Methods and compositions for the treatment of polycystic kidney disease

Abstract

Provided herein are methods for the treatment of polycystic kidney disease, including autosomal dominant polycystic kidney disease, using modified oligonucleotides that target miR-17.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 253,933, filed October 8, 2021, which is incorporated by reference herein in its entirety for all purposes.

[0002] Provided herein are compositions and methods for the treatment of polycystic kidney disease. [Background technology]

[0003] Polycystic kidney disease is characterized by the accumulation of numerous fluid-filled cysts in the kidney. These cysts are covered by a single layer of epithelial cells called the cystic epithelium. Over time, the cysts increase in size due to increased cell proliferation and active secretion of fluid by the cystic epithelium. The enlarged cysts compress the surrounding normal tissue, resulting in decreased 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 areas of fibrosis containing atrophic tubules. Polycystic kidney disease can also cause cysts to develop in the liver and elsewhere in the body.

[0004] Several genetic disorders can result in polycystic kidney disease (PKD). The various forms of PKD are distinguished by the mode of inheritance, e.g., autosomal dominant or autosomal recessive inheritance, organ involvement and extrarenal phenotypic presentation, age of onset of end-stage renal disease (e.g., at birth, in childhood, or in adulthood, etc.), and underlying genetic mutations associated with the disease. See Kurschat et al., 2014, Nature Reviews Nephrology, 10:687-699. Summary of the Invention

[0005] Embodiment 1. A modified oligonucleotide, comprising, in the 5' to 3' direction: (N”) p -(N) r -(N')q wherein each N" is independently a modified or unmodified nucleoside; p is 0 to 14, and if p is not 0, then (N") p is complementary to an equal length portion of the nucleobase sequence of miR-17, (N) r each N is independently a modified or unmodified nucleotide, (N r is 5'-AGCACUUU-3', N' is a nucleoside containing a modified sugar moiety; q is 0 or 1, and when q is 1, the nucleobase of N' is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that said purine nucleobase does not have a hydrogen bond acceptor at position 6; A compound comprising the modified oligonucleotide, or a pharma- ceutically acceptable salt thereof, wherein each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. Embodiment 2. (N) r The structure of A S G S C M A F C F U F U M U S wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside; A nucleoside followed by the subscript "F" is a 2'-fluoronucleoside; The compound of embodiment 1, wherein the nucleoside followed by the subscript "S" is an S-cEt nucleoside. Embodiment 3. The compound of embodiment 1 or 2, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 4. The compound of any one of embodiments 1-3, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 5. A compound according to any one of embodiments 1 to 4, wherein q is 1. Embodiment 6. A compound according to any one of embodiments 1 to 4, wherein q is 0. Embodiment 7. A compound according to any one of embodiments 1 to 6, wherein p is 0. Embodiment 8. The compound of any one of embodiments 1-6, wherein p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14. Embodiment 9. The (N") p The compound of embodiment 8, wherein the nucleobase sequence of has no more than one mismatch to the nucleobase sequence of miR-17 (SEQ ID NO: 1). Embodiment 10. The (N") p The compound of embodiment 8, wherein the nucleobase sequence of has no mismatches to the nucleobase sequence of miR-17 (SEQ ID NO: 1). Embodiment 11. The (N") p 11. The compound of any one of embodiments 8, 9, or 10, wherein 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. Embodiment 12. The compound of any one of embodiments 1-5 or 7-11, wherein said N' nucleobase is a purine nucleobase that does not have a hydrogen bond acceptor at the 6-position. Embodiment 13. The compound of embodiment 12, wherein the N' nucleobase is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine. Embodiment 14. The compound of any one of embodiments 1 to 13, wherein the sugar moiety of N' is not a 2'-O-methyl sugar. Embodiment 15. The compound of any one of embodiments 1-14, wherein said sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar. Embodiment 16. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S A S -3', and each cytosine is an unmethylated cytosine. Embodiment 17. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S U S -3', and each cytosine is an unmethylated cytosine. Embodiment 18. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S C S -3', and each cytosine is an unmethylated cytosine. Embodiment 19. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S -3', and each cytosine is an unmethylated cytosine. Embodiment 20. The compound of any one of embodiments 1 to 19, wherein the compound consists of the modified oligonucleotide. Embodiment 21. The compound of any one of embodiments 1 to 20, wherein the pharma- ceutically acceptable salt is a sodium salt. Embodiment 22. Structure: [ka] wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that said purine nucleobase does not have a hydrogen bond acceptor at position 6, or a pharma- ceutically acceptable salt thereof. Embodiment 23. The modified oligonucleotide of embodiment 22, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine. Embodiment 24. The modified oligonucleotide of embodiment 22 or 23, wherein the pharma- ceutically acceptable salt is a sodium salt. Embodiment 25. Structure: [ka] wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that said purine nucleobase does not have a hydrogen bond acceptor at the 6-position. Embodiment 26. The modified oligonucleotide of embodiment 25, wherein B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine. Embodiment 27. Structure: [ka] or a pharma- ceutically acceptable salt thereof. Embodiment 28. The modified oligonucleotide of embodiment 27, wherein the pharma- ceutically acceptable salt is a sodium salt. Embodiment 29. Structure: [ka] A modified oligonucleotide having the formula: Embodiment 30. 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, and a pharma- ceutically acceptable diluent. Embodiment 31. The pharmaceutical composition of embodiment 30, wherein the pharma- ceutically acceptable diluent is an aqueous solution. Embodiment 32. The pharmaceutical composition of embodiment 31, wherein the aqueous solution is saline. Embodiment 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, which is a lyophilized composition. Embodiment 34. A pharmaceutical composition consisting essentially of a compound according to any one of embodiments 1-21, or a modified oligonucleotide according to any one of embodiments 22-29, in saline. Embodiment 35. A method for inhibiting the activity of one or more members of the miR-17 family in a cell, comprising contacting the cell with a compound according to any one of embodiments 1 to 21 or a modified oligonucleotide according to any one of embodiments 22 to 29. Embodiment 36. A method for inhibiting the activity of one or more members of the miR-17 family in a subject, comprising administering to the subject a compound according to any one of embodiments 1 to 21, a modified oligonucleotide according to any one of embodiments 22 to 29, or a pharmaceutical composition according to any one of embodiments 30 to 34. Embodiment 37 The method of embodiment 36, wherein the subject has a disease associated with miR-17. Embodiment 38. A method of treating polycystic kidney disease, comprising administering to a subject in need thereof a modified oligonucleotide comprising, in a 5' to 3' direction: (N”) p -(N) r -(N') q wherein each N" is independently a modified or unmodified nucleoside; p is 0 to 14, and if p is not 0, then (N") p is complementary to an equal length portion of the nucleobase sequence of miR-17, (N) reach N is independently a modified or unmodified nucleotide, (N r is 5'-AGCACUUU-3', N' is a nucleoside containing a modified sugar moiety; q is 0 or 1, and when q is 1, the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that said purine nucleobase does not have an H-bond acceptor at position 6; administering a compound comprising the modified oligonucleotide, or a pharma- ceutically acceptable salt thereof, wherein each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. Embodiment 39.(N) r The structure of A S G S C M A F C F U F U M U S wherein the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside; The method of embodiment 38, wherein a nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside and a nucleoside followed by the subscript "S" is an S-cEt nucleoside. Embodiment 40 The method of embodiment 38 or 39, wherein at least one internucleoside linkage is a phosphorothioate internucleoside linkage. Embodiment 41 The method of any one of embodiments 38 to 40, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage. The method of any one of embodiments 38 to 41, wherein embodiment 42.q is 1. The method of any one of embodiments 38 to 41, wherein embodiment 43.q is 0. Embodiment 44. The method of any one of embodiments 38 to 43, wherein p is 0. The method of 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. Embodiment 46. The (N") p 46. ​​The method of embodiment 45, wherein the nucleobase sequence of has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1). Embodiment 47. The (N") p The compound of embodiment 45, wherein the nucleobase sequence of has no mismatches to the nucleobase sequence of miR-17 (SEQ ID NO: 1). Embodiment 48. The (N") p 48. The compound of embodiment 47, wherein 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. Embodiment 49. The method of any one of embodiments 38 to 42 or 44 to 48, wherein the nucleobase at N' is a purine nucleobase that does not have a hydrogen bond acceptor at the 6-position. Embodiment 50. The method of embodiment 49, wherein the N' nucleobase is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine. Embodiment 51. The method of any one of embodiments 38 to 50, wherein the sugar moiety of N' is not a 2'-O-methyl sugar. Embodiment 52. The compound of any one of embodiments 38-51, wherein said sugar moiety of N' is a 2'-O-methoxyethyl sugar or an S-cEt sugar. Embodiment 53. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S A S40. The method of embodiment 39, wherein each cytosine is -3' and an unmethylated cytosine. Embodiment 54. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S U S 40. The method of embodiment 39, wherein each cytosine is -3' and an unmethylated cytosine. Embodiment 55. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S C S 40. The method of embodiment 39, wherein each cytosine is -3' and an unmethylated cytosine. Embodiment 56. The structure of the modified oligonucleotide is: 5'-A S G S C M A F C F U F U M U S 40. The method of embodiment 39, wherein each cytosine is -3' and an unmethylated cytosine. Embodiment 57. The method of any one of embodiments 38 to 56, wherein the compound consists of the modified oligonucleotide. Embodiment 58. The method of any one of embodiments 38 to 57, wherein the pharma- ceutically acceptable salt is a sodium salt. Embodiment 59. A method of treating polycystic kidney disease, comprising administering to a subject in need thereof a compound of the structure: [ka] or a pharma- ceutically acceptable salt thereof. Embodiment 60. The method of embodiment 59, wherein the pharma- ceutically acceptable salt is a sodium salt. Embodiment 61 The method of embodiment 60, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharma- ceutically acceptable diluent. Embodiment 62. The method of embodiment 61, wherein the pharma- ceutically acceptable diluent is a sterile aqueous solution. Embodiment 63. The method of embodiment 62, wherein the sterile aqueous solution is saline. Embodiment 64. A method of treating polycystic kidney disease, comprising administering to a subject in need thereof a compound of the structure: [ka] The method comprises administering a modified oligonucleotide having the formula: Embodiment 65. The method of embodiment 64, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharma- ceutically acceptable diluent. Embodiment 66. The method of embodiment 65, wherein the pharma- ceutically acceptable diluent is a sterile aqueous solution. Embodiment 67. The method of embodiment 66, wherein the sterile aqueous solution is saline. Embodiment 68. The method of any one of embodiments 38 to 67, wherein the subject has polycystic kidney disease. Embodiment 69. The method of any one of embodiments 38-67, wherein the subject is suspected of having polycystic kidney disease. Embodiment 70. The method of any one of embodiments 38-68, wherein the subject has been diagnosed with polycystic kidney disease using clinical, histopathological, and / or genetic criteria. Embodiment 71. The method of any one of embodiments 38 to 70, wherein the subject is determined to have reduced levels of polycystin-1 (PC1) and / or polycystin-2 (PC2) in the kidneys, urine, or blood of the subject prior to administration of the compound, the modified oligonucleotide, or the pharmaceutical composition. Embodiment 72. The method of any one of embodiments 38-71, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease. Embodiment 73. The method of any one of embodiments 38-71, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease. Embodiment 74. The method of any one of embodiments 38 to 73, wherein the subject has a mutation selected from a mutation in the PKD1 gene or a mutation in the PKD2 gene. Embodiment 75. The method of any one of embodiments 38 to 74, wherein the subject has an increased total kidney volume. Embodiment 76. The method of any one of embodiments 38 to 75, wherein the subject has hypertension. Embodiment 77. The method of any one of embodiments 38 to 76, wherein the subject has impaired renal function. Embodiment 78 The method of any one of embodiments 38-77, wherein said administering reduces total kidney volume in said subject. Embodiment 79. The method of any one of embodiments 38-78, wherein said administering slows the rate of increase in total kidney volume in said subject. Embodiment 80. The method of embodiment 78 or 79, wherein the total kidney volume is height-adjusted total kidney volume. Embodiment 81 The method of any one of embodiments 38-80, wherein said administering slows the rate of decline in glomerular filtration rate in said subject. Embodiment 82 The method of any one of embodiments 38-81, wherein said administering increases the glomerular filtration rate in said subject. Embodiment 83 The method of embodiment 81 or 82, wherein said glomerular filtration rate is an estimated glomerular filtration rate. Embodiment 84. The method of any one of embodiments 38-83, wherein said administering slows the growth of cysts in the kidneys and / or liver of the subject. Embodiment 85. The administering comprises: a) improving renal function in said subject; b) slowing the deterioration of renal function in said subject; c) reducing renal pain in said subject; d) slowing the increase in renal pain in said subject; e) delaying the onset of renal pain in said subject; f) reducing hypertension in said subject; g) slowing the progression of hypertension in said subject; h) delaying the onset of hypertension in said subject; i) reducing fibrosis of the kidney in the subject; j) slowing the progression of fibrosis of the kidney in the subject; k) delaying the onset of end stage renal disease in said subject; l) delaying the time to dialysis in said subject; m) delaying the time to kidney transplant in said subject; and / or n) The method according to any one of embodiments 38 to 84, wherein the life expectancy of the subject is improved. Embodiment 86. The administering comprises: a) reducing albuminuria in said subject; b) slowing the worsening of albuminuria in said subject; c) delaying the onset of albuminuria in said subject; d) reducing hematuria in said subject; e) slowing the progression of hematuria in said subject; f) delaying the onset of hematuria in said subject; g) reducing blood urea nitrogen levels in said subject; h) reducing serum creatinine levels in said subject; i) improving creatinine clearance in said subject; j) reducing the albumin:creatinine ratio in said subject; k) increasing urinary polycystin-1 (PC1) in said subject; l) increasing urinary polycystin-2 (PC2) in said subject; m) reducing urinary neutrophil gelatinase-associated lipocalin (NGAL) protein in said subject; and / or n) The method according to any one of embodiments 38 to 85, wherein kidney injury molecule-1 (KIM-1) protein is reduced in the urine of the subject. 87. a) measuring total kidney volume in said subject; b) measuring hypertension in said subject; c) measuring renal pain in said subject; d) measuring urinary polycystin-1 (PC1) in said subject; e) measuring urinary polycystin-2 (PC2) in said subject; f) measuring fibrosis in the kidney in the subject; g) measuring blood urea nitrogen levels in said subject; h) measuring serum creatinine level in said subject; i) measuring creatinine clearance in said subject; j) measuring albuminuria in said subject; k) measuring the albumin:creatinine ratio in said subject; l) measuring the glomerular filtration rate in said subject; m) measuring neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject; and / or The method according to any one of embodiments 38 to 86, comprising: n) measuring kidney injury molecule-1 (KIM-1) protein in the urine of the subject. Embodiment 88. The method of any one of embodiments 38 to 87, comprising administering at least one additional therapy, wherein said at least one additional therapy is an antihypertensive agent. Embodiment 89. The method of any one of embodiments 38 to 87, comprising 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, glucosylceramide synthase inhibitors, antihyperglycemic agents, dialysis, and kidney transplantation. Embodiment 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. Embodiment 91. The method of embodiment 89, wherein the angiotensin II receptor blocker (ARB) is selected from candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, and eprosartan. Embodiment 92. The method of embodiment 89, wherein the vasopressin receptor 2 antagonist is tolvaptan. Embodiment 93 The method of embodiment 89, wherein the aldosterone receptor antagonist is spironolactone. Embodiment 94. The method of embodiment 89, wherein the kinase inhibitor is selected from bosutinib and KD019. Embodiment 95. The method of embodiment 89, wherein the mTOR inhibitor is selected from everolimus, rapamycin, and sirolimus. Embodiment 96 The method of embodiment 89, wherein the hormone analog is selected from somatostatin and adrenocorticotropic hormone. Embodiment 97. The method of embodiment 89, wherein the glucosylceramide synthase inhibitor is benglustat. Embodiment 98. The method of embodiment 89, wherein the antihyperglycemic agent is metformin. Embodiment 99. The method of any one of embodiments 38-96, comprising administering a therapeutically effective amount of the compound. Embodiment 100. The method of any one of embodiments 38 to 99, wherein the subject is a human subject. Embodiment 101. A modified oligonucleotide for use in therapy, comprising, in the 5' to 3' direction: (N”) p -(N) r -(N') q wherein each N" is independently a modified or unmodified nucleoside; p is 0 to 14, and if p is not 0, then (N") p is complementary to an equal length portion of the nucleobase sequence of miR-17, (N) r each N is independently a modified or unmodified nucleotide, (N r is 5'-AGCACUUU-3', N' is a nucleoside containing a modified sugar moiety; q is 0 or 1, and when q is 1, the nucleobase of N' is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that said purine nucleobase does not have an H-bond acceptor at position 6; A compound comprising the modified oligonucleotide, or a pharma- ceutically acceptable salt thereof, wherein each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. Embodiment 102. The compound of embodiment 101, wherein the therapy is a therapy for polycystic kidney disease. Embodiment 103. The compound of embodiment 102, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). Embodiment 104. The compound of embodiment 102, wherein the polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). Embodiment 105. A compound according to any one of embodiments 1 to 22, a modified oligonucleotide according to any one of embodiments 23 to 29, or a pharmaceutical composition according to any one of embodiments 30 to 33, for use in therapy. [Brief description of the drawings]

