Modified oligonucleotides for treatment of polycystic kidney disease

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 495,576, filed April 12, 2023, and U.S. Provisional Patent Application No. 63 / 563,881, filed March 11, 2024, which are incorporated herein by reference in their entirety for any purpose. Technical Field

[0003] This article provides a method for treating polycystic kidney disease using compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof.

[0004] sequence list

[0005] This application includes a sequence list submitted electronically in XML format. The XML copy created on March 21, 2024, is named “01138-0046-00PCT.xml” and has a size of 104,766 bytes. The information in the electronic sequence list is incorporated herein by reference in its entirety. Background Technology

[0006] Polycystic kidney disease (PKD) is characterized by the accumulation of numerous fluid-filled cysts in the kidneys. These cysts are lined with a single layer of epithelial cells called cyst epithelium. Over time, the cysts increase in size due to increased cell proliferation and the active secretion of fluid by the cyst epithelium. The enlarged cysts compress surrounding normal tissue, leading to a decline in kidney function. The disease eventually progresses to end-stage renal disease, requiring dialysis or a kidney transplant. At this stage, the cysts may be surrounded by fibrotic areas containing atrophic tubules. PKD can also cause cysts to appear in the liver and other parts of the body.

[0007] Many genetic disorders can lead to PKD. Different forms of PKD are distinguished by their mode of inheritance, such as autosomal dominant or autosomal recessive inheritance; extrarenal organ involvement and phenotype presentation; age of onset of end-stage renal disease, such as at birth, in childhood, or in adulthood; and underlying gene mutations associated with the disease. See, for example, Kurschat et al., 2014, Nature Reviews Nephrology, 10:687-699. Summary of the Invention

[0008] This disclosure relates to a method for treating polycystic kidney disease (PKD), optionally for treating autosomal dominant polycystic kidney disease (ADPKD), comprising administering to a subject in need a therapeutically effective amount of a modified oligonucleotide or a pharmaceutically acceptable salt thereof.

[0009] Implementation Scheme 1. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide or a pharmaceutically acceptable salt thereof to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg.

[0010] The modified oligonucleotide described therein has a 5'-A structure. S G S C M A F C F U F U M U S A S -3',

[0011] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine in the nucleoside is unmethylated cytosine.

[0012] Implementation Scheme 2. The method as described in Implementation Scheme 1, wherein the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a dose of 1 mg / kg, 2 mg / kg or 3 mg / kg.

[0013] Implementation Scheme 3. The method as described in Implementation Scheme 1 or 2, wherein the pharmaceutically acceptable salt is a sodium salt.

[0014] Implementation Scheme 4. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg, wherein the modified oligonucleotide has the following structure:

[0015]

[0016] Or its pharmaceutically acceptable salt.

[0017] Implementation Scheme 5. The method of Implementation Scheme 4, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg or 3 mg / kg.

[0018] Implementation Scheme 6. The method as described in Implementation Scheme 4 or 5, wherein the pharmaceutically acceptable salt is a sodium salt.

[0019] Implementation Scheme 7. The method of any one of Implementation Schemes 1 to 6, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0020] Implementation Scheme 8. The method as described in Implementation Scheme 7, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0021] Implementation Scheme 9. The method as described in Implementation Scheme 8, wherein the sterile aqueous solution is a saline solution.

[0022] Implementation Scheme 10. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg, wherein the modified oligonucleotide has the following structure:

[0023]

[0024] Implementation Scheme 11. The method of Implementation Scheme 10, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg or 3 mg / kg.

[0025] Implementation Scheme 12. The method of implementation scheme 10 or 11, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

[0026] Implementation Scheme 13. The method as described in Implementation Scheme 12, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

[0027] Implementation Scheme 14. The method as described in Implementation Scheme 13, wherein the sterile aqueous solution is a saline solution.

[0028] Implementation Scheme 15. The method of any one of Implementation Schemes 1 to 14, wherein the subject suffers from polycystic kidney disease.

[0029] Implementation Scheme 16. The method of any one of Implementation Schemes 1 to 15, wherein the subject has been diagnosed with polycystic kidney disease using clinical, histopathological and / or genetic criteria.

