratio, serum creatinine, or serum BUN, demonstrating that the results observed with RGLS4329 and RG-NG-1015 are specific for miR-17 inhibition.

[0574] Table 17: Efficacy of RG-NG-1015 in ADPKD mouse model

[0575]

[0576] The efficacy of RG-NG-1015, alone and in combination with tolvaptan, was also evaluated in the Pcy / DBA mouse model of PKD. Pcy / DBA mice exhibit slowly progressive PKD caused by missense mutations in the Nphp3 gene, leading to renal wasting disease in human adolescents (Takahashi et al., J Am Soc Nephrol 1991, 1:980-989; Olbrich et al., NatGenet 2003, 34:455-459). In Pcy mice, cysts originate from the distal tubules, and by 30 weeks of age, the entire nephron segment is extensively occupied by cysts, which accompany disease progression and typically result in ESRD (Nagao et al., Exp Anim 2012, 61:477-488). In particular, male Pcy / DBA mice have been used to characterize the pharmacological properties of many investigational products for the treatment of ADPKD, including tolvaptan and RGLS4326 (first-generation anti-miR-17) (Aihara et al., J Pharmacol ExpTher 2014 May;349(2):258-67 and Lee et al., Nat. Commun., 2019, 10, 4148). Studies in these mice typically involve initiating treatment at approximately 5 weeks of age and continuing until 15–30 weeks of age.

[0577] like Figure 6A and Figure 6B As outlined in the study, five groups of male Pcy / DBA mice (n=13 / treatment group) were treated subcutaneously with PBS or RG-NG-1015 at doses of 25 mg / kg, 5 mg / kg, 1 mg / kg, or 0.2 mg / kg every two weeks (Q2W). Two groups of male Pcy / DBA mice (n=13 / group) were also treated subcutaneously with RG-NG-1015 at doses of 50 mg / kg every four weeks (Q4W) or 12.5 mg / kg weekly (QW). Four additional groups of male Pcy / DBA mice (n=13 / group) were treated subcutaneously with PBS or a combination of RG-NG-1015 at doses of 25 mg / kg, 5 mg / kg, or 1 mg / kg and tolvaptan in a randomly consumed diet at doses of 0.3% (w / w) every two weeks. One group of male WT-BDA / 2J mice receiving subcutaneous injections of PBS at doses of 2 weeks was included in the study as a normal range reference. Mice were randomized to treatment groups at 5 weeks of age, and treatment began at 6 weeks of age and lasted for 17 weeks. Mice were sacrificed 7 days after the last treatment. Kidney weight, body weight, renal cyst index, and urinary Ngal / creatinine ratio (Ngal / Cr) were measured. Urinary Ngal / Cr is a biomarker of kidney damage.

[0578] like Figures 6C to 6EAs shown in Tables 18 to 20, RG-NG-1015 was effective against PKD in the Pcy / DBA mouse model at various doses and regimens, and also provided an additive or synergistic effect when used in combination with tolvaptan. In particular, RG-NG-1015 treatment significantly reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index in Pcy / DBA mice in a dose-dependent manner (Tables 18 and 20). Figures 6C to 6E Additionally, in Pcy / DBA mice, RG-NG-1015 treatment with similar total doses (212.5–250 mg per mouse over the duration of the study) but with different dosing regimens (including QW, Q2W, and Q4W) reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index to similar levels (Table 19). Figures 6C to 6E In Pcy / DBA mice, treatment with tolvaptan alone reduced mean KW / BW, urinary Ngal / Cr and renal cyst index, and the combination of RG-NG-1015 and tolvaptan further reduced mean KW / BW, urinary Ngal / Cr and renal cyst index (Table 20). Figures 6C to 6E As indicated by Bliss additive analysis, the observed drug combinations had synergistic, primarily additive, and less than additive effects on KW / BW, urinary Ngal / Cr, and renal cyst index, respectively (Table 20).

[0579]

[0580]

[0581] Example 8: Metabolites of RG-NG-1015

[0582] In vitro and in vivo studies were conducted to investigate the metabolism of RG-NG-1015. For both in vitro and in vivo samples, tissue samples were homogenized in a thawing buffer on ice, and RG-NG-1015 and / or metabolites were isolated from plasma, tissue homogenates, or urine via liquid-liquid extraction and solid-phase extraction steps. Calibration samples containing known amounts of RG-NG-1015 were extracted in parallel with test tissue homogenates, plasma, or urine samples. The molecular weights (MW) of RG-NG-1015 and potential metabolites were calculated from MS signals and compared with theoretical values.