[0006] [Figure 1] Purine nucleobase structures. [Diagram 2] A, Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on kidney-to-body weight ratio. B, Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on blood urea nitrogen (BUN) levels. C, Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on blood creatinine levels. [Diagram 3] Maximum tolerated dose (MTD) study and comparative dose evaluation of RG-NG-1001, RGLS4326, and RG-NG-1017. Male C57BL / 6J mice aged 6-7 weeks were administered a single intracerebroventricular (ICV) injection of various dose levels of RG-NG-1001 and RGLS4326 (anti-miR-17 oligos that inhibit AMPA-R) and RG-NG-1017 (anti-miR-17 oligos that do not inhibit AMPA-R, RG-NG-1017) in 4 μL volumes and monitored for 7 days. Mouse mortality is shown for the three different compounds at different doses. [Figure 4A-4F] We describe the activity of RG-NG-1015 and RGLS4326 against miR-17 (4A), miR-20a (4B), miR-93 (4C), and miR106(a) (4D) luciferase sensor activity in HeLa cells in vitro. We describe the activity of RG-NG-1015 and RGLS4326 against luciferase sensors containing the full-length 3' untranslated regions (UTRs) of the miR-17 direct target genes PKD1 (4E) and PKD2 (4F). [Figure 5A-5D]The pharmacokinetics and target binding (measured by miPSA) of RGLS4326 and RG-NG-1015 were measured following a single subcutaneous dose in C57BL6 mice. Plasma concentrations (5A), tissue concentrations (5B), kidney target binding (5C), and liver target binding (5D) are shown. [Figures 6A-6E] The effect of RG-NG-1015 at various doses and regimens, and in combination with tolvaptan, on the Pcy / DBA mouse model of PKD was measured. The dosing schedule is shown in Figure 6A, and the key to the graphs of Figures 6C-6E is shown in Figure 6B. Kidney weight / body weight (6C), cyst area (%) (6D), and urinary Ngal / Cr (6E) are shown. Error bars represent standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, (ns)p>0.05 compared to Pcy vehicle-treated group; one-way ANOVA Bonferroni's multiple comparison test. Compared with tolvaptan alone, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001, (ns)p>0.05; one-way ANOVA Sadik's multiple comparisons test. Compared with dose-matched RG-NG-1015 alone, $p<0.05, $$p<0.01, $$$p<0.001, $$$$p<0.001, (ns)p>0.05; one-way ANOVA Sadik's multiple comparisons test. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Unless a special definition is provided, the nomenclature used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the procedures and techniques thereof, are well known and commonly used in the art. In the event of a plurality of definitions for a term herein, the definition in this section shall prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients. Certain 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, which are incorporated herein by reference for any purpose. Where permitted, all patents, patent applications, published applications and publications, GENBANK sequences, websites, and other published materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise noted. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may change, but equivalent information may be found by searching the Internet. Reference thereto evidences the availability and public dissemination of such information.

[0008] Before the present compositions and methods are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0009] definition "Polycystic kidney disease" or "PKD" is a cystic kidney disease characterized by the accumulation of numerous fluid-filled cysts within the kidneys. Multiple cysts form in at least one kidney, often leading to enlargement of the affected kidney(s) and progressive loss of renal function.

[0010] "Polycystic kidney disease marker" means a medical parameter used to assess the severity of polycystic kidney disease, renal function, and / or response of a subject with polycystic kidney disease to treatment. Non-limiting examples of polycystic kidney disease markers include total kidney volume, high blood pressure, glomerular filtration rate, and renal pain.

[0011] "Marker of renal function" means a medical parameter used to assess renal function in a subject. Non-limiting examples of markers of renal function include glomerular filtration rate, blood urea nitrogen level, and serum creatinine level.

[0012] "Autosomal dominant polycystic kidney disease" or "ADPKD" is a polycystic kidney disease caused by one or more genetic mutations in the PKD1 and / or PKD2 genes. Eighty-five percent of ADPKD cases are caused by mutations in PKD1, located on chromosome 16, and the majority of the remaining ADPKD cases are caused by mutations in PKD2, located on chromosome 4.

[0013] "Autosomal recessive polycystic kidney disease" or "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 will die from complications of intrauterine renal disease, and approximately one-third of survivors will develop end-stage renal disease (ESRD) within 10 years.

[0014] "Nephronophthisis" or "NPHP" means an autosomal recessive cystic kidney disease characterized by corticomedullary cysts, tubular basement membrane destruction, and tubulointerstitial nephropathy.

[0015] "Total renal volume" or "TKV" is a measurement of total renal volume. Total renal volume may be determined by magnetic resonance imaging (MRI), computed tomography (CT) scan, or ultrasound (US) imaging, and the volume may be calculated by standard methodologies such as the ellipsoid volume equation (for ultrasound) or determined by quantitative stereology or boundary tracing (for CT / MRI).

[0016] "Height-adjusted total kidney volume" or "HtTKV" is a measure of total kidney volume per unit of height. Patients with HtTKV values ​​of 600 ml / m or greater are predicted to develop stage 3 chronic kidney disease within 8 years.

[0017] "Renal pain" means clinically significant renal pain requiring medical emergence, pharmacological treatment (narcotic or last resort analgesics), or invasive intervention.

[0018] "Worsening hypertension" means a change in blood pressure that requires initiation or increase in hypertension treatment.

[0019] "Fibrosis" refers to the formation or development of excess fibrous connective tissue in an organ or tissue. In certain embodiments, fibrosis occurs as a repair or reactive process. In certain embodiments, fibrosis occurs in response to damage or injury. The term "fibrosis" should be understood as the formation or development of excess fibrous connective tissue in an organ or tissue as a repair or reactive process, as opposed to the formation of fibrous tissue as a normal component of the organ or tissue.

[0020] "Hematuria" refers to the presence of red blood cells in the urine.

[0021] "Albuminuria" refers to the presence of excess albumin in urine, including but not limited to normoalbuminuria, hypernormoalbuminuria, microalbuminuria and macroalbuminuria. Normally, the glomerular filtration permeability barrier, which consists of podocytes, glomerular basement membrane and endothelial cells, prevents serum proteins from leaking into the urine. Albuminuria may reflect damage to the glomerular filtration permeability barrier. Albuminuria may be calculated from a 24-hour urine sample, an overnight urine sample, or a spot urine sample.

[0022] "Hyper-normal albuminuria" means elevated albuminuria characterized by (i) urinary excretion of 15 to <30 mg of albumin per 24 hours and / or (ii) an albumin / creatinine ratio of 1.25 to 2.5 mg / mmol (or 10 to <20 mg / g) in men and 1.75 to <3.5 mg / mmol (or 15 to <30 mg / g) in women.

[0023] "Microalbuminuria" means elevated albuminuria characterized by (i) urinary excretion of 30-300 mg of albumin per 24 hours and / or (ii) an albumin / creatinine ratio of 2.5-25 mg / mmol (or 20-200 mg / g) in men and 3.5 to <35 mg / mmol (or 30-300 mg / g) in women.

[0024] "Macro albuminuria" means elevated albuminuria characterized by urinary excretion of more than 300 mg of albumin per 24 hours, and / or (ii) an albumin / creatinine ratio of >25 mg / mmol (or >200 mg / g) in men and >35 mg / mmol (or >300 mg / g) in women.

[0025] "Albumin / creatinine ratio" means the ratio of urinary albumin (mg / dL) per urinary creatinine (g / dL), expressed in mg / g. In certain embodiments, the albumin / creatinine ratio may be calculated from a spot urine sample and used as an estimate of albumin excretion over a 24-hour period.

[0026] "Glomerular filtration rate" or "GFR" refers to the flow rate of filtered fluid through the kidney, and is used as an indicator of renal function in a subject.In certain embodiments, the GFR of a subject is determined by calculating estimated glomerular filtration rate.In certain embodiments, the GFR of a subject is measured directly in the subject using inulin method.

[0027] "Estimated glomerular filtration rate" or "eGFR" refers to a measurement of how well the kidneys filter creatinine and is used to estimate glomerular filtration rate. Direct measurement of GFR is complicated, so eGFR is frequently used in clinical practice. Normal results are between 90 and 120 mL / min / 1.73 m 2 can range from 60 mL / min / 1.73 m for 3 months or more. 2 Levels below 15 mL / min / 1.73 m may be indicative of chronic kidney disease. 2 Levels below this level may be indicative of renal failure.

[0028] "Proteinuria" refers to the presence of excess serum protein in urine. Proteinuria can be characterized by the excretion of more than 250 mg of protein in urine per 24 hours and / or a urinary protein to creatinine ratio of 0.20 mg / mg or greater. Elevated serum proteins associated with proteinuria include, but are not limited to, albumin.

[0029] "Blood urea nitrogen level" or "BUN level" means a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea in the urea cycle as a waste product of protein digestion, and urea is removed from the blood by the kidneys. Normal human adult blood contains 7-21 mg of urea nitrogen per 100 ml (7-21 mg / dL) of blood. Measurement of blood urea nitrogen level is used as an indicator of kidney health. If the kidneys cannot normally remove urea from the blood, a subject's BUN level is elevated.

[0030] "Elevation" means an increase in a medical parameter that is considered to be clinically relevant. A medical professional can determine whether an increase is clinically significant.

[0031] "End stage renal disease (ESRD)" means complete or near complete failure of kidney function.

[0032] "Quality of life" refers to the extent to which a subject's physical, psychological, and social functioning is impaired by a disease and / or treatment for the disease. Quality of life may be decreased in subjects with polycystic kidney disease.

[0033] "Renal dysfunction" refers to a decrease in renal function as compared to normal renal function.

[0034] "Slow the deterioration of" "Slow the deterioration" means reducing the rate at which a medical condition progresses toward an advanced state.

[0035] "Delay to dialysis" means maintaining sufficient renal function so that the need for dialysis treatment is delayed.

[0036] "Delay to kidney transplant" means maintaining sufficient renal function so that the need for a kidney transplant is delayed.

[0037] "Improving life expectancy" means extending the lifespan of a subject by treating one or more symptoms of a disease in the subject.

[0038] "Subject" means a human or non-human animal selected for treatment or therapy.

[0039] By "subject in need thereof" is meant a subject identified as being in need of therapy or treatment.

[0040] By "subject suspected of having" is meant a subject who exhibits one or more clinical indicators of a disease.

[0041] By "miR-17-associated disease" is meant a disease or condition that is modulated by the activity of one or more miR-17 family members.

[0042] "Administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0043] "Parenteral administration" means administration via infusion or injection. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

[0044] "Subcutaneous administration" means administration just below the skin.

[0045] "Intravenous administration" means administration into a vein.

[0046] "Administered simultaneously" refers to the co-administration of two or more agents in any manner in which both pharmacological effects are manifested in the patient at the same time. Concurrent administration does not require that both agents be administered in a single pharmaceutical composition, in the same dosage form, or by the same route of administration. The effects of both agents do not have to be manifested at the same time. The effects need only overlap for a period of time, and do not have to be coextensive.

[0047] "Duration" refers to the period of time that an activity or event continues. In certain embodiments, a treatment period is the period during which a dose of a pharmaceutical agent or pharmaceutical composition is administered.

[0048] "Therapy" refers to a method of treating a disease. In certain embodiments, therapy includes, but is not limited to, administering one or more pharmaceutical agents to a subject with a disease.