[0030] Implementation Scheme 17. The method of any one of Implementation Schemes 1 to 16, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

[0031] Implementation Scheme 18. The method as described in Implementation Scheme 17, wherein the subject has a Mayo imaging classification of ADPKD 1C, 1D, or 1E.

[0032] Implementation Scheme 19. The method as described in any one of Implementation Schemes 1 to 18, wherein prior to administration of the modified oligonucleotide, the subject's estimated glomerular filtration rate (eGFR) is between 30 and 90 mL / min / 1.73 m 2 between.

[0033] Implementation Scheme 20. The method of any one of Implementation Schemes 1 to 19, wherein prior to administration of the modified oligonucleotide, the subject is determined to have reduced polycystin-1 (PC1) and / or polycystin-2 (PC2) levels in the subject's kidneys, urine, or blood.

[0034] Implementation Scheme 21. The method of any one of Implementation Schemes 1 to 20, wherein the subject has a mutation selected from a mutation in the PKD1 gene or a mutation in the PKD2 gene.

[0035] Implementation Scheme 22. The method of any one of Implementation Schemes 1 to 21, wherein the subject has an increased total kidney volume.

[0036] Implementation Scheme 23. The method of any one of Implementation Schemes 1 to 22, wherein the subject suffers from hypertension.

[0037] Implementation Scheme 24. The method of any one of Implementation Schemes 1 to 23, wherein the subject has impaired renal function.

[0038] Implementation Scheme 25. The method of any one of Implementation Schemes 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 1 mg / kg.

[0039] Implementation Scheme 26. The method of any one of Implementation Schemes 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 2 mg / kg.

[0040] Implementation Scheme 27. The method of any one of Implementation Schemes 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 3 mg / kg.

[0041] Implementation Scheme 28. The method of any one of Implementation Schemes 1 to 27, wherein the method comprises applying the modified oligonucleotide once every 2 weeks.

[0042] Implementation Scheme 29. The method of any one of Implementation Schemes 1 to 28, wherein the method comprises applying the modified oligonucleotide at least 7 times.

[0043] Implementation Scheme 30. The method of any one of Implementation Schemes 1 to 29, wherein the modified oligonucleotide is administered subcutaneously.

[0044] Implementation Scheme 31. The method of any one of Implementation Schemes 1 to 30, wherein the treatment reduces the total kidney volume of the subject.

[0045] Implementation Scheme 32. The method of any one of Implementation Schemes 1 to 31, wherein the treatment slows the rate of increase in the total kidney volume of the subject.

[0046] Implementation Scheme 33. The method as described in Implementation Scheme 31 or 32, wherein the total kidney volume is the total kidney volume adjusted for height (htTKV).

[0047] Implementation Scheme 34. The method of any one of Implementation Schemes 1 to 33, wherein the treatment slows the rate of decline in the glomerular filtration rate of the subject.

[0048] Implementation Scheme 35. The method of any one of Implementation Schemes 1 to 34, wherein the treatment increases the glomerular filtration rate of the subject.

[0049] Implementation Scheme 36. The method as described in Implementation Scheme 34 or 35, wherein the glomerular filtration rate is an estimated glomerular filtration rate.

[0050] Implementation Scheme 37. The method of any one of Implementation Schemes 1 to 36, wherein the treatment inhibits or slows the increase in cyst growth in the kidneys and / or liver of the subject.

[0051] Implementation Scheme 38. The method of Implementation Scheme 37, wherein the treatment inhibits or slows the increase in total cyst volume, number and / or size distribution.

[0052] Implementation Scheme 39. The method as described in any one of Implementation Schemes 1 to 38, wherein the treatment:

[0053] a) Improve or slow the rate of decrease in the subject's creatinine clearance;

[0054] b) Reduce or slow the rate of increase in the albumin:creatinine ratio of the subject;

[0055] c) Reduce or slow the rate of increase in the subject's blood urinary nitrogen (BUN) level;

[0056] d) Reduce or slow the rate of increase in the subject's serum creatinine (SCr) level;

[0057] e) Increase polycystic protein-1 (PC1) in the urine of the subjects;

[0058] f) Increase polycystic protein-2 (PC2) in the urine of the subjects;

[0059] g) Reduce or slow the rate of increase of neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subjects; and / or