[0583] The in vitro metabolic stability of RG-NG-1015 was assessed in mouse, monkey, and human tissues (i.e., kidney and liver lysates) and serum. RG-NG-1015 was incubated at 37 °C for 24 h in these matrices at a concentration of 5 µM with kidney and liver homogenates (corresponding to 307 µg / g tissue) or serum samples (corresponding to 15.3 µg / mL). RG-NG-1015 and metabolites were then extracted and analyzed by HPLC-TOF.

[0584] In vivo metabolism of RG-NG-1015 was evaluated in the liver and kidneys following a single administration to CD-1 mice, and in plasma, tissues, and urine following single and / or repeated administrations to monkeys. CD-1 mice received a single SC dose of 2000 mg / kg of RG-NG-1015, and monkeys received up to five weekly SC doses of 15 mg / kg, 75 mg / kg, or 150 mg / kg of RG-NG-1015. RG-NG-1015 and its metabolites were then extracted and analyzed by HPLC-TOF.

[0585] RG-NG-1015 undergoes sequential hydrolysis from both the 3' and 5' ends to produce chain-shortened metabolites (see Table 21). Nine potential metabolites were identified, as described in Table 21 below: 5' N-1, 5' N-2, 5' N-3, 5' N-4, 3' N-1, 3' N-2, 3' N-3, 3' N-4, and 3' N-5. All metabolites differ from RG-NG-1015 in that they involve the sequential removal of terminal nucleotides and termination at hydroxyl groups at both the 3' and 5' ends. No 5' short polymers (from N-5 to N-8) or 3' short polymers (from N-6 to N-8) were observed.

[0586] Table 21: Sequences, exact mass, m / z, and charge state of RG-NG-1015 and its potential metabolites

[0587]

[0588] This disclosure relates to the following implementation plan.

[0589] 1. A compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0590] (N'') p -(N) r -(N') q

[0591] Each N'' is independently a modified or unmodified nucleoside;

[0592] p is between 0 and 14; where if p is not 0, then (N'') p The nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0593] (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3';

[0594] N' is a nucleoside containing a modified sugar moiety;

[0595] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; and

[0596] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof.

[0597] 2. The compound as described in embodiment 1, wherein (N) r The structure is as follows:

[0598] A S G S C M A F C F U F U M U S

[0599] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside;

[0600] The nucleoside followed by the subscript "F" is a 2'-fluoronucleoside; and

[0601] Nucleosides followed by the subscript "S" are S-cEt nucleosides.

[0602] 3. The compound as described in embodiment 1 or 2, wherein at least one nucleoside link is a thiophosphate nucleoside link.

[0603] 4. The compound as described in any one of embodiments 1 to 3, wherein the internucleotide linkage is a thiophosphate internucleotide linkage.

[0604] 5. The compound as described in any one of embodiments 1 to 4, wherein q is 1.

[0605] 6. The compound as described in any one of embodiments 1 to 4, wherein q is 0.

[0606] 7. The compound as described in any one of embodiments 1 to 6, wherein p is 0.

[0607] 8. The compound as described in any one of embodiments 1 to 6, wherein p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0608] 9. The compound as described in embodiment 8, wherein (N'') p The nucleobase sequence of the nucleotide has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0609] 10. The compound as described in embodiment 8, wherein (N'') p The nucleobase sequence of the sample does not mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0610] 11. The compound as described in any one of embodiments 8, 9, or 10, wherein (N'') p The nucleobase sequences are selected from CUACCUGCACUGUA (SEQ ID NO: 7), CUACCUGCACUGU (SEQ ID NO: 8), CUACCUGCACUG (SEQ ID NO: 9), CUACCUGCACU (SEQ ID NO: 10), CUACCUGCAC (SEQ ID NO: 11), CUACCUGCA, CUACCUGC, CUACCUG, CUACCU, CUACC, CUAC, CUA, CU, and C.

[0611] 12. The compound as described in any one of embodiments 1 to 5 or 7 to 11, wherein the nucleobase of N' is a purine nucleobase that does not have a hydrogen bond acceptor at position 6.

[0612] 13. The compound as described in embodiment 12, wherein the nucleobase of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0613] 14. The compound of any one of embodiments 1 to 13, wherein the sugar moiety of N' is not a 2'-O-methyl sugar.