[0049] "Treating" refers to the application of one or more specific procedures used to alleviate at least one indicator of a disease. In certain embodiments, the specific procedure is administering one or more pharmaceutical agents. In certain embodiments, treating PKD includes, but is not limited to, reducing total kidney volume, improving kidney function, reducing hypertension, and / or reducing kidney pain.

[0050] "Ameliorate" means that the severity of at least one indicator of a condition or disease is reduced. In certain embodiments, amelioration includes delaying or slowing the progression of one or more indicators of a condition or disease. The severity of an indicator can be determined by subjective or objective measures known to those skilled in the art.

[0051] "At risk of developing" refers to a condition in which a subject is predisposed to developing a condition or disease.In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed with the condition or disease.In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but does not exhibit a sufficient number of symptoms to be diagnosed with the condition or disease.

[0052] "Preventing occurrence" means preventing the onset of a condition or disease in a subject at risk of developing the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives a treatment similar to that received by a subject who already has the disease or condition.

[0053] "Delaying onset" means delaying the onset of a condition or disease in a subject at risk of developing the disease or condition. In certain embodiments, a subject at risk of developing a disease or condition receives a treatment similar to that received by a subject who already has the disease or condition.

[0054] "Dose" refers to a specific amount of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that is not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reactions in an individual. In certain embodiments, a dose is administered as a slow infusion.

[0055] "Dosage unit" refers to the form in which a pharmaceutical product is provided. In certain embodiments, the dosage unit is a vial containing lyophilized oligonucleotide. In certain embodiments, the dosage unit is a vial containing reconstituted oligonucleotide.

[0056] "Therapeutically effective amount" refers to an amount of a pharmaceutical agent that confers a therapeutic benefit on an animal.

[0057] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, including pharmaceutical agents. For example, a pharmaceutical composition can include a sterile aqueous solution.

[0058] By "pharmaceutical product" is meant a substance that produces a therapeutic effect when administered to a subject.

[0059] "Active pharmaceutical ingredient" means the substance in a pharmaceutical composition that produces a desired effect.

[0060] "Pharmaceutically acceptable salt" refers to a physiologically and pharma- ceutically acceptable salt of a compound provided herein, i.e., a salt that retains the desired biological activity of the compound and does not have undesired toxicological effects when administered to a subject. Non-limiting exemplary pharma-ceutically acceptable salts of the compounds provided herein include sodium and potassium salt forms. As used herein, the terms "compound," "oligonucleotide," and "modified oligonucleotide" include pharma-ceutically acceptable salts thereof, unless otherwise specified.

[0061] By "saline" is meant an aqueous solution of sodium chloride.

[0062] "Improvement of organ function" refers to a change in organ function toward normal range. In certain embodiments, organ function is assessed by measuring molecules found in the subject's blood or urine. In certain embodiments, improvement of renal function is measured by a reduction in blood urea nitrogen, a reduction in proteinuria, a reduction in albuminuria, and the like.

[0063] By "acceptable safety profile" is meant a pattern of side effects that is within clinically acceptable limits.

[0064] "Side effects" refers to physiological responses resulting from a treatment other than the desired effect. In certain embodiments, side effects include, but are not limited to, injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. Such side effects may be detected directly or indirectly. For example, an increase in serum aminotransferase levels may indicate hepatotoxicity or liver function abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or liver function abnormalities.

[0065] As used herein, the term "blood" includes whole blood and blood fractions such as serum and plasma.

[0066] "Anti-miR" means an oligonucleotide having a nucleobase sequence complementary to a microRNA. In certain embodiments, an anti-miR is a modified oligonucleotide.

[0067] "Anti-miR-17" refers to a modified oligonucleotide having a nucleobase sequence complementary to one or more miR-17 family members. In certain embodiments, anti-miR-17 is fully complementary (i.e., 100% complementary) to one or more miR-17 family members. In certain embodiments, anti-miR-17 is at least 80%, at least 85%, at least 90%, or at least 95% complementary to one or more miR-17 family members.

[0068] By "miR-17" is meant the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO:1).

[0069] By "miR-20a" is meant the mature miRNA having the nucleobase sequence 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' (SEQ ID NO: 2).

[0070] By "miR-20b" is meant the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUCAUAGUGCAGGUAG-3' (SEQ ID NO:3).

[0071] By "miR-93" is meant the mature miRNA having the nucleobase sequence 5'-CAAAGUGCUGUUCGUGCAGGUAG-3' (SEQ ID NO:4).

[0072] By "miR-106a" is meant the mature miRNA having the nucleobase sequence 5'-AAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO:5).

[0073] By "miR-106b" is meant the mature miRNA having the nucleobase sequence 5'-UAAAGUGCUGACAGUGCAGAU-3' (SEQ ID NO:6).

[0074] By "miR-17 seed sequence" is meant the nucleobase sequence 5'-AAAGUG-3 that is present in each miR-17 family member.

[0075] By "miR-17 family member" is meant a mature miRNA having a nucleobase sequence that includes a miR-17 seed sequence and is selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0076] "miR-17 family" refers to the group of the following miRNAs, each of which has a nucleobase sequence that includes a miR-17 seed sequence: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0077] "Target nucleic acid" means a nucleic acid to which an oligomeric compound is designed to hybridize.

[0078] "Targeting" refers to the process of design and selection of a nucleobase sequence that will hybridize to a target nucleic acid.

[0079] By "targeted to" is meant having a nucleobase sequence that allows for hybridization to a target nucleic acid.

[0080] "Modulation" refers to a variation in function, amount, or activity. In certain embodiments, modulation refers to an increase in function, amount, or activity. In certain embodiments, modulation refers to a decrease in function, amount, or activity.

[0081] By "expression" is meant any of the functions and steps by which a gene's coded information is converted into structures present and operating within a cell.

[0082] "Nucleobase sequence" means the order of contiguous nucleobases in an oligomeric compound or nucleic acid, typically listed in the 5' to 3' direction, without regard to any sugar, linkage, and / or nucleobase modifications.

[0083] "Contiguous nucleobases" means nucleobases that are immediately adjacent to each other within a nucleic acid.

[0084] "Nucleobase complementarity" means the capacity of two nucleobases to non-covalently pair through hydrogen bonding.

[0085] "Complementary" means that one nucleic acid can hybridize to another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide that can hybridize to a target nucleic acid.

[0086] "Fully complementary" means that each nucleobase of an oligonucleotide can pair with a nucleobase at each corresponding position in a target nucleic acid. In certain embodiments, an oligonucleotide is fully complementary to a microRNA (also referred to as 100% complementary). That is, each nucleobase of an oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. A modified oligonucleotide can have a number of linked nucleosides that are fully complementary to a microRNA and are shorter than the length of the microRNA. For example, an oligonucleotide having 16 linked nucleosides, where each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA, is fully complementary to a microRNA. In certain embodiments, an oligonucleotide in which each nucleobase has complementarity to a nucleobase in a region of a microRNA stem-loop sequence is fully complementary to a microRNA stem-loop sequence.

[0087] "Percent complementarity" refers to the percentage of nucleobases of an oligonucleotide that are complementary to a portion of the target nucleic acid that is equal in length. Percent complementarity is calculated by dividing the number of nucleobases of an oligonucleotide that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

[0088] "Percentage identity" refers to the number of nucleobases in a first nucleic acid that are identical to the nucleobases at corresponding positions in a second nucleic acid divided by the total number of nucleobases in the first nucleic acid.In certain embodiments, the first nucleic acid is a microRNA and the second nucleic acid is a microRNA.In certain embodiments, the first nucleic acid is an oligonucleotide and the second nucleic acid is an oligonucleotide.

[0089] "Hybridize" refers to the annealing of complementary nucleic acids which occurs by virtue of nucleobase complementarity.

[0090] By "mismatch" is meant a nucleobase of a first nucleic acid that is not capable of Watson-Crick pairing with a nucleobase at a corresponding position in a second nucleic acid.

[0091] "Identical" in the context of nucleobase sequences means having the same nucleobase sequence, regardless of sugar, linkage, and / or nucleobase modifications and regardless of the methylation state of any pyrimidines present.

[0092] "MicroRNA" means an endogenous non-coding RNA that is 18-25 nucleobases in length and is the product of cleavage of a pre-microRNA by the enzyme Dicer. Examples of mature microRNAs can be found in the microRNA database known as miRBase (microrna.sanger.ac.uk / ). In certain embodiments, microRNA is abbreviated as "miR."

[0093] By "microRNA-regulated transcript" is meant a transcript that is regulated by a microRNA.

[0094] "Seed match sequence" means a nucleobase sequence that is complementary to and the same length as the seed sequence.

[0095] "Oligomeric compound" means a compound comprising a plurality of linked monomeric subunits. Oligomeric compounds include oligonucleotides.

[0096] By "oligonucleotide" is meant a compound comprising multiple linked nucleosides, each of which, independently of each other, can be modified or unmodified.

[0097] "Naturally occurring internucleoside linkage" means a 3' to 5' phosphodiester linkage between nucleosides.

[0098] By "natural sugar" is meant a sugar found in DNA (2'-H) or RNA (2'-OH).

[0099] "Internucleoside linkage" means a covalent bond between adjacent nucleosides.

[0100] "Linked nucleosides" means nucleosides linked by a covalent bond.

[0101] "Nucleobase" means a heterocyclic moiety capable of non-covalent pairing with another nucleobase.

[0102] "Nucleoside" means a nucleobase linked to a sugar moiety.

[0103] "Nucleotide" means a nucleoside that has a phosphate group covalently linked to the sugar portion of the nucleoside.

[0104] A compound that comprises a modified oligonucleotide that is composed of a plurality of linked nucleosides means a compound that comprises a modified oligonucleotide that has a specific number of linked nucleosides.Thus, the compound may comprise additional substituents or conjugates.Unless otherwise specified, the modified oligonucleotide is not hybridized to a complementary strand, and the compound does not comprise any additional nucleosides beyond those of the modified oligonucleotide.

[0105] "Modified oligonucleotide" means a single-stranded oligonucleotide having one or more modifications compared to naturally occurring termini, sugars, nucleobases, and / or internucleoside linkages. Modified oligonucleotides can contain unmodified nucleosides.

[0106] "Modified nucleoside" means a nucleoside having any alteration from a naturally occurring nucleoside. A modified nucleoside can have a modified sugar and an unmodified nucleobase. A modified nucleoside can have a modified sugar and a modified nucleobase. A modified nucleoside can have a natural sugar and a modified nucleobase. In certain embodiments, a modified nucleoside is a bicyclic nucleoside. In certain embodiments, a modified nucleoside is a non-bicyclic nucleoside.

[0107] By "modified internucleoside linkage" is meant any variation from a naturally occurring internucleoside linkage.

[0108] By "phosphorothioate internucleoside linkage" is meant a linkage between nucleosides in which one of the non-bridging atoms is a sulfur atom.

[0109] By "modified sugar moiety" is meant a substitution and / or any change from a natural sugar.

[0110] "Unmodified nucleobase" means the naturally occurring heterocyclic bases of RNA or DNA, the purine bases being adenine (A) and guanine (G) and the pyrimidine bases being thymine (T), cytosine (C) (such as 5-methylcytosine), and uracil (U).

[0111] "5-methylcytosine" means a cytosine with a methyl group attached to the 5 position.

[0112] "Unmethylated cytosine" means a cytosine that does not have a methyl group attached to the 5-position.

[0113] By "modified nucleobase" is meant any nucleobase that is not an unmodified nucleobase.

[0114] "Sugar moiety" means a naturally occurring furanosyl or modified sugar moiety.

[0115] By "modified sugar moiety" is meant a substituted sugar moiety or sugar surrogate.

[0116] By "2'-O-methyl sugar" or "2'-OMe sugar" is meant a sugar having an O-methyl modification at the 2' position.

[0117] By "2'-O-methoxyethyl sugar" or "2'-MOE sugar" is meant a sugar having an O-methoxyethyl modification at the 2' position.

[0118] "2'-fluoro" or "2'-F" refers to a sugar having a fluoro modification at the 2' position.

[0119] "Bicyclic sugar moiety" means a modified sugar moiety comprising a 4-7 membered ring (such as, but not limited to, a furanosyl) that includes a bridge linking two atoms of the 4-7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4-7 membered ring is a sugar ring. In certain embodiments, the 4-7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2'-carbon and the 4'-carbon of the furanosyl. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

[0120] By "Locked Nucleic Acid (LNA) sugar moiety" is meant a substituted sugar moiety that contains a (CH2)-O bridge between the 4' and 2' furanose ring atoms.

[0121] By "ENA sugar moiety" is meant a substituted sugar moiety containing a (CH2)2-O bridge between the 4' and 2' furanose ring atoms.

[0122] "Constrained ethyl (cEt) sugar moiety" means a substituted sugar moiety comprising a CH(CH3)-O bridge between the 4' and 2' furanose ring atoms. In certain embodiments, the CH(CH3)-O bridge is constrained in the S orientation. In certain embodiments, the CH(CH3)-O is constrained in the R orientation.

[0123] By "S-cEt sugar moiety" is meant a substituted sugar moiety that contains an S-constrained CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0124] By "R-cEt sugar moiety" is meant a substituted sugar moiety that contains an R-constrained CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0125] "2'-O-methyl nucleoside" means a 2'-modified nucleoside having a 2'-O-methyl sugar modification.

[0126] "2'-O-methoxyethyl nucleoside" means a 2'-modified nucleoside having a 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleosides can contain modified or unmodified nucleobases.

[0127] "2'-fluoro nucleoside" means a 2'-modified nucleoside having a 2'-fluoro sugar modification. 2'-Fluoro nucleosides can contain modified or unmodified nucleobases.

[0128] "Bicyclic nucleoside" means a 2'-modified nucleoside having a bicyclic sugar moiety. Bicyclic nucleosides can have modified or unmodified nucleobases.

[0129] "cEt nucleoside" means a nucleoside that includes a cEt sugar moiety. A cEt nucleoside can include a modified or unmodified nucleobase.

[0130] By "S-cEt nucleoside" is meant a nucleoside that includes an S-cEt sugar moiety.

[0131] By "R-cEt nucleoside" is meant a nucleoside that includes an R-cEt sugar moiety.

[0132] By "β-D-deoxyribonucleoside" is meant a naturally occurring DNA nucleoside.