[0060] h) Reduce or slow the rate of increase of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

[0061] Implementation Scheme 40. The method as described in any one of Implementation Schemes 1 to 39, wherein the application:

[0062] a) Reduce or slow the rate of increase of monocyte chemoattractant protein-1 (MCP-1) in the urine of the subjects;

[0063] b) Reduce or slow the rate of increase of β-2 microglobulin (B2M) in the urine of the subjects;

[0064] c) Reduce or slow the rate of increase of complement cleavage products C3a and / or Bb in the plasma of the subject;

[0065] d) Reduce or slow the rate of increase in serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the subjects;

[0066] e) Reduce or slow the rate of increase of serum and pro-AVP in the subjects;

[0067] f) Reduce or slow the rate of increase in serum N-acetyl-1-methylhistidine in the subjects; and / or

[0068] g) Reduce or slow the rate of increase in acute-phase proteins in the subject.

[0069] Implementation Scheme 41. The method of any one of Implementation Schemes 1 to 40, wherein the treatment results in little or no CNS damage in the subject.

[0070] Implementation Scheme 42. The method of Implementation Scheme 41, wherein the treatment results in little or no change in the subject's ataxia assessment and rating scale (SARA) test score.

[0071] Implementation Scheme 43. The method as described in any one of Implementation Schemes 1 to 42, comprising:

[0072] a) Measure the height-adjusted total kidney volume (HtTKV) of the subject;

[0073] b) Measure polycystic protein-1 (PC1) in the urine of the subjects;

[0074] c) Measure polycystic protein-2 (PC2) in the urine of the subjects;

[0075] d) Measure the blood urea nitrogen (BUN) level of the subjects;

[0076] e) Measure the serum creatinine (SCr) level of the subjects;

[0077] f) Measure the creatinine clearance rate of the subjects;

[0078] g) Measure the urine albumin:creatinine ratio (UACR) of the subject;

[0079] h) Measure the estimated glomerular filtration rate (eGFR) of the subject;

[0080] i) Measure the level of neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subjects;

[0081] j) Measure the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects;

[0082] k) Measure the level of monocyte chemoattractant protein-1 (MCP-1) in the urine of the subjects;

[0083] l) Measure β-2 microglobulin (B2M) in the urine of the subjects;

[0084] m) Measure the serum insulin-like growth factor-binding protein acid unstable subunit (IGFALS) of the subjects;

[0085] n) Measure the serum and peptide (CT-proAVP) of the subjects;

[0086] o) Measure the serum N-acetyl-1-methylhistidine of the subject;

[0087] p) Measure the complement cleavage products C3a and / or Bb in the plasma of the subjects; and / or

[0088] q) Measure the total cyst volume, number, and / or size distribution of the subjects; and / or

[0089] r) Measure the subject's SARA test score.

[0090] Implementation Scheme 44. The method of any one of Implementation Schemes 1 to 43, wherein the subject is a human subject.

[0091] Implementation Scheme 45. The method of any one of Implementation Schemes 1 to 44, having acceptable safety and tolerability characteristics.

[0092] Implementation Scheme 46. A modified oligonucleotide or a pharmaceutically acceptable salt thereof for use in the treatment of polycystic kidney disease, wherein said modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3',

[0093] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine in the nucleoside is unmethylated cytosine.

[0094] The modified oligonucleotide is administered at doses of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg.

[0095] Implementation Scheme 47. A modified oligonucleotide for use as described in Implementation Scheme 46, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg or 3 mg / kg.

[0096] Implementation Scheme 48. A modified oligonucleotide for use as described in Implementation Scheme 46 or 47, wherein the pharmaceutically acceptable salt is a sodium salt.

[0097] Implementation Scheme 49. A modified oligonucleotide for use as described in any one of Implementation Schemes 46 to 48, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

[0098] Implementation Scheme 50. A modified oligonucleotide for use as described in any one of Implementation Schemes 46 to 49, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

[0099] Implementation Scheme 51. A modified oligonucleotide for use as described in any one of Implementation Schemes 46 to 50, wherein the modified oligonucleotide is applied once every two weeks.

[0100] Implementation Scheme 52. A modified oligonucleotide for use as described in any one of Implementation Schemes 46 to 51, wherein the modified oligonucleotide is applied at least seven times.