[0614] 15. The compound of any one of embodiments 1 to 14, wherein the sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar.

[0615] 16. The compound of embodiment 2, wherein the modified oligonucleotide has a 5'-A structure. S G S CM A F C F U F U M U S A S -3', where each cytosine is a nonmethylated cytosine.

[0616] 17. The compound of embodiment 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S U S -3', where each cytosine is a nonmethylated cytosine.

[0617] 18. The compound of embodiment 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S C S -3', where each cytosine is a nonmethylated cytosine.

[0618] 19. The compound of embodiment 2, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S -3', where each cytosine is a nonmethylated cytosine.

[0619] 20. The compound of any one of embodiments 1 to 19, wherein the compound comprises the modified oligonucleotide.

[0620] 21. The compound of any one of embodiments 1 to 20, wherein the pharmaceutically acceptable salt is a sodium salt.

[0621] 22. A modified oligonucleotide having the following structure:

[0622]

[0623] Wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6; or a pharmaceutically acceptable salt thereof.

[0624] 23. The modified oligonucleotide as described in embodiment 22, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine and isoguanosine.

[0625] 24. The modified oligonucleotide as described in embodiment 22 or 23, wherein the pharmaceutically acceptable salt is a sodium salt.

[0626] 25. A modified oligonucleotide having the following structure:

[0627]

[0628] Where B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have a hydrogen bond acceptor at position 6.

[0629] 26. The modified oligonucleotide as described in embodiment 25, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0630] 27. A modified oligonucleotide having the following structure:

[0631]

[0632] Or its pharmaceutically acceptable salt.

[0633] 28. The modified oligonucleotide as described in embodiment 27, wherein the pharmaceutically acceptable salt is a sodium salt.

[0634] 29. A modified oligonucleotide having the following structure:

[0635] .

[0636] 30. A pharmaceutical composition comprising a compound of any one of embodiments 1 to 21 or a modified oligonucleotide of any one of embodiments 22 to 29, and a pharmaceutically acceptable diluent.

[0637] 31. The pharmaceutical composition of embodiment 30, wherein the pharmaceutically acceptable diluent is an aqueous solution.

[0638] 32. The pharmaceutical composition as described in embodiment 31, wherein the aqueous solution is a saline solution.

[0639] 33. A pharmaceutical composition comprising a compound according to any one of embodiments 1 to 21 or a modified oligonucleotide according to any one of embodiments 22 to 29, wherein the composition is a lyophilized composition.

[0640] 34. A pharmaceutical composition comprising substantially a compound of any one of embodiments 1 to 21 or a modified oligonucleotide of any one of embodiments 22 to 29 in a saline solution.

[0641] 35. A method for inhibiting the activity of one or more members of the miR-17 family in cells, the method comprising contacting the cells with a compound of any one of embodiments 1 to 21 or a modified oligonucleotide of any one of embodiments 22 to 29.

[0642] 36. A method for inhibiting the activity of one or more members of the miR-17 family in a subject, the method comprising administering to the subject a compound of any one of embodiments 1 to 21, a modified oligonucleotide of any one of embodiments 22 to 29, or a pharmaceutical composition of any one of embodiments 30 to 34.

[0643] 37. The method as described in embodiment 36, wherein the subject suffers from a miR-17-related disease.

[0644] 38. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0645] (N'') p -(N) r -(N') q

[0646] Each N'' is independently a modified or unmodified nucleoside;

[0647] p is between 0 and 14; where if p is not 0, then (N'') p The nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0648] (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3';

[0649] N' is a nucleoside containing a modified sugar moiety;

[0650] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have an H bond acceptor at position 6; and

[0651] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof.

[0652] 39. The method as described in implementation scheme 38, wherein (N) r The structure is as follows:

[0653] A S G S C M A F C F U F U M U S

[0654] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside;

[0655] The nucleoside followed by the subscript "F" is 2'-fluoronucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside.

[0656] 40. The method as described in embodiment 38 or 39, wherein at least one nucleoside link is a phosphate thioside link.

[0657] 41. The method as described in any one of embodiments 38 to 40, wherein the internucleotide link is a phosphate thioside link.

[0658] 42. The method as described in any one of embodiments 38 to 41, wherein q is 1.

[0659] 43. The method as described in any one of embodiments 38 to 41, wherein q is 0.