[0133] By "β-D-ribonucleoside" is meant a naturally occurring RNA nucleoside.

[0134] By "LNA nucleoside" is meant a nucleoside that includes an LNA sugar moiety.

[0135] "ENA nucleoside" means a nucleoside that includes an ENA sugar moiety.

[0136] By "hydrogen bond acceptor" is meant a component of a hydrogen bond that does not donate a shared hydrogen atom.

[0137] "Hydrogen bond donor" means a bond or a molecule that donates a hydrogen atom for a hydrogen bond.

[0138] overview Polycystic kidney disease (PKD) is a genetic form of kidney disease in which fluid-filled cysts develop in the kidney, leading to kidney failure and often end-stage renal disease. Certain forms of PKD are also characterized by kidney enlargement. Excessive growth of cysts is the defining pathological feature of PKD. In managing PKD, the primary goals of treatment are to manage symptoms such as hypertension and infections, preserve kidney function, and prevent the development of end-stage renal disease (ESRD), thereby improving the life expectancy of subjects with PKD.

[0139] miR-17 has been identified as a target for the treatment of PKD. RGLS4326, an anti-miR-17 compound, was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney- and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and derepresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. A Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) was initiated in October 2020.

[0140] Following the initiation of Phase 1b clinical trials, nonclinical toxicity studies revealed CNS-related findings, including abnormal gait, reduced motor activity, and / or weakness at high doses of RGLS4326 in mice. RGLS4326 was found to be an antagonist of AMPA receptors (AMPA-Rs), which are glutamate receptors and ion channels on excitatory synapses in the central nervous system (CNS) that mediate fast excitatory neurotransmission and are therefore important components of all neuronal networks. Antagonism of AMPA receptors could explain the CNS-mediated findings observed at high doses of RGLS4326 in nonclinical toxicity models. Although such CNS-related findings were not observed in human subjects, it is nevertheless preferable to avoid antagonism of AMPA receptors. Therefore, a library of anti-miR-17 compounds was screened to identify compounds with physicochemical and pharmacological properties comparable to RGLS4326 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.

[0141] compound A modified oligonucleotide, the modified oligonucleotide comprising, in the 5' to 3' direction: (N”) p -(N) r -(N') q wherein each N" is independently a modified or unmodified nucleoside, p is 0 to 14, and when p is not 0, (N" p is complementary to an equal length portion of the nucleobase sequence of miR-17, (N) r Each N is independently a modified or unmodified nucleotide, (N ror a pharma- ceutical acceptable salt thereof.

[0142] The atoms of a purine nucleobase are: [ka] As shown in the structure of , they are numbered from 1 to 9 according to the standard numbering convention for nucleobases.

[0143] The atoms or groups bound to nucleobase ring atoms have the same number as the ring atoms to which they are attached.

[0144] Certain nucleobases, such as guanosine and inosine, contain a hydrogen bond acceptor at position 6. The hydrogen bond acceptor at position 6 of guanosine is the oxygen attached to the carbon at position 6. The hydrogen bond acceptor at position 6 of inosine is the oxygen attached to the carbon at position 6.

[0145] Purine nucleobases that do not have hydrogen bond acceptors at position 6 include, but are not limited to, 2-aminopurine, 2,6-diaminopurine, isoguanosine, and adenosine.The NH2 present at position 6 of each of 2,6-diaminopurine, isoguanosine, and adenosine functions as a hydrogen bond donor.The 6-position of 2-aminopurine does not have a substituent, and therefore lacks a hydrogen bond acceptor or donor.

[0146] In certain embodiments, (N) r The structure of A S G S C M A F CF U F U M U S where a nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, a nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, and a nucleoside followed by the subscript "S" is an S-cEt nucleoside.

[0147] In certain embodiments, at least one internucleoside linkage is a phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage is a phosphorothioate internucleoside linkage.

[0148] In certain embodiments, q is 1. In certain embodiments, q is 0. In certain embodiments, p is 0. In certain embodiments, p is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14.

[0149] In certain embodiments, (N″) p has no more than one mismatch with the nucleobase sequence of miR-17 (SEQ ID NO: 1). In certain embodiments, (N") p has no mismatches to the nucleobase sequence of miR-17 (SEQ ID NO: 1). In certain embodiments, (N") p The nucleic acid base 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.

[0150] In certain embodiments, the nucleobase of N' is a purine nucleobase that does not have a hydrogen bond acceptor at position 6. In certain embodiments, the nucleobase of N' is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0151] In certain embodiments, the sugar moiety at N' is not a 2'-O-methyl sugar.In certain embodiments, the sugar moiety at N' is a 2'-O-methoxyethyl sugar or a S-cEt sugar.

[0152] In certain embodiments, the structure of the modified oligonucleotide is S G S C M A F C F U F U M U S A S -3', and each cytosine is an unmethylated cytosine. In certain embodiments, the structure of the modified oligonucleotide is S G S C M A F C F U F U M U S U S -3', and each cytosine is an unmethylated cytosine. In certain embodiments, the structure of the modified oligonucleotide is S G S C M A F C F U F U M U S C S -3', and each cytosine is an unmethylated cytosine. The structure of the modified oligonucleotide is S G S C M A F C F U F U M U S 3. The compound of claim 2, wherein each cytosine is -3' and is an unmethylated cytosine.

[0153] In certain embodiments, the compound consists of a modified oligonucleotide.

[0154] In certain embodiments, the pharma- ceutically acceptable salt is a sodium salt.

[0155] structure: [ka] Provided herein are modified oligonucleotides having the formula: wherein B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at position 6, or a pharma- ceutically acceptable salt thereof. In certain embodiments, B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0156] structure: [ka] Provided herein are modified oligonucleotides having the formula: where B is a uridine nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In certain embodiments, B is selected from adenosine, 2-aminopurine, 2,6-diaminopurine, and isoguanosine.

[0157] The modified oligonucleotide has the structure: [ka] Provided herein is a modified oligonucleotide designated RG-NG-1015, which is

[0158] Also provided herein are pharma- ceutically acceptable salts of the modified oligonucleotide RG-NG-1015. Thus, in some embodiments, the modified oligonucleotide has the following structure: [ka] or a pharma- ceutically acceptable salt thereof. A non-limiting exemplary pharma- ceutically acceptable salt of RG-NG-1015 is a modified oligonucleotide having the structure: [ka] has.

[0159] In some embodiments, pharma- ceutically acceptable salts of modified oligonucleotides contain fewer cationic counterions (Na) than phosphorothioate and / or phosphodiester linkages per molecule. + etc.) (i.e., some phosphorothioate and / or phosphodiester linkages are protonated). In some embodiments, a pharma- ceutically acceptable salt of RG-NG-1015 contains fewer than eight cationic counterions (Na + That is, in some embodiments, a pharma- ceutically acceptable salt of RG-NG-1015 may contain, on average, 1, 2, 3, 4, 5, 6, or 7 cationic counterions per molecule of RG-NG-1015, with the remaining phosphorothioate groups being protonated.

[0160] Specific Use Provided herein are methods for inhibiting the activity of one or more miR-17 family members in a cell, comprising contacting the cell with a compound provided herein comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0161] Provided herein is a method for inhibiting the activity of one or more members of the miR-17 family in a subject, comprising administering to the subject a pharmaceutical composition provided herein.In certain embodiments, the subject has a disease associated with one or more members of the miR-17 family.

[0162] Provided herein is a method for treating polycystic kidney disease (PKD), comprising administering to a subject in need thereof a compound provided herein, comprising a nucleobase sequence complementary to a miR-17 seed sequence. In certain embodiments, the subject has polycystic kidney disease. In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis (NPHP). In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD).

[0163] In certain embodiments, the subject has a disorder characterized by multiple non-renal indicators and polycystic kidney disease. Such disorders include, for example, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). Thus, provided herein is a method for treating polycystic kidney disease (PKD), comprising administering to a subject a compound provided herein comprising a nucleobase sequence complementary to a miR-17 seed sequence, the subject having Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). Provided herein is a method for treating polycystic kidney disease (PKD), comprising administering to a subject a compound provided herein comprising a nucleobase sequence complementary to a miR-17 seed sequence, the subject being suspected of having Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0164] In certain embodiments, the 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 failure in 50% of patients by age 60, eventually resulting in end-stage renal disease. ADPKD patients may require lifelong dialysis and / or kidney transplants. ADPKD is the most frequent genetic cause of renal failure. Excessive growth of cysts is the characteristic pathological feature of ADPKD. In the management of PKD, the primary goal of treatment is to preserve renal function and prevent the development of end-stage renal disease (ESRD), thereby improving the life expectancy of subjects with PKD. Total kidney volume generally increases steadily in ADPKD patients, and the increase correlates with a decline in renal function. Provided herein are methods for treating ADPKD, comprising administering to a subject having or suspected of having ADPKD a compound provided herein comprising a nucleobase sequence complementary to a miR-17 seed sequence.

[0165] In certain embodiments, polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). ARPKD is caused by mutations in PKHD1 gene and is the cause of chronic kidney disease in children. Although the typical renal phenotype of ARPKD is kidney enlargement, ARPKD has a significant effect on other organs, especially the liver. Patients with ARPKD progress to end-stage renal disease and require kidney transplantation as early as 15 years old. Provided herein is a method for treating ARPKD, comprising administering to a subject having or suspected of having ARPKD a compound provided herein, comprising a nucleic acid base sequence complementary to miR-17 seed sequence.

[0166] In certain embodiments, the polycystic kidney disease is nephronophthisis (NPHP). Nephronophthisis is an autosomal recessive cystic kidney disease that is a frequent 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, for example, NPHP1, have been identified in patients with NPHP. Provided herein is a method for treating NPHP, comprising administering to a subject having or suspected of having NPHP a compound provided herein, comprising a nucleobase sequence complementary to miR-17 seed sequence.

[0167] In certain embodiments, the subject with polycystic kidney disease has Jobert syndrome and related disorders (JSRD). JSRD includes a wide range of characteristic features, including brain, retinal, and skeletal abnormalities. Certain subjects with JSRD have polycystic kidney disease in addition to the features of JSRD. Thus, provided herein is a method for treating polycystic kidney disease in a subject with JSRD, comprising administering to the subject with JSRD a compound provided herein, comprising a nucleobase sequence complementary to a miR-17 seed sequence. In certain embodiments, the subject is suspected of having JSRD.

[0168] In certain embodiments, the subject with polycystic kidney disease has Meckel's syndrome (MKS). MKS is a disorder with severe signs and symptoms in many parts of the body, including the central nervous system, skeletal system, liver, kidneys, and heart. The common feature of MKS is the presence of multiple fluid-filled cysts in the kidneys, which cause the kidneys to become enlarged. Thus, provided herein is a method for treating MKS, comprising administering to a subject with MKS a compound provided herein, which comprises a nucleobase sequence complementary to miR-17 seed sequence. In certain embodiments, the subject is suspected of having MKS.

[0169] In certain embodiments, the subject with polycystic kidney disease has Bardet-Biedl syndrome (BBS). BBS is a disorder that affects many parts of the body, including the eyes, heart, kidneys, liver, and digestive system. A hallmark of BBS is the presence of renal cysts. Thus, provided herein is a method for treating polycystic kidney disease in a subject with BBS, comprising administering to the subject with BBS a compound provided herein comprising a nucleobase sequence complementary to a miR-17 seed sequence. In certain embodiments, the subject is suspected of having BBS.

[0170] In certain embodiments, the subject is diagnosed with PKD prior to administration of the compound comprising the modified oligonucleotide. Diagnosis of PKD can be achieved by evaluation of parameters including, but not limited to, the subject's family history, clinical features (including but not limited to, hypertension, albuminuria, hematuria, and impaired GFR), renal imaging studies (including but not limited to, MRI, ultrasound, and CT scan), and / or histological analysis.

[0171] In certain embodiments, the diagnosis of PKD comprises screening for mutations in one or more of the PKD1 or PKD2 genes. In certain embodiments, the diagnosis of ARPKD comprises screening for mutations in the PKHP1 gene. In certain embodiments, the diagnosis of NPHP comprises screening for one or more mutations in one or more of the NPHP1, NPHP2, NPHP3, NPHP4, NPHP5, NPHP6, NPHP7, NPHP8, or NPHP9 genes. In certain embodiments, the diagnosis of JSRD comprises screening for mutations in the NPHP1, NPHP6, AHI1, MKS3, or RPGRIP1L genes. In certain embodiments, the diagnosis of MKS comprises screening for mutations in the NPHP6, MKS3, RPGRIP1L, NPHP3, CC2D2A, BBS2, BBS4, BBS6, or MKS1 genes. In certain embodiments, diagnosis of BBS involves screening for mutations in the BBS2, BBS4, BBS6, MKS1, BBS1, BBS3, BBS5, BBS7, BBS7, BBS8, BBS9, BBS10, BBS11, or BBS12 genes.

[0172] In certain embodiments, the subject has an increased total kidney volume. In certain embodiments, the total kidney volume is height-adjusted total kidney volume (HtTKV). In certain embodiments, the subject has hypertension. In certain embodiments, the subject has renal dysfunction. In certain embodiments, the subject is in need of improved renal function. In certain embodiments, the subject is identified as having renal dysfunction.

[0173] In certain embodiments, the level of one or more miR-17 family members is increased in the kidney of a subject with PKD. In certain embodiments, prior to administration, the subject is determined to have an increased level of one or more miR-17 family members in the kidney. The level of the miR-17 family members may be measured from a kidney biopsy. In certain embodiments, prior to administration, the subject is determined to have an increased level of one or more miR-17 family members in the subject's urine or blood. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) or polycystin-2 (PC2) in the subject's urine. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) or polycystin-2 (PC2) in the subject's urine. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) and / or polycystin-2 (PC2) in the subject's urine.

[0174] In any of the embodiments provided herein, the subject may undergo certain tests to diagnose the subject's polycystic kidney disease, e.g., to determine the cause of polycystic kidney disease, to assess the extent of the subject's polycystic kidney disease, and / or to determine the subject's response to treatment. Such tests may evaluate markers of polycystic kidney disease. Certain tests, such as glomerular filtration rate and blood urea nitrogen levels, are also indicators of kidney function. Markers of polycystic disease include, but are not limited to, measuring total kidney volume in a subject, measuring high blood pressure in a subject, assessing renal pain in a subject, measuring fibrosis in a subject, measuring polycystin-1 (PC1) in the urine of a subject, measuring polycystin-2 (PC2) in the urine of a subject, measuring blood urea nitrogen levels in a subject, measuring serum creatinine levels in a subject, measuring creatinine clearance in a subject, measuring albuminuria in a subject, measuring the albumin:creatinine ratio in a subject, measuring glomerular filtration rate in a subject, measuring hematuria in a subject, measuring NGAL protein in the urine of a subject, and / or measuring KIM-1 protein in the urine of a subject. 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 (such as whole blood or serum).