[0101] Implementation Scheme 53. Use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of polycystic kidney disease, wherein said modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3',

[0102] The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine in the nucleoside is unmethylated cytosine.

[0103] The modified oligonucleotides are formulated for administration at doses of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg.

[0104] Implementation Scheme 54. Use as described in Implementation Scheme 53, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

[0105] Implementation Scheme 55. Use as described in Implementation Scheme 53 or 54, wherein the pharmaceutically acceptable salt is a sodium salt.

[0106] Implementation Scheme 56. Use as described in any one of Implementation Schemes 53 to 55, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

[0107] Implementation Scheme 57. The use as described in any one of Implementation Schemes 53 to 56, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

[0108] Implementation Scheme 58. The use as described in any one of Implementation Schemes 53 to 57, wherein the modified oligonucleotide is applied at least once every two weeks.

[0109] Implementation Scheme 59. The use as described in any one of Implementation Schemes 53 to 58, wherein the modified oligonucleotide is applied at least seven times. Attached Figure Description

[0110] Figure 1 Purine nucleobase structure.

[0111] Figures 2A to 2C Efficacy of .RG-NG-1015 in the Pkd1-F / RC model of PKD. Effects of treatment on (2A) kidney-to-body weight ratio, (2B) blood urea nitrogen (BUN) level and (2C) serum creatinine level.

[0112] Figure 3 Maximum tolerated dose (MTD) studies and comparative dose assessments of RG-NG-1001, RGLS4326, and RG-NG-1017. Six- to seven-week-old male C57BL / 6J mice were administered RG-NG-1001 and RGLS4326 (an anti-miR-17 oligomer that inhibits AMPA-R) and RG-NG-1017 (an anti-miR-17 oligomer that does not inhibit AMPA-R; RG-NG-1017) via a single intraventricular (ICV) injection at different dose levels and monitored for seven days. Mice mortality was indicated for different doses of the three different compounds.

[0113] Figures 4A to 4F This study describes the in vitro evaluation of the activities of RG-NG-1015 and RGLS4326 on miR-17 (4A), miR-20a (4B), miR-93 (4C), and miR106(a)(4D) luciferase sensors in HeLa cells. The study also describes the evaluation of the activities of RG-NG-1015 and RGLS4326 on luciferase sensors containing the full-length 3' untranslated region (UTR) of the miR-17 direct target genes PKD1 (4E) and PKD2 (4F).

[0114] Figures 5A to 5D The pharmacokinetics and target binding of RGLS4326 and RG-NG-1015 were measured in C57BL6 mice after a single subcutaneous administration (as measured by miPSA). Plasma concentrations (5A), tissue concentrations (5B), renal target binding (5C), and hepatic target binding (5D) are shown.

[0115] Figures 6A to 6E The effects of RG-NG-1015 at different doses and regimens, and in combination with tolvaptan, on a Pcy / DBA mouse model of PKD were measured. The dosing schedule is shown in [date / time]. Figure 6A In the middle, and Figures 6C to 6EThe key to the chart in the middle is shown in Figure 6B The figures show kidney weight / body weight (6C), cyst area (%) (6D), and urinary Ngal / Cr (6E). Error bars represent standard deviations. Compared with the Pcy mediator treatment group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, (ns)p>0.05; univariate ANOVA Bonferroni multiple comparison test. Compared with the tolvaptan alone treatment group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001, (ns)p>0.05; univariate ANOVA Sadik multiple comparison test. Compared with the dose-matched RG-NG-1015 treatment alone, $p<0.05, $$p<0.01, $$p<0.001, $$$p<0.001, (ns)p>0.05; univariate ANOVA Sadik multiple comparison test.