[0660] 44. The method as described in any one of embodiments 38 to 43, wherein p is 0.

[0661] 45. The method as described in any one of embodiments 38 to 43, wherein p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0662] 46. ​​The method as described in implementation scheme 45, wherein (N'') p The nucleobase sequence of the nucleotide has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0663] 47. The compound as described in embodiment 45, wherein (N'')p The nucleobase sequence of the sample does not mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1).

[0664] 48. The compound as described in embodiment 47, wherein (N'') p The nucleobase sequences are selected from CUACCUGCACUGUA (SEQ ID NO: 7), CUACCUGCACUGU (SEQ ID NO: 8), CUACCUGCACUG (SEQ ID NO: 9), CUACCUGCACU (SEQ ID NO: 10), CUACCUGCAC (SEQ ID NO: 11), CUACCUGCA, CUACCUGC, CUACCUG, CUACCU, CUACC, CUAC, CUA, CU, and C.

[0665] 49. The method as described in any one of embodiments 38 to 42 or 44 to 48, wherein the nucleobase of N' is a purine nucleobase that does not have a hydrogen bond acceptor at position 6.

[0666] 50. The method as described in embodiment 49, wherein the nucleobase of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0667] 51. The method of any one of embodiments 38 to 50, wherein the sugar portion of N' is not a 2'-O-methyl sugar.

[0668] 52. The compound of any one of embodiments 38 to 51, wherein the sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar.

[0669] 53. The method of embodiment 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3', and each cytosine is a nonmethylated cytosine.

[0670] 54. The method of embodiment 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M US U S -3', where each cytosine is a nonmethylated cytosine.

[0671] 55. The method of embodiment 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S C S -3', where each cytosine is a nonmethylated cytosine.

[0672] 56. The method of embodiment 39, wherein the modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S -3', where each cytosine is a nonmethylated cytosine.

[0673] 57. The method of any one of embodiments 38 to 56, wherein the compound comprises the modified oligonucleotide.

[0674] 58. The method of any one of embodiments 38 to 57, wherein the pharmaceutically acceptable salt is a sodium salt.

[0675] 59. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a modified oligonucleotide having the following structure:

[0676]

[0677] Or its pharmaceutically acceptable salt.

[0678] 60. The method as described in embodiment 59, wherein the pharmaceutically acceptable salt is a sodium salt.

[0679] 61. The method of embodiment 60, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0680] 62. The method of embodiment 61, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0681] 63. The method of embodiment 62, wherein the sterile aqueous solution is a saline solution.

[0682] 64. A method for treating polycystic kidney disease, the method comprising administering to a subject in need a modified oligonucleotide having the following structure:

[0683] .

[0684] 65. The method of embodiment 64, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0685] 66. The method of embodiment 65, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0686] 67. The method of embodiment 66, wherein the sterile aqueous solution is a saline solution.

[0687] 68. The method of any one of embodiments 38 to 67, wherein the subject suffers from polycystic kidney disease.

[0688] 69. The method of any one of embodiments 38 to 67, wherein the subject is suspected of having polycystic kidney disease.

[0689] 70. The method of any one of embodiments 38 to 68, wherein the subject has been diagnosed with polycystic kidney disease using clinical, histopathological and / or genetic criteria.

[0690] 71. The method of any one of embodiments 38 to 70, wherein prior to administration of the compound, the modified oligonucleotide, or the pharmaceutical composition, the subject is determined to have reduced polycystin-1 (PC1) and / or polycystin-2 (PC2) levels in the subject's kidneys, urine, or blood.

[0691] 72. The method of any one of embodiments 38 to 71, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease.

[0692] 73. The method of any one of embodiments 38 to 71, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease.

[0693] 74. The method of any one of embodiments 38 to 73, wherein the subject has a mutation selected from PKD1 gene mutation or PKD2 gene mutation.

[0694] 75. The method as described in any one of embodiments 3838 to 74, wherein the subject has an increased total kidney volume.

[0695] 76. The method as described in any one of embodiments 38 to 75, wherein the subject suffers from hypertension.

[0696] 77. The method of any one of embodiments 38 to 76, wherein the subject has impaired renal function.

[0697] 78. The method of any one of embodiments 38 to 77, wherein the administration reduces the total kidney volume of the subject.

[0698] 79. The method of any one of embodiments 38 to 78, wherein the administration slows the rate of increase in total kidney volume in the subject.