[0175] Markers for polycystic kidney disease are determined by laboratory testing. Reference ranges for individual markers may vary from laboratory to laboratory. Variation may be due to, for example, differences in the specific assays used. Thus, the upper and lower limits of normal distribution of a marker in a population (also known as upper limit of normal (ULN) and lower limit of normal (LLN) respectively) may vary from laboratory to laboratory. For any particular marker, a medical professional may determine which levels outside of the normal distribution are clinically relevant and / or indicative of disease. For example, a medical professional may determine the glomerular filtration rate, which may indicate a decrease in the rate of renal function in subjects with polycystic kidney disease.

[0176] In certain embodiments, administration of a compound provided herein results in one or more clinically beneficial outcomes. In certain embodiments, administration improves renal function in a subject. In certain embodiments, administration slows the rate of decline in renal function in a subject. In certain embodiments, administration reduces total kidney volume in a subject. In certain embodiments, administration slows the rate of increase in total kidney volume in a subject. In certain embodiments, administration reduces height-adjusted total kidney volume (HtTKV). In certain embodiments, administration slows the rate of increase in HtTKV.

[0177] In certain embodiments, the administration increases polycystin-1 (PC1) in the subject's urine. In certain embodiments, the administration increases polycystin-2 (PC2) in the subject's urine. In certain embodiments, the administration increases polycystin-1 (PC1) and polycystin-2 (PC2) in the subject's urine.

[0178] In certain embodiments, administration inhibits the growth of cysts in the subject. In certain embodiments, administration slows the rate of increase in the growth of cysts in the subject. In some embodiments, the cysts are present in the kidney of the subject. In some embodiments, the cysts are present in an organ other than the kidney, for example, the liver.

[0179] In certain embodiments, administration reduces renal pain in the subject. In certain embodiments, administration delays the increase of renal pain in the subject. In certain embodiments, administration delays the onset of renal pain in the subject.

[0180] In certain embodiments, administration reduces hypertension in the subject. In certain embodiments, administration delays the worsening of hypertension in the subject. In certain embodiments, administration delays the onset of hypertension in the subject.

[0181] In certain embodiments, the administration reduces fibrosis in the subject's kidney.In certain embodiments, the administration slows the progression of fibrosis in the subject's kidney.

[0182] In certain embodiments, administration delays the onset of end stage renal disease in the subject. In certain embodiments, administration delays the subject's time to dialysis. In certain embodiments, administration delays the subject's time to kidney transplant. In certain embodiments, administration improves the subject's life expectancy.

[0183] In certain embodiments, administration reduces albuminuria in the subject. In certain embodiments, administration delays the worsening of albuminuria in the subject. In certain embodiments, administration delays the onset of albuminuria in the subject. In certain embodiments, administration reduces hematuria in the subject. In certain embodiments, administration delays the worsening of hematuria in the subject. In certain embodiments, administration delays the onset of hematuria in the subject. In certain embodiments, administration reduces blood urea nitrogen levels in the subject. In certain embodiments, administration reduces serum creatinine levels in the subject. In certain embodiments, administration improves creatinine clearance in the subject. In certain embodiments, administration reduces the albumin:creatinine ratio in the subject.

[0184] In certain embodiments, the administration improves the glomerular filtration rate in the subject. In certain embodiments, the administration slows down the rate of decline of the glomerular filtration rate in the subject. In certain embodiments, the glomerular filtration rate is an estimated glomerular filtration rate (eGFR). In certain embodiments, the glomerular filtration rate is a measured glomerular filtration rate (mGFR).

[0185] In certain embodiments, the administration reduces neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject. In certain embodiments, the administration reduces kidney injury molecule-1 (KIM-1) protein in the urine of the subject.

[0186] In any of the embodiments provided herein, the subject may be subjected to certain tests to assess the extent of disease in the subject. Such tests include, but are not limited to, measuring total kidney volume in the subject, measuring high blood pressure in the subject, measuring renal pain in the subject, measuring fibrosis in the kidney of the subject, measuring blood urea nitrogen level in the subject, measuring serum creatinine level in the subject, measuring blood creatinine clearance in the subject, measuring albuminuria in the subject, measuring albumin:creatinine ratio in the subject, measuring glomerular filtration rate in the subject (glomerular filtration rate is estimated or measured), measuring neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subject, and / or measuring kidney injury molecule-1 (KIM-1) protein in the urine of the subject.

[0187] In certain embodiments, subjects with polycystic kidney disease experience a reduced quality of life. For example, subjects with polycystic kidney disease may experience kidney pain, which may reduce the subject's quality of life. In certain embodiments, administration improves the subject's quality of life.

[0188] In any of the embodiments provided herein, the subject is a human subject. In certain embodiments, the human subject is an adult. In certain embodiments, the adult is at least 21 years old. In certain embodiments, the human subject is a pediatric subject, i.e., the subject is less than 21 years old. The pediatric population may be defined by a regulatory agency. In certain embodiments, the human subject is an adolescent. In certain embodiments, the adolescent is at least 12 years old and less than 21 years old. In certain embodiments, the human subject is a child. In certain embodiments, the child is at least 2 years old and less than 12 years old. In certain embodiments, the human subject is an infant. In certain embodiments, the infant is at least 1 month old and less than 2 years old. In certain embodiments, the subject is a newborn. In certain embodiments, the newborn is less than 1 month old.

[0189] Any of the compounds described herein may be for use in therapy. Any of the compounds provided herein may be for use in the treatment of polycystic kidney disease. In certain embodiments, the polycystic kidney disease is autosomal dominant polycystic kidney disease. In certain embodiments, the polycystic kidney disease is autosomal recessive polycystic kidney disease. In certain embodiments, the polycystic kidney disease is nephronophthisis. In certain embodiments, the subject has Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0190] Any of the modified oligonucleotides described herein can be for use in therapy. Any of the modified oligonucleotides provided herein can be for use in the treatment of polycystic kidney disease.

[0191] Any of the compounds provided herein may be for use in the preparation of a medicament. Any of the compounds provided herein may be for use in the preparation of a medicament for the treatment of polycystic kidney disease.

[0192] Any of the modified oligonucleotides provided herein can be for use in the preparation of a medicament. Any of the modified oligonucleotides provided herein can be for use in the preparation of a medicament for the treatment of polycystic kidney disease.

[0193] Any of the pharmaceutical compositions provided herein can be for use in treating polycystic kidney disease.

[0194] Certain additional therapies Treatment for polycystic kidney disease or any of the conditions listed herein may include two or more therapies.Thus, in certain embodiments, a method for treating a subject having or suspected having polycystic kidney disease is provided herein, comprising administering a compound provided herein that comprises a nucleic acid sequence complementary to miR-17 seed sequence, and administering at least one therapy.

[0195] In certain embodiments, the at least one additional therapy comprises a pharmaceutical agent. In certain embodiments, the pharmaceutical agent is an antihypertensive agent. Antihypertensive agents are used to control the blood pressure of a subject.

[0196] In certain embodiments, the pharmaceutical agent is a vasopressin receptor 2 antagonist. In certain embodiments, the vasopressin receptor 2 antagonist is tolvaptan.

[0197] In certain embodiments, the pharmaceutical agent comprises an angiotensin II receptor antagonist (ARB). In certain embodiments, the angiotensin II receptor antagonist is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, or eprosartan.

[0198] In certain embodiments, the pharmaceutical agent comprises an angiotensin II converting enzyme (ACE) inhibitor, hi certain embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, or ramipril.

[0199] In certain embodiments, the pharmaceutical agent is a diuretic.In certain embodiments, the pharmaceutical agent is a calcium channel blocker.

[0200] In certain embodiments, the pharmaceutical agent is a glucosylceramide synthase inhibitor. In certain embodiments, the glucosylceramide synthase inhibitor is benglustat.

[0201] In certain embodiments, the pharmaceutical agent is an antihyperglycemic agent. In certain embodiments, the antihyperglycemic agent is a biguanide. In certain embodiments, the biguanide is metformin.

[0202] In certain embodiments, the pharmaceutical agent is a kinase inhibitor. In certain embodiments, the kinase inhibitor is bosutinib or KD019.

[0203] In certain embodiments, the pharmaceutical agent is an adrenergic receptor antagonist.

[0204] In certain embodiments, the pharmaceutical agent is an aldosterone receptor antagonist. In certain embodiments, the aldosterone receptor antagonist is spironolactone. In certain embodiments, the spironolactone is administered at a dose ranging from 10 to 35 mg daily. In certain embodiments, the spironolactone is administered at a dose of 25 mg daily.

[0205] In certain embodiments, the pharmaceutical agent is a mammalian target of rapamycin (mTOR) inhibitor. In certain embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

[0206] In certain embodiments, the pharmaceutical agent is a hormone analog, hi certain embodiments, the hormone analog is somatostatin or adrenocorticotropic hormone.

[0207] In certain embodiments, the pharmaceutical agent is an anti-fibrotic agent. In certain embodiments, the anti-fibrotic agent is a modified oligonucleotide complementary to miR-21.

[0208] In certain embodiments, the additional therapy is dialysis. In certain embodiments, the additional therapy is a kidney transplant.

[0209] In certain embodiments, the pharmaceutical agent comprises an anti-inflammatory agent. In certain embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In certain embodiments, the steroidal anti-inflammatory agent is a corticosteroid. In certain embodiments, the corticosteroid is prednisone. In certain embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory agent. In certain embodiments, the non-steroidal anti-inflammatory agent is ibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

[0210] In certain embodiments, the pharmaceutical agent is one that blocks one or more responses to a fibrogenic signal.

[0211] In certain embodiments, the additional therapy may be medications that boost the body's immune system, including low-dose cyclophosphamide, thymostimulin, vitamins, and nutritional supplements (e.g., antioxidants including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea), as well as vaccines, e.g., immune stimulating complexes (ISCOMs), including vaccine formulations that combine multimeric presentation of antigens and adjuvants.

[0212] In certain embodiments, additional therapy is selected to treat or alleviate the side effects of one or more pharmaceutical compositions provided herein.Such side effects include, but are not limited to, injection site reaction, liver function abnormality, renal function abnormality, liver toxicity, nephrotoxicity, central nervous system abnormality, and myopathy.For example, an increase in aminotransferase level in serum can indicate liver toxicity or liver function abnormality.For example, an increase in bilirubin can indicate liver toxicity or liver function abnormality.

[0213] Specific microRNA nucleobase sequence 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 nucleobase sequence that includes the miR-17 seed sequence, which is the nucleobase sequence 5'-AAAGUG-3', or the nucleobase sequence at positions 2-7 of SEQ ID NO:1. Additionally, each member of the miR-17 family shares some nucleobase sequence identity outside of the seed region. Thus, modified oligonucleotides that include a nucleobase sequence complementary to the miR-17 seed sequence may target other microRNAs of the miR-17 family in addition to miR-17. In certain embodiments, the modified oligonucleotide targets two or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotide targets three or more microRNAs of the miR-17 family. In certain embodiments, the modified oligonucleotide targets four or more microRNAs of the miR-17 family. In certain embodiments, modified oligonucleotide targets five or more microRNAs of miR-17 family.In certain embodiments, modified oligonucleotide targets six microRNAs of miR-17 family.For example, modified oligonucleotide with nucleobase sequence 5'-AGCACUUU-3' targets all members of miR-17 family.

[0214] In certain embodiments, a modified oligonucleotide comprises the nucleobase sequence 5'-CACUUU-3'. In certain embodiments, a modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUU-3'.

[0215] In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-GCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at position 6. In certain embodiments, the modified oligonucleotide has the nucleobase sequence 5'-AGCACUUUX-3', where X is a uracil nucleobase, a cytosine nucleobase, or a purine nucleobase, with the proviso that the purine nucleobase does not have a hydrogen bond acceptor at the 6-position.

[0216] In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUUA-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUUU-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACUU-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCACU-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCAC-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-AGCA-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-GCACUUUA-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUUA-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-ACUUUA-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CUUUA-3'. In certain embodiments, a modified oligonucleotide comprises the nucleobase sequence 5'-AAGCACUUUA-3'.

[0217] In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUUT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTUT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACUTT-3'. In certain embodiments, the modified oligonucleotide comprises the nucleobase sequence 5'-CACTTU-3'.

[0218] In certain embodiments, each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. In certain embodiments, at least one cytosine is an unmethylated cytosine. In certain embodiments, each cytosine is an unmethylated cytosine. In certain embodiments, at least one cytosine is a 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.

[0219] In certain embodiments, the number of linked nucleosides of the modified oligonucleotide is shorter than the length of the target microRNA. The modified oligonucleotide has a number of linked nucleosides shorter than the length of the target microRNA, and each nucleobase of the modified oligonucleotide is complementary to the nucleobase at the corresponding position of the target microRNA, and is considered to be a modified oligonucleotide having a nucleobase sequence that is fully complementary (also referred to as 100% complementary) to the region of the target microRNA sequence. For example, the modified oligonucleotide is composed of 9 linked nucleosides, and each nucleobase is complementary to the corresponding position of miR-17, and is fully complementary to miR-17.

[0220] In certain embodiments, the modified oligonucleotide has a nucleobase sequence with one mismatch with respect to the nucleobase sequence of the target microRNA. In certain embodiments, the modified oligonucleotide has a nucleobase sequence with two mismatches with respect to the nucleobase sequence of the target microRNA. In certain such embodiments, the modified oligonucleotide has a nucleobase sequence with no more than two mismatches with respect to the nucleobase sequence of the target microRNA. In certain such embodiments, the mismatched nucleobases are consecutive. In certain such embodiments, the mismatched nucleobases are not consecutive.

[0221] The sequence listing accompanying this application identifies each nucleic acid base sequence as either "RNA" or "DNA", as appropriate, although in practice those sequences may be modified by a combination of chemical modifications as specified herein. Those skilled in the art will readily appreciate that the designations such as "RNA" or "DNA" in the sequence listing to describe modified oligonucleotides are somewhat arbitrary. For example, modified oligonucleotides containing nucleosides containing 2'-O-methoxyethyl sugar moieties and thymine bases may be described as DNA residues in the sequence listing, even though the nucleoside is modified and is not a naturally occurring DNA nucleoside.

[0222] Thus, the nucleic acid sequences provided in the sequence listing are intended to encompass nucleic acids containing any combination of natural or modified RNA and / or DNA, including, but not limited to, such nucleic acids having modified nucleobases. As a further example, and without limitation, a modified oligonucleotide having the nucleobase sequence "ATCGATCG" in the sequence listing, whether modified or unmodified, can be used with an RNA base, such as, but not limited to, one having the sequence "AUCGAUCG", as well as those having some DNA bases and some RNA bases, such as "AUCGATCG", and me C indicates 5-methylcytosine, "AT me The invention encompasses any oligonucleotide containing such compounds, including oligonucleotides with other modified bases such as "AACGAUCG".

[0223] Specific Modifications In certain embodiments, the oligonucleotides provided herein may contain one or more modifications to the nucleobase, sugar, and / or internucleoside linkage, and thus may be modified oligonucleotides. For example, modified nucleobases, sugars, and / or internucleoside linkages may be selected over unmodified forms for desirable properties such as enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases.

[0224] In certain embodiments, a modified oligonucleotide comprises one or more modified nucleosides.

[0225] In certain embodiments, the modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, the sugar-modified nucleoside may further comprise a natural or modified heterocyclic base moiety, and / or may be linked to another nucleoside via a natural or modified internucleoside linkage, and / or may comprise further modifications independent of the sugar modification. In certain embodiments, the sugar-modified nucleoside is a 2'-modified nucleoside, in which the sugar ring is modified at the 2' carbon from natural ribose or 2'-deoxy-ribose.

[0226] In certain embodiments, the 2'-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.

[0227] Nucleosides containing such bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. In certain embodiments, bicyclic nucleosides include, as shown below: (A) α-L-methyleneoxy (4'-CH2-O-2') BNAs, (B) β-D-methyleneoxy (4'-CH2-O-2') BNAs, (C) ethyleneoxy (4'-(CH2)2-O-2') BNAs, (D) aminooxy (4'-CH2-ON(R)-2') BNAs, (E) oxyamino (4'-CH2-N(R)-O-2') BNAs, (F) methyl(methyleneoxy) (4'-CH( (CH3)-O-2') BNAs (also referred to as constrained ethyl or cEt), (G) methylene-thio (4'-CH2-S-2') BNAs, (H) methylene-amino (4'-CH2-N(R)-2') BNAs, (I) methyl carbocyclic (4'-CH2-CH(CH3)-2') BNAs, (J) c-MOE (4'-CH(CH2-OMe)-O-2') BNAs, and (K) propylene carbocyclic (4'-(CH2)3-2') BNAs. [ka] where Bx is a nucleobase moiety and R is independently H, a protecting group, or C1-C 12 It is an alkyl.

[0228] In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from F, OCF3, O-CH3 (also referred to as "2'-OMe"), OCH2CH2OCH3 (also referred to 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.

[0229] In certain embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from F, O-CH3, and OCH2CH2OCH3.

[0230] In certain embodiments, the sugar modified nucleoside is a 4'-thio modified nucleoside. In certain embodiments, the sugar modified nucleoside is a 4'-thio-2'-modified nucleoside. A 4'-thio modified nucleoside has a β-D ribonucleoside with a 4'-O replaced with a 4'-S. A 4'-thio-2'-modified nucleoside is a 4'-thio modified nucleoside with a 2'-OH replaced with a 2' substituent. Suitable 2'-substituents include 2'-OCH3, 2'-OCH2CH2OCH3, and 2'-F.

[0231] In certain embodiments, the modified oligonucleotide comprises one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage comprises a phosphorus atom.

[0232] In certain embodiments, the modified oligonucleotide comprises at least one phosphorothioate internucleoside linkage. In certain embodiments, each internucleoside linkage of the modified oligonucleotide is a phosphorothioate internucleoside linkage.

[0233] In certain embodiments, the modified oligonucleotide comprises one or more modified nucleobases.In certain embodiments, the modified nucleobases are selected from 5-hydroxymethylcytosine, 7-deazaguanine, and 7-deazaadenine.In certain embodiments, the modified nucleobases are selected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.In certain embodiments, the modified nucleobases are 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.

[0234] In certain embodiments, modified nucleobase is a polycyclic heterocycle.In certain embodiments, modified nucleobase is a tricyclic heterocycle.In certain embodiments, modified nucleobase is a phenoxazine derivative.In certain embodiments, phenoxazine can be further modified to form the nucleobase known in the art as G-clamp.

[0235] In certain embodiments, modified oligonucleotides are conjugated to one or more moieties that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety. In certain embodiments, the moiety is a lipid moiety. Additional moieties for conjugation include carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In certain embodiments, the carbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In certain embodiments, the conjugation group is directly attached to the oligonucleotide. In certain embodiments, the conjugate group is attached to the modified oligonucleotide by a linking moiety selected from amino, azido, hydroxyl, carboxylic acid, thiol, unsaturation (e.g., double or triple bond), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, the substituent is selected from hydroxyl, amino, alkoxy, azido, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0236] In certain 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, for example, nuclease stability. Stabilizing groups include cap structures. These end modifications can protect the modified oligonucleotide from exonuclease degradation and aid in intracellular delivery and / or localization. The cap can be at the 5' end (5' cap), or at the 3' end (3' cap), or at both ends. Cap structures include, for example, reverse deoxy abasic caps.

[0237] Certain Pharmaceutical Compositions Provided herein is a pharmaceutical composition comprising a compound or modified oligonucleotide provided herein and a pharma- ceutically acceptable diluent. In certain embodiments, the pharma- ceutically acceptable diluent is an aqueous solution. In certain embodiments, the aqueous solution is saline. As used herein, a pharma- ceutically acceptable diluent is understood to be a sterile diluent. Suitable routes of administration include, but are not limited to, intravenous and subcutaneous administration. In certain embodiments, administration is intravenous administration. In certain embodiments, administration is subcutaneous administration. In certain embodiments, administration is oral administration.

[0238] In certain embodiments, the pharmaceutical composition is administered in the form of a dosage unit, e.g., in certain embodiments, the dosage unit is in the form of a tablet, capsule, or bolus injection.

[0239] In certain embodiments, the pharmaceutical product is a modified oligonucleotide that has been prepared with a suitable diluent, adjusted to pH 7.0-9.0 with an acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified oligonucleotide is then reconstituted with a suitable diluent, e.g., an aqueous solution such as water, or a physiologically compatible buffer such as saline, Hank's solution, or Ringer's solution. The reconstituted product is administered as a subcutaneous injection or intravenous infusion. The lyophilized formulation may be packaged in 2 mL type I clear glass vials (ammonium sulfate treated), stoppered with bromobutyl rubber closures, and sealed with an aluminum overseal.

[0240] In certain embodiments, the pharmaceutical compositions provided herein may further contain other auxiliary ingredients conventionally found in pharmaceutical compositions at their art-established usage levels.Thus, for example, the compositions may contain additional compatible pharma- ceutical active materials, such as, for example, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents.

[0241] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials useful for physically formulating various dosage forms of the compositions provided herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers, including, but not limited to, excipients such as alcohol, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In various embodiments, such materials, when added, should not unduly interfere with the biological activity of the components of the compositions provided herein. The formulations are sterilized and, if necessary, can be mixed with auxiliary agents that do not adversely interact with the oligonucleotide(s) of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, flavorings, and / or aromatic substances. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent, allowing for the preparation of highly concentrated solutions.

[0242] Lipid moieties have been used in nucleic acid therapy in a variety of ways. In one method, nucleic acid is introduced into preformed liposomes or lipoplexes made from a mixture of cationic and neutral lipids. In another method, DNA complexes with mono- or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of pharmaceuticals to muscle tissue.

[0243] In certain embodiments, the pharmaceutical compositions provided herein comprise a polyamine compound or lipid moiety complexed with a nucleic acid. In certain embodiments, such preparations comprise one or more compounds each individually having a structure defined by formula (Z) or a pharma- ceutically acceptable salt thereof, [ka] In the formula, each a and X b is independently for each occurrence, C 1~6 alkylene, n is 0, 1, 2, 3, 4, or 5, each R is independently H, at least n+2 of the R moieties of at least about 80% of the molecules of the compound of formula (Z) in the preparation are not H, m is 1, 2, 3, or 4, and Y is O, NR 2 , or S and R 1 is alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents; R 2is H, alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents, with the proviso that when n is 0, at least n+3 R moieties are not H. Such preparations are described in PCT Publication WO / 2008 / 042973, which is incorporated by reference in its entirety for its disclosure of lipid preparations. Certain additional preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (May 01, 2008), which is incorporated by reference in its entirety for its disclosure of lipid preparations.

[0244] In certain embodiments, the pharmaceutical compositions provided herein are prepared using known techniques, including, but not limited to, mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or tabletting processes.

[0245] In certain embodiments, the pharmaceutical compositions provided herein are solid (e.g., powders, tablets, and / or capsules). In certain such embodiments, solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using ingredients known in the art, including, but not limited to, starches, sugars, diluents, granulating agents, lubricants, binders, and disintegrants.

[0246] In certain embodiments, the pharmaceutical compositions provided herein are formulated as depot preparations. Certain such depot preparations typically have a longer duration of action than non-depot preparations. In certain embodiments, such preparations are administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. In certain embodiments, depot preparations are prepared using suitable polymers or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or as poorly soluble derivatives, e.g., as poorly soluble salts.

[0247] In certain 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 are useful for the preparation of certain pharmaceutical compositions, including pharmaceutical compositions that comprise hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethylsulfoxide, are used.

[0248] In certain 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 particular tissue or cell type. For example, in certain embodiments, the pharmaceutical compositions comprise liposomes coated with tissue-specific antibodies.

[0249] In certain embodiments, the pharmaceutical compositions provided herein comprise sustained release systems. A non-limiting example of such sustained release systems is a semipermeable matrix of solid hydrophobic polymers. In certain embodiments, sustained release systems may release pharmaceutical agents over hours, days, weeks, or months, depending on their chemical nature.

[0250] Certain pharmaceutical compositions for injection are presented in unit dosage form, for example, in ampoules or in multi-dose containers.

[0251] In certain embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a modified oligonucleotide, which in certain embodiments is an amount sufficient to prevent, reduce, or ameliorate a disease symptom, or prolong the survival of the subject being treated.

[0252] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, upon in vivo administration, the prodrug is chemically converted to a biologically, pharma- ceutical, or therapeutically more active form of the oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain cases, the prodrug is more bioavailable (e.g., by oral administration) than the corresponding active form. In certain cases, the prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrug is less water soluble than the corresponding active form. In certain cases, such prodrugs have superior permeability across cell membranes where aqueous solubility impairs mobility. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In certain cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In certain such embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0253] In certain embodiments, prodrugs are produced by modifying a pharma- ceutical active compound so that the active compound is regenerated upon in vivo administration. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, mask side effects or toxicity, improve the taste of a drug, or modify other characteristics or properties of a drug. With knowledge of in vivo pharmacodynamic processes and drug metabolism, a person skilled in the art can design a prodrug of a compound once he knows a pharma- ceutical active compound (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392).

[0254] Additional routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In certain embodiments, intrathecal pharmaceutical agents are administered to achieve local exposure rather than systemic exposure. For example, pharmaceutical compositions can be injected directly into the area where effect is desired (e.g., into the liver).

[0255] Specific Kit Kits are also provided. In some embodiments, the kits include one or more compounds comprising the modified oligonucleotides disclosed herein. In some embodiments, the kits can be used to administer the compounds to a subject.

[0256] In certain embodiments, the kit includes a pharmaceutical composition ready for administration. In certain embodiments, the pharmaceutical composition is present in a vial. A plurality of vials, such as 10, may be present, for example, in a dispensing pack. In some embodiments, the vial is manufactured to be available in a syringe. The kit may also include instructions for using the compound.

[0257] In some embodiments, the kit comprises a pharmaceutical composition that is present in a pre-filled syringe (e.g., a single dose syringe with a 27 gauge, ½ inch needle with a needle guard, etc.) rather than in a vial. A plurality of pre-filled syringes, such as 10, can be present, for example, in a dispensing pack. The kit can also include instructions for administering the compound, including the modified oligonucleotides disclosed herein.

[0258] In some embodiments, the kit comprises a modified oligonucleotide provided herein as a lyophilized formulation and a pharma- ceutically acceptable diluent. In preparation for administration to a subject, the lyophilized formulation is reconstituted in the pharma- ceutically acceptable diluent.

[0259] In some embodiments, in addition to compounds comprising modified oligonucleotides disclosed herein, the kits may further comprise one or more of a syringe, an alcohol swab, a cotton ball, and / or a gauze pad.

[0260] Specific Experimental Model In certain embodiments, 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 for such experimental models to evaluate pharmaceutical agents provided herein.

[0261] Generally, modified oligonucleotide is first tested in cultured cells.Suitable cell types include those related to the cell type that is desired to deliver modified oligonucleotide in vivo.For example, suitable cell types for the study of the method described herein include primary or cultured cells.

[0262] In certain embodiments, the degree to which modified oligonucleotides interfere with the activity of one or more miR-17 family members is evaluated in cultured cells. In certain embodiments, inhibition of microRNA activity can be evaluated by measuring the level of one or more predicted or verified microRNA-regulated transcripts. Inhibition of microRNA activity can result in an increase in miR-17 family member-regulated transcripts and / or proteins encoded by miR-17 family member-regulated transcripts (i.e., miR-17 family member-regulated transcripts are derepressed). Additionally, in certain embodiments, a particular phenotypic outcome can be measured.

[0263] Several animal models are available to those skilled in the art for the study of one or more miR-17 family members in models of human disease. Models of polycystic kidney disease include, but are not limited to, models with mutations and / or deletions in Pkd1 and / or Pkd2, as well as models with mutations in other genes. Non-limiting exemplary models of PKD with mutations and / or deletions in Pkd1 and / or Pkd2 include hypomorphic models, such as models with missense mutations in Pkd1, and models with reduced or unstable expression of Pkd2, inducible conditional knockout models, and conditional knockout models. Non-limiting exemplary models of PKD with mutations in genes other than Pkd1 and Pkd2 include models with mutations in Pkhd1, Nek8, Kif3a, and / or Nphp3. PKD models are reviewed, for example, in 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.