[0699] 80. The method as described in embodiment 78 or 79, wherein the total kidney volume is a height-adjusted total kidney volume.

[0700] 81. The method of any one of embodiments 38 to 80, wherein the administration slows the rate of decline of the glomerular filtration rate in the subject.

[0701] 82. The method of any one of embodiments 38 to 81, wherein the administration increases the glomerular filtration rate in the subject.

[0702] 83. The method as described in embodiment 81 or 82, wherein the glomerular filtration rate is an estimated glomerular filtration rate.

[0703] 84. The method of any one of embodiments 38 to 83, wherein the administration slows the increase of cyst growth in the kidneys and / or liver of the subject.

[0704] 85. The method as described in any one of embodiments 38 to 84, wherein the application:

[0705] a) Improved renal function in the subjects;

[0706] b) Delay the deterioration of the subject's renal function;

[0707] c) Reduce kidney pain in the subjects;

[0708] d) Reduced the increase in kidney pain in the subjects;

[0709] e) Delay the onset of kidney pain in the subjects;

[0710] f) Lower the high blood pressure of the subjects;

[0711] g) To slow the worsening of hypertension in the subjects;

[0712] h) Delay the onset of hypertension in the subjects;

[0713] i) Reduce fibrosis in the kidneys of the subjects;

[0714] j) Slowing down the progression of fibrosis in the kidneys of the subjects;

[0715] k) Delay the onset of end-stage renal disease in the subjects;

[0716] l) Delay the dialysis time of the subjects;

[0717] m) Delaying the time for the subject to undergo kidney transplantation; and / or

[0718] n) Improve the life expectancy of the subjects.

[0719] 86. The method as described in any one of embodiments 38 to 85, wherein the application:

[0720] a) Reduce the albuminuria of the subjects;

[0721] b) Slowing the progression of albuminuria in the subjects;

[0722] c) Delay the onset of albuminuria in the subjects;

[0723] d) Reduce the hematuria of the subjects;

[0724] e) To slow the worsening of hematuria in the subjects;

[0725] f) Delay the onset of hematuria in the subject;

[0726] g) Reduce the blood urea nitrogen level of the subject;

[0727] h) Reduce the serum creatinine level of the subjects;

[0728] i) Improve the creatinine clearance rate of the subjects;

[0729] j) Reduce the albumin:creatinine ratio of the subjects;

[0730] k) Increase polycystic protein-1 (PC1) in the urine of the subjects;

[0731] l) Increase polycystic protein-2 (PC2) in the urine of the subjects;

[0732] m) Reduce neutrophil gelatinase-associated lipocalin (NGAL) in the urine of the subjects; and / or

[0733] n) Reduce the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

[0734] 87. The method as described in any one of embodiments 38 to 86, the method comprising:

[0735] a) Measure the total kidney volume of the subject;

[0736] b) Measure the subject's blood pressure;

[0737] c) Measure the kidney pain of the subject;

[0738] d) Measure polycystic protein-1 (PC1) in the urine of the subjects;

[0739] e) Measure polycystic protein-2 (PC2) in the urine of the subjects;

[0740] f) Measure the fibrosis of the subject's kidneys;

[0741] g) Measure the blood urea nitrogen level of the subject;

[0742] h) Measure the serum creatinine level of the subjects;

[0743] i) Measure the creatinine clearance rate of the subjects;

[0744] j) Measure the albuminuria of the subjects;

[0745] k) Measure the albumin:creatinine ratio of the subject;

[0746] l) Measure the glomerular filtration rate of the subject;

[0747] m) Measure neutrophil gelatinase-associated lipocalin (NGAL) in the urine of the subjects; and / or

[0748] n) Measure the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

[0749] 88. The method of any one of embodiments 38 to 87, wherein the method includes administering at least one additional therapy, wherein the at least one additional therapy is an antihypertensive agent.

[0750] 89. The method of any one of embodiments 38 to 87, wherein the method comprises administering at least one additional therapy selected from: angiotensin II converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), diuretics, calcium channel blockers, kinase inhibitors, adrenergic receptor antagonists, vasodilators, benzodiazepines, renin inhibitors, aldosterone receptor antagonists, endothelin receptor blockers, mammalian target of rapamycin (mTOR) inhibitors, hormone analogs, vasopressin receptor 2 antagonists, aldosterone receptor antagonists, glucose-ceramide synthase inhibitors, antihyperglycemic agents, dialysis, and kidney transplantation.