[0264] Specific quantitative assays In certain embodiments, microRNA levels are quantified in cells or tissues in vitro or in vivo. In certain embodiments, the changes in microRNA levels are measured by microarray analysis. In certain embodiments, the changes in microRNA levels are measured by one of several commercially available PCR assays, such as TaqMan® MicroRNA Assay (Applied Biosystems).

[0265] Modulation of microRNA activity by anti-miRs or microRNA mimics can be assessed by microarray profiling of mRNAs. The sequences of mRNAs that are modulated (either increased or decreased) by anti-miRs or microRNA mimics are searched for microRNA seed sequences to compare the modulation of mRNAs that are targets of the microRNA with the modulation of mRNAs that are not targets of the microRNA. In this way, the interaction of the anti-miR with its target microRNA, or the microRNA mimic with its target, can be assessed. In the case of anti-miRs, mRNAs with increased expression levels are screened for mRNA sequences that contain a seed match with the microRNA to which the anti-miR is complementary.

[0266] Modulation of microRNA activity by anti-miR compounds can be assessed by measuring the levels of the messenger RNA targets of the microRNA, either by measuring the levels of the messenger RNA itself or the protein transcribed therefrom. Antisense inhibition of a microRNA generally results in an increase in the levels of the messenger RNA and / or protein of the messenger RNA target of the microRNA, i.e., anti-miR treatment results in derepression of one or more target messenger RNAs. EXAMPLES

[0267] The following examples are presented in order to more fully illustrate some embodiments of the invention, but they should in no way be construed as limiting the broad scope of the invention.

[0268] Those skilled in the art will readily adopt the principles underlying this discovery to design a variety of compounds without departing from the spirit of the present invention.

[0269] Example 1: The role of miR-17 in PKD The miR-17 family members of the miR-17~92 cluster of microRNAs are upregulated in mouse models of PKD. Genetic deletion of the miR-17~92 cluster in mouse models of PKD reduces renal cyst growth, improves renal function, and prolongs survival (Patel et al., PNAS, 2013;110(26): 10765-10770). The miR-17~92 cluster contains six different microRNAs, each with a distinct sequence: miR-17, miR-18a, miR-19a, miR-19-b-1, and miR-92a-1.

[0270] The miR-17~92 cluster contains two microRNAs, miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Each member of this family shares seed sequence identity and 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 found in the miR-106a~363 cluster on the human X chromosome, and miR-93 and miR-106b are found in the miR-106b~25 cluster on human chromosome 7. The sequences of the miR-17 family members are shown in Table 1. [Table 1]

[0271] The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney- and collecting duct-derived cysts, displaces miR-17 from translationally active polysomes, and derepresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in human in vitro ADPKD models and multiple PKD mouse models after subcutaneous administration. A Phase 1 single ascending dose (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a Phase 1 multiple ascending dose (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.

[0272] Nonclinical toxicology studies following the initiation of Phase 1 MAD clinical trials revealed central nervous system (CNS)-related findings at high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or weakness. 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 for possible interactions with RGLS4326. RGLS4326 was found to be an antagonist of AMPA glutamate receptors, with a 50% inhibitory concentration (IC50) of 4.6 μM (14.2 μg / mL) based on ligand binding, and a functional IC50 of 300-600 nM (0.9-1.8 μg / mL) based on patch clamp activity. AMPA receptors are ion channels on excitatory synapses in the CNS that mediate fast excitatory neurotransmission and are therefore critical components of all neuronal networks. Such interactions with AMPA receptors could explain the CNS-mediated findings observed with high doses of RGLS4326 in nonclinical toxicity models.

[0273] Example 2: Screening for anti-miR-17 compounds with reduced AMPA receptor binding RGLS4326 has the following sequence and chemical modification pattern: S G S C M A F C F U F U M U S G S where the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is an S-cEt nucleoside, each cytosine is an unmethylated cytosine, and all linkages are phosphorothioate linkages. Chemical modification and length variants of RGLS4326 were designed and screened to identify compounds that retain the potency and pharmacokinetic profile of RGLS4326 and exhibit reduced binding to AMPA receptors (AMPA-R).

[0274] Against RGLS4326, a library of compounds was designed with various chemical modifications, nucleobase sequences, and lengths. [Table 2]

[0275] The activity of the anti-miR-17 compounds was evaluated by binding to AMPA-Rs present on rat brain synaptic membranes in the presence of increasing concentrations of the anti-miR-17 compounds. 3 Anti-miR-17 compounds with affinity for the AMPA-R were evaluated in a radioligand binding assay measuring the binding of [H]AMPA ligands. 3 H]AMPA ligand binds to [ 3 H] competes with the binding of AMPA ligands.

[0276] 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). 5.0 nM of ligand [ 3 [H]AMPA, 1.0 mM of the non-specific ligand L-glutamate, and uM concentrations of anti-miR compounds were incubated for 90 minutes with synaptic membranes prepared from Wistar rat cerebral cortex. The compounds shown in Table 2 were tested in three experiments. Anti-miRs 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, as they were demonstrated to bind to AMPA-R and inhibit its activity. 3 The amount of [H]AMPA ligand was quantified by radioligand binding and is shown in Tables 3, 4, and 5. As shown by the data, the compounds differ in their ability to inhibit the binding of radiolabeled ligand to AMPA-R. [Table 3] [Table 4] [Table 5]

[0277] To assess the functional antagonism of anti-miR-17 oligonucleotides against AMPA-R, specific oligonucleotides were tested using the manual whole-cell patch clamp technique to record membrane currents as a measure of AMPA-R activity.

[0278] Manual whole-cell patch clamp studies were performed by Metrion Biosciences (Cambridge, UK). Whole-cell voltage clamp experiments were performed at room temperature (18-21 °C) using an EPC10 patch clamp amplifier with Patchmaster software (HEKA Elektronik). Glass patch pipettes were fabricated from borosilicate glass capillaries (Harvard Apparatus) to resistances of 1.4-2.5 MΩ. Membrane currents were recorded using the whole-cell patch clamp technique. ChanTest® GluA1 / GluA4 EZCells were clamped at a holding potential of -80 mV and membrane currents delivered by 10 μM (S)-AMPA were elicited using a VC38 perfusion system (ALA Scientific Instruments). Minimum current amplitude values ​​were measured at each application of 10 μM (S)-AMPA. The fractional change in current amplitude produced by each concentration of compound was calculated compared to the control current (pre-compound) and expressed as a percentage change (% inhibition) for each cell. The compounds tested are shown in Table 6. RGLS4326 was tested in a separate study from all other compounds in Table 6.

[0279] As shown in Table 6, compared to RGLS4326, compounds RG-NG-1015, RG-NG-1016, and RG-NG-1017 demonstrated reduced functional antagonism against AMPA-Rs based on manual whole-cell patch clamp studies in human ChanTest® GluA1 / GluA4 EZ-Cells. [Table 6]

[0280] Example 3: Relationship between nucleobase properties and AMPA-R binding As shown by AMPA-R binding and whole-cell patch clamp studies, the presence of guanosine at the 3' end of the anti-miR-17 oligonucleotide, at a position complementary to the first nucleotide of miR-17, affects the functional antagonism of AMPA-R. Like guanosine, adenosine is a purine, but adenosine did not inhibit AMPA-R. Because guanosine and adenosine are similar with respect to several properties, except for hydrogen bonding, differences in hydrogen bonding at positions 1, 2, and 6 of the purine base were evaluated. The purine nucleobases tested are shown in FIG. 1 and Table 7. In the "Purine Position" column of Table 7, "A" indicates the position of a purine that is a hydrogen acceptor, and "D" indicates the position of a purine that is a hydrogen donor. In the "Purine Position" column of Table 7, "N" indicates the natural position that is neither a hydrogen acceptor nor a donor. Various 2'-sugar moieties on the purine nucleobases were also tested to evaluate the effect of the 2'-sugar moiety chemistry on the ability of the purine nucleobase to inhibit the sugar AMPA-R. [Table 7]

[0281] The compounds were tested in the radioligand binding assay described herein to determine the binding activity of the anti-miR-17 compounds, 3 The ability of the oligonucleotides to bind and compete with the H]AMPA ligand was determined. 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 nucleobase of the oligonucleotide. For example, compounds with guanosine or inosine at the 3'-terminal resulted in inhibition of ligand binding to AMPA-R. Compounds with a 3'-terminal nucleobase with a hydrogen bond acceptor at the purine 6 position, such as RG-NG-1037 and RG-NG-1039, were less likely to inhibit ligand binding to AMPA-R. [Table 8]

[0282] Example 4: Anti-miR-17 compounds with reduced AMPA-R binding and inhibition did not exhibit CNS toxicity in high dose studies. RG-NG-1015, RG-NG-1016, and RG-NG-1017 were tested in a high dose mouse toxicity study. Each compound was tested at a single dose of 2000 mg / kg and at increasing doses (100, 450, and 2000 mg / kg). As shown in Table 9, increasing doses of RG-NG-1001 and RGLS4326 resulted in ataxia, coma, and in the case of RGLS4326, unconsciousness at the highest dose, and no CNS toxicity was observed for RG-NG-1015, RG-NG-1016, or RG-NG-1017. [Table 9]

[0283] Example 5: Maximum Tolerated Dose (MTD) Study and Comparative Dosage Evaluation of Different Compounds Data from the following studies further support that AMPA-R antagonism is responsible for the CNS toxicity and mortality observed in previous toxicity studies of RGLS4326.

[0284] Study 1: Maximum Tolerated Dose (MTD) Study and Comparative Dose Evaluation of RG-NG-1017, RGLS4326, and RG-NG-1001 The compounds (RG-NG-1017, RGLS4326, RG-NG-1001) were evaluated in a pilot maximum tolerated dose (MTD) study (described below). RG-NG-1017, RGLS4326, and RG-NG-1001 were initially evaluated at four dose levels each. RG-NG-1017 was included for evaluation as a non-AMPA-R binding compound compared to RGLS4326 and RG-NG-1001, which bind to AMPA-R. 6-7 week old C57Bl / 6J male mice (Jackson Laboratories) were used in this study. Mice were randomly assigned to treatment groups and blinded to the study. Animals were acclimated for over 5 days and housed on a 12-h light / dark cycle (lights on at 7 am). No more than four mice were housed in each cage in a ventilated cage rack system. Diet consisted of standard rodent chow and water ad libitum.

[0285] MTD Pilot Study The following parameters were used in this study: 1. Route(s) of administration: Intracerebroventricular (ICV) dosing of RG-NG-1017, RG-NG-1001, and RGLS4326 2. Dose volume(s): 4 μL 3. Formulation(s): Vehicle, Ca 2+ and Mg 2+ dPBS free 4.Dosage frequency: once 5. Test period: 8 days 6. Number of groups: 3 7. Number of animals per group: (2-4 per group) 8. Total number of animals: 54

[0286] For ICV administration, mice were anesthetized and positioned for injection. The skin over the skull was incised and a small hole was drilled into the skull over the target using a microdrill. Stereotaxic coordinates were anteroposterior (AP) -0.4mm, mediolateral (ML) + / -1.0-1.5mm, dorsoventral (DV) -3.0mm for injections from the anterior of the skull into both the left and right lateral ventricles (Hironaka et al, 2015). Animals were injected unilaterally with 4 μl into the right lateral ventricle. Compounds were injected over 1-2 minutes and the needle was left in place for 0.5-1 minute before being removed. The incision was closed with sutures, wound clips, or VetBond.

[0287] After ICV treatment (day 0), animals were monitored for 7 days with daily health checks, body weights, and mortality recorded. On day 7, brains and kidneys were collected, fixed (10% formalin) and preserved pending tissue types.

[0288] Results of the MTD study are shown in Table 10 and Figure 3. All animal deaths were reported to occur within the first 5-8 hours after ICV injection. Mice injected with 2.5 μg of RG4326 were reported to show some immediate signs of respiratory distress and were provided with a heating pad. RG-NG-1017 (a non-AMPA-R binding compound) was well tolerated at high doses and there was no MTD established for this compound (0 deaths at 600 μg, 100 μg, or 50 μg; 1 death at 300 μg). For RG4326 and RG-NG-1001 (e.g., 600, 300, 100 μg), 100% mortality was observed at high doses, plus 100% mortality was observed at 50 μg and 25 μg for both AMPA-R binding compounds. The RG-NG-1001 MTD was not reached in this study and was predicted to be below 2.5 μg. The MTD of RG4326 was predicted to be approximately 2.5 < 5.0 μg by ICV. All animals were reported to fully recover by the second day of observation. [Table 10]

[0289] Maximum tolerated dose (MTD) study of RGLS4326 A second MTD study of RGL4326 by ICV was performed to evaluate dose selection for evaluating the compound in disease models (Table 11). A different mouse strain was evaluated in this study (Swiss:Rjorl male mice, 5 weeks old, sourced from Janvier). Mice were placed under isoflurane anesthesia (5% for induction, 2% for maintenance, <100% O2) and given 5 mg / kg sc carprofen (Rimadyl®). They were then placed in a stereotaxic 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 stereotaxically placed in the left lateral ventricle at the following coordinates: +0.5, L±0.7 mm, V=-2.7 mm posterior to the parietal. After a 2-minute delay to allow brain tissue to slide over the cannula, 4 μL of a solution containing 0.625 mg / mL RG4326 was slowly injected over a 2-minute period. After injection, the cannula was left in place for an additional 5 minutes to prevent the solution from flowing back along the cannula track. Mice were administered 5 mg / kg sc carprofen (Rimadyl®) at 24 and 48 hours after surgery. Mice were monitored for 3-7 days after surgery (starting 24 hours after ICV administration) and weighed daily to ensure health. For mice monitored over 7 days, weights were measured on days 1 and 7 after surgery to ensure health. [Table 11]

[0290] In study 1, six mice were injected with 4 μL of a 0.625 mg / mL solution (total of 2.5 μg per ICV; Table 10). At the end of anesthesia, the mice remained lying on one side. They were quiet and scratching for the first few hours after surgery. No toxic effects were observed at 24, 48, or 72 hours in the six mice that were administered. In study 2, four mice were injected with four different doses of RGLS4326 (0.75, 1.0, 1.25, and 1.875 mg / mL, in a volume of 4 μL). One mouse that received the highest dose (1.875 mg / mL, i.e., 7.5 μg / mouse) died approximately 24 hours after ICV injection. All other mice were in good health until the end of the pilot study (7 days after administration).

[0291] The combined results of Studies 1 and 2 demonstrated that RG4326 was generally well tolerated in study subjects, but only at significantly lower doses than RG-NG-1017 (see Figure 3).

[0292] Table 12 summarizes the MTD data for RGLS4326 in the mouse models from Studies 1 and 2. Based on these results from Study 2, RGLS4326 was predicted to have an MTD of approximately 4 μg in the Swiss:Rjorl mouse strain. [Table 12]

[0293] In summary, compounds RG-NG-1017, RGLS4326 and RG-NG-1001 were evaluated across two MTD studies, illustrating significant differences in tolerability between non-AMPA-R binding compounds (RG-NG-1017) and AMPA-R binding compounds (RGLS4326, RG-NG-1001) (see Figure 3). Despite one death at the 300 μg ICV dose, no deaths occurred at the higher tested dose of 600 μg, so the MTD for RG-NG-1017 was not established. Additionally, no effect on mortality was observed at doses of 100 and 50 μg for RG-NG-1017. In comparison, no clear effect on mortality was evident for the AMPA-R binding compounds RGLS4326 and RG-NG-1001, with no animals surviving across the tested dose range of 25 μg to 600 μg. A trend towards improved survival was seen with lower doses of RGLS4326 (10 μg), with 50% survival in RGLS4326-treated animals at 5 μg, and 100% survival at 2.5 μg. Similarly, in the case of RG-NG-1001 (which exhibits stronger AMPA-R binding compared to RGLS4326), 100% mortality was evident at the lower dose of 5 μg, with a trend towards improved survival at 2.5 μg. The RGLS43426 results from Study 1 were further confirmed in a second MTD study (Study 2) utilizing a different mouse strain. In this study, it was found that there may be slight differences in the tolerability of RGLS4326 between strains, with survival observed to only be affected in mice at the highest dose of 7.5 μg versus 5 μg in Study 1 using C57 / Bl / 6J. However, these results still support that the MTD for AMPA-R-binding RGLS4326 occurs at approximately 2.5 μg and between 5-7.5 μg (depending on the strain), compared with the significantly higher MTD (at least >40-fold, or more) for non-AMPA-R-binding RG-NG-1017 (Figure 3).

[0294] Example 6: In vitro and in vivo efficacy of anti-miR-17 compounds The in vitro potency of certain compounds was evaluated using a miR-17 luciferase sensor assay that uses a luciferase reporter vector for miR-17 in tandem with two perfectly complementary miR-17 binding sites in the 3'-UTR of the luciferase gene. HeLa cells were co-transfected with the luciferase reporter vector and an exogenous miR-17 expression vector that acts to suppress the luciferase signal. HeLa cells were then treated individually with anti-miR-17 oligonucleotides at concentrations of 0.045, 0.137, 0.412, 1.23, 3.70, 11.1, 33.3, 100, 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 showed similar EC 50 levels inhibited miR-17 function and derepressed miR-17 luciferase reporter activity. [Table 13]

[0295] As shown in Figure 4, RG-NG-1015 inhibited miR-17, as well as miR-20a, miR-106a, and miR-93 in luciferase assays in HeLa cells, with similar EC20 activity compared to RGLS4326 in vitro. 50 had value.

[0296] RG-NG-1015 also exhibited similar EC 50 We also derepressed luciferase enzymes containing the full-length 3' untranslated regions (UTRs) of miR-17 direct target genes PKD1 and PKD2 with the same expression levels.

[0297] The activity of specific compounds was evaluated using the mouse miR-17 pharmacodynamic signature (miR-17 PD-Sig), which consists of the expression of 18 unique miR-17 target genes normalized by six reference housekeeping genes, providing an unbiased and comprehensive assessment of miR-17 activity. The mouse miR-17 PD-Sig score was the calculated average of the individual log2 fold changes (normalized by six housekeeping genes) of the 18 genes compared to mock transfection (Lee et al., Nat. Commun., 2019, 10, 4148).

[0298] As shown in Table 13, the tested oligonucleotides had similar EC 50 RGLS4326 inhibited miR-17 function and derepressed the expression of multiple direct miR-17 target genes (measured by miR-17 PD signature) in normal and PKD kidney cell lines (both mouse and human) with values. The PD-Sig of RGLS4326 in mIMCD3 cells (77.2, indicated by "*") was not generated in this experiment. The values ​​in Table 14 are those reported by Lee et al., Nat. Commun., 2019, 10, 4148. Blank cells in the table indicate that the compound was not tested in the particular cell line. [Table 14]

[0299] In vivo efficacy was evaluated using microRNA polysome shift assay (miPSA). This assay was used to determine the extent to which compounds directly bind to miR-17 targets in normal and PKD mouse kidneys. miPSA relies on the principle that active miRNAs bind to their mRNA targets in translationally active high molecular weight (HMW) polysomes, whereas inhibited miRNAs reside in low molecular weight (LMW) polysomes. Treatment with anti-miR results in the shift of microRNAs from HMW polysomes to LMW polysomes. Thus, miPSA provides a direct measurement of the engagement of microRNA targets by complementary anti-miRs (Androsavich et al., Nucleic Acids Research, 2015, 44: e13).

[0300] Wild-type mice were administered a single dose of 0.3 mg / kg, 3 mg / kg, or 30 mg / kg. Kidney tissue was collected 7 days later and subjected to miPSA. The mean displacement scores for each treatment are shown in Table 15 (PBS, n=17; RGLS4326 30 mg / kg, n=10; all other treatments, n=4-5). The oligonucleotides tested displaced miR-17 from translationally active polysomes (measured by miPSA) in normal mouse kidneys. [Table 15]

[0301] Furthermore, as shown in Table 16 and Figures 5A-5D, RGLS4326 and RG-NG-1015 have similar pharmacokinetic and target engagement (as measured by miPSA) profiles following a single subcutaneous administration in C57BL6 mice. [Table 16]

[0302] Example 7: Efficacy of RG-NG-1015 in an experimental model of ADPKD The efficacy of RG-NG-1015 was evaluated in the KspCre / Pkd1F / RC (Pkd1-F / RC) mouse model. Pkd1-F / RC is an orthologous ADPKD model that contains a germline hypomorphic Pkd1 mutation (the mouse equivalent of human PKD1-R3277C (RC mutation) on one allele and loxP sites flanking Pkd1 exons 2 and 4 on the other allele). KspCre-mediated recombination was used to delete the floxed Pkd1 exons and generate compound mutant mice with a renal tubule-specific somatic null mutation on one allele and a germline hypomorphic mutation on the other. This is an aggressive but long-lived model of ADPKD (Hajarnis et al., Nat. Commun., 2017, 8, 14395).

[0303] On days 8, 10, 12, and 15 of age, sex-matched 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), or RG-NG-1015 at a dose of 20 mg / kg (n=8), or PBS (n=8). At 18 days of age, mice were sacrificed and kidney weight, body weight, cystic index, serum creatinine level, and blood urea nitrogen (BUN) level were measured. BUN level is a marker of renal function. Higher BUN levels correlate with decreased renal function, so decreased BUN levels are an indicator of decreased renal injury and damage as well as improved function. Statistical significance was calculated by one-way ANOVA with Dunnett's multiple correction.

[0304] The results are shown in Table 17 and Figure 2 (****=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) was significantly lower in Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively, than the mean KW / BW ratio of Pkd1-F / RC mice administered PBS (Figure 2A). The mean BUN level was significantly reduced in Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively, compared to mice treated with PBS (Figure 2B). Compared to mice treated with PBS, mean serum creatinine levels in Pkd1-F / RC mice were decreased in mice treated with RGLS4326 and RG-NG-1015, respectively, but the decrease was not statistically significant (Figure 2C). Treatment with the control oligonucleotide RG5124 did not decrease kidney-to-body weight ratio, serum creatinine, or serum BUN, illustrating that the results observed with RGLS4329 and RG-NG-1015 were specific to inhibition of miR-17. [Table 17]

[0305] The efficacy of RG-NG-1015 was also evaluated in the Pcy / DBA mouse model of PKD alone and in combination with tolvaptan. Pcy / DBA mice exhibit slowly progressive PKD caused by a missense mutation in the Nphp3 gene, which is involved in adolescent nephronophthisis in humans (Takahashi et al., J Am Soc Nephrol 1991, 1:980-989; Olbrich et al., Nat Genet 2003, 34:455-459). In Pcy mice, cysts originate from the distal ducts and entire nephron segments are sporadically occupied by cysts with disease progression by 30 weeks of age, often with the development of ESRD (Nagao et al., Exp Anim 2012, 61:477-488). In particular, male Pcy / DBA mice have been used to characterize the pharmacological profiles of many investigational products for ADPKD treatment, including the first generation anti-miR-17, tolvaptan and RGLS4326 (Aihara et al., J Pharmacol Exp Ther 2014 May;349(2):258-67 and Lee et al., Nat. Commun., 2019,10,4148). Studies in these mice typically involve initiation of treatment at approximately 5 weeks of age and continue until 15-30 weeks of age.

[0306] As outlined in Figures 6A and 6B, five groups of male Pcy / DBA mice (n=13 per treatment group) were treated subcutaneously with PBS or RG-NG-1015 at 25, 5, 1, or 0.2 mg / kg once every two weeks (Q2W). Two groups of male Pcy / DBA mice (n=13 per group) were also treated with RG-NG-1015 at 50 mg / kg once every four weeks (Q4W) or 12.5 mg / kg once a week (QW). Another four groups of male Pcy / DBA mice (n=13 per group) were treated subcutaneously with PBS or RG-NG-1015 at 25, 5, or 1 mg / kg Q2W, in combination with 0.3% (w / w feed) tolvaptan as appropriate. A group of male WT-BDA / 2J mice receiving subcutaneous injections of PBS Q2W were included in the study as a normal range reference. Mice were randomized into treatment groups at 5 weeks of age, treatment was started at 6 weeks of age for 17 weeks, and sacrificed 7 days after the last treatment. Kidney weight, body weight, renal cystic index, and urinary Ngal to creatinine ratio (Ngal / Cr) were measured. Urinary Ngal / Cr is a marker of kidney damage.

[0307] As seen in Figures 6C-6E and Tables 18-20, RG-NG-1015 is effective in the Pcy / DBA mouse model of PKD at various doses and regimens, and also provides additive or synergistic effects when used in combination with tolvaptan. Notably, 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 (Table 18 and Figures 6C-6E). In addition, RG-NG-1015 treatment with similar total doses (a total of 212.5-250 mg per mouse over the study period) but different dosing regimens (including QW, Q2W, and Q4W) reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index at similar levels in Pcy / DBA mice (Table 19; Figures 6C-6E). Treatment with tolvaptan alone reduced mean KW / BW, urinary Ngal / Cr and renal cyst index in Pcy / DBA mice, and the combination of RG-NG-1015 + tolvaptan further reduced mean KW / BW, urinary Ngal / Cr and renal cyst index (Table 20; Figures 6C-6E). The observed effects of drug combinations on KW / BW, urinary Ngal / Cr and renal cyst index were synergistic, primarily additive and less than additive, respectively, as shown by Bliss additivity analysis (Table 20). [Table 18] [Table 19] [Table 20]

[0308] Example 8: Metabolites of RG-NG-1015 In vitro and in vivo studies were performed to investigate the metabolism of RG-NG-1015. For both in vitro and in vivo samples, tissue samples were homogenized in lysis buffer on ice, and RG-NG-1015 and / or metabolites were isolated from plasma, tissue homogenate, or urine via liquid-liquid and solid-phase extraction steps. Calibration samples containing known amounts of RG-NG-1015 were extracted in parallel with the test tissue homogenate, plasma, or urine samples. The molecular weights (MW) of RG-NG-1015 and potential metabolites were calculated from the MS signals and compared to theoretical values.

[0309] The in vitro metabolic stability of RG-NG-1015 was evaluated in mouse, monkey, and human tissues (i.e., kidney and liver lysates) and serum. RG-NG-1015 was incubated 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) for 24 hours at 37° C. RG-NG-1015 and metabolites were then extracted and analyzed by HPLC-TOF.

[0310] In vivo metabolism was assessed in liver and kidney after a single dose of RG-NG-1015 in CD-1 mice, and in plasma, tissues, and urine after single and / or repeated administration to monkeys. CD-1 mice received a single SC dose of 2000 mg / kg RG-NG-1015, and monkeys received SC doses of 15, 75, or 150 mg / kg RG-NG-1015 for up to 5 weeks. RG-NG-1015 and metabolites were then extracted and analyzed by HPLC-TOF.

[0311] RG-NG-1015 undergoes sequential hydrolysis from both the 3' and 5' ends to generate chain shortened metabolites (see Table 21). Nine potential metabolites were identified: 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, as shown in Table 21 below. All metabolites differ from RG-NG-1015 by sequential removal of terminal nucleotides, terminating with hydroxyl groups at the 3' and 5' ends. No 5' terminal shortmers (N-5 to N-8) or 3' terminal shortmers (N-6 to N-8) were observed. [Table 21]

Claims

1. structure: 【Chemistry 1】 A modified oligonucleotide having [a specific characteristic], or a pharmaceutically acceptable salt thereof.

2. The modified oligonucleotide according to claim 1, wherein the pharmaceutically acceptable salt is a sodium salt.

3. structure: 【Chemistry 2】 Modified oligonucleotides having [specific characteristics].

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 physiological saline.

7. A pharmaceutical composition comprising the modified oligonucleotide described in 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 physiological saline.

10. A pharmaceutical composition comprising the modified oligonucleotide described in Claim 3 and a pharmaceutically acceptable diluent.

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

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

13. A lyophilized pharmaceutically active pharmaceutically active oligonucleotide comprising the modified oligonucleotide described in claim 1.

14. A lyophilized pharmaceutically active composition comprising the modified oligonucleotide described in Claim 2.

15. A lyophilized pharmaceutically active composition comprising the modified oligonucleotide described in Claim 3.

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