[0751] 90. The method of embodiment 89, wherein the angiotensin II converting enzyme (ACE) inhibitor is selected from captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, and ramipril.

[0752] 91. The method of embodiment 89, wherein the angiotensin II receptor blocker (ARB) is selected from candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, and eprosartan.

[0753] 92. The method of embodiment 89, wherein the vasopressin receptor 2 antagonist is tolvaptan.

[0754] 93. The method of embodiment 89, wherein the aldosterone receptor antagonist is spironolactone.

[0755] 94. The method of embodiment 89, wherein the kinase inhibitor is selected from besutinib and KD019.

[0756] 95. The method of embodiment 89, wherein the mTOR inhibitor is selected from everolimus, rapamycin, and sirolimus.

[0757] 96. The method of embodiment 89, wherein the hormone analogue is selected from somatostatin and adrenocorticotropic hormone.

[0758] 97. The method of embodiment 89, wherein the glucose-ceramide synthase inhibitor is venulstat.

[0759] 98. The method of embodiment 89, wherein the antihyperglycemic agent is metformin.

[0760] 99. The method as described in any one of embodiments 38 to 96, wherein the method comprises administering a therapeutically effective amount of the compound.

[0761] 100. The method as described in any one of embodiments 38 to 99, wherein the subject is a human subject.

[0762] 101. A compound comprising a modified oligonucleotide, wherein the modified oligonucleotide has the following structure in the 5' to 3' direction:

[0763] (N'') p -(N) r -(N') q

[0764] Each N'' is independently a modified or unmodified nucleoside;

[0765] p is between 0 and 14; where if p is not 0, then (N'') p The nucleobase sequence of [the nucleobase sequence] is complementary to the same length portion of the nucleobase sequence of miR-17.

[0766] (N) r Each N is independently a modified or unmodified nucleotide, and (N) r The nucleobase sequence is 5'-AGCACUUU-3';

[0767] N' is a nucleoside containing a modified sugar moiety;

[0768] q is 0 or 1; where if q is 1, then the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, provided that the purine nucleobase does not have an H bond acceptor at position 6; and

[0769] Each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine; or pharmaceutically acceptable salts thereof, which are used in therapy.

[0770] 102. The compound as described in embodiment 101, wherein the therapy is a treatment for polycystic kidney disease.

[0771] 103. The compound of embodiment 102, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

[0772] 104. The compound of embodiment 102, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD).

[0773] 105. The compound of any one of embodiments 1 to 22, the modified oligonucleotide of any one of embodiments 23 to 29, or the pharmaceutical composition of any one of embodiments 30 to 33, for use in a therapy.

Claims

1. A modified oligonucleotide or a pharmaceutically acceptable salt thereof, said modified oligonucleotide having the following structure: 。 2. The modified oligonucleotide according to claim 1, wherein the pharmaceutically acceptable salt is a sodium salt.

3. A modified oligonucleotide having the following structure: 。 4. A pharmaceutical composition comprising the modified oligonucleotide according to claim 1 and a pharmaceutically acceptable diluent.

5. The pharmaceutical composition according to claim 4, wherein the pharmaceutically acceptable diluent is an aqueous solution.

6. The pharmaceutical composition according to claim 5, wherein the aqueous solution is a saline solution.

7. A pharmaceutical composition comprising the modified oligonucleotide according to claim 2 and a pharmaceutically acceptable diluent.

8. The pharmaceutical composition according to claim 7, wherein the pharmaceutically acceptable diluent is an aqueous solution.

9. The pharmaceutical composition according to claim 8, wherein the aqueous solution is a saline solution.

10. A pharmaceutical composition comprising the modified oligonucleotide according to claim 3 and a pharmaceutically acceptable diluent.

11. The pharmaceutical composition of claim 10, wherein the pharmaceutically acceptable diluent is an aqueous solution.

12. The pharmaceutical composition according to claim 11, wherein the aqueous solution is a saline solution.

13. A pharmaceutical composition comprising the modified oligonucleotide according to claim 1, wherein the pharmaceutical composition is a lyophilized composition.

14. A pharmaceutical composition comprising the modified oligonucleotide according to claim 2, wherein the pharmaceutical composition is a lyophilized composition.

15. A pharmaceutical composition comprising the modified oligonucleotide according to claim 3, wherein the pharmaceutical composition is a lyophilized composition.

Images