[0116] Figures 7A to 7D Urinary polycystic protein-1 (PC1) was measured in healthy patients and patients with chronic kidney disease and ADPKD. Figure 7A ) and polycystin-2 (PC2; Figure 7C ) levels (shown as PC1 / CD133 or PC2 / CD133 ratios). HV: healthy volunteers; CKD: chronic kidney disease, including T1D (type 1 diabetes), T2D (type 2 diabetes), AKF (acute renal failure), HT (hypertension), and COPD patient samples of CKD stages 2–4 (chronic obstructive pulmonary disease); ADPKD: autosomal dominant polycystic kidney disease, where the Mayo classification was based on htTKV and age; CD133: Prominin-1 has been shown to colocalize with PC1 and PC2 on urinary exosome-like vesicles in ADPKD patient samples (Hogan et al., J Am Soc Nephrol. 2009 Feb; 20(2):278–288). *p-values, using Dunnett-corrected univariate ANOVA compared to HV. Urinary PC1 () levels were measured at baseline with placebo and 1 mg / kg and 2 mg / kg RG-NG-1015 (RGLS8429). Figure 7B ) and PC2 ( Figure 7D ) Level (displayed as PC1 / CD133 or PC2 / CD133 ratio) and average D85-D113 value.

[0117] Figures 8A to 8C The concentration of RG-NG-1015 (RGLS8429) at 2 mg / kg was measured. Figure 8A ), 1 mg / kg RG-NG-1015 ( Figure 8B ) and placebo ( Figure 8C The absolute change of the urinary PC1 / CD133 ratio relative to the individual baseline.

[0118] Figures 9A to 9C Measure 2 mg / kg RG-NG-1015 ( Figure 9A ), 1 mg / kg RG-NG-1015 ( Figure 9B ) and placebo ( Figure 9C The absolute change of the urinary PC2 / CD133 ratio relative to the individual baseline.

[0119] Figures 10A to 10B Urinary PC1 / CD133 levels were compared between 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. Figure 10A ) and PC2 / CD133 ( Figure 10B Exploratory regression analysis was performed on the absolute change in PC1 / CD133 ratios relative to baseline. Solid circles represent RG-NG-1015 treatment (1 mg / kg Q2W x7), and solid squares represent RG-NG-1015 treatment (2 mg / kg Q2W x7). Hollow circles represent placebo (cohort 1+2). For RG-NG-1015 (1 mg / kg): #, Change in PC1 relative to baseline; statistical significance based on the PC1 / CD133 ratio on days 85 and 86, using the Wilcoxon paired signed-rank test. #, Change in PC2 relative to baseline; statistical significance based on the PC2 / CD133 ratio on day 113, using the Wilcoxon paired signed-rank test. For RG-NG-1015 (2 mg / kg): #, Change in PC1 relative to baseline; statistical significance based on the PC1 / CD133 ratio on days 57, 86, 99, and 113, using the Wilcoxon paired signed-rank test. # Change in PC2 relative to baseline; statistical significance based on the PC2 / CD133 ratio on day 57 using the Wilcoxon paired signed-rank test. Exploratory regression analysis was performed using nonlinear regression (second-order polynomial). One subject with an absolute increase in PC1 / CD133 = 3.85 and PC2 / CD133 = 0.033 on day 113 was excluded for curve fitting purposes.

[0120] Figures 11A to 11B Measure urinary PC1 / CD133 levels at 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. Figure 11A ) and PC2 / CD133 ( Figure 11BThe percentage change in the polycystic protein ratio relative to baseline is shown in the group mean plot. Urinary polycystic protein levels were measured before, during, and 28 days after the last (and 7th) dose of RG-NG-1015 administered every two weeks at doses of 1 mg / kg and 2 mg / kg over 113 days. Urinary PC1 levels are shown in the figure. Figure 11A In the middle, and the urinary PC2 level was shown in Figure 11B In the diagram, solid circles represent RG-NG-1015 treatment (1 mg / kg Q2W x7) (total N = 9 subjects, Mayo classification 1C / 1D / 1E = 5 / 3 / 1). Hollow circles represent placebo cohort 1+2 (Q2W x7) (total N = 6 subjects, Mayo classification 1C / 1D / 1E = 1 / 4 / 1). Solid squares represent RG-NG-1015 treatment (2 mg / kg Q2W x7) (total N = 11 subjects, Mayo classification 1C / 1D / 1E = 5 / 4 / 1).

[0121] Figures 12A to 12B The mean change in polycystic ovary syndrome (PCOS) levels from baseline after 3 months of Q2W administration of 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo is shown (mean of all available measurements between day 85 and day 116). Figure 12A The absolute change in the urinary PC1 / CD133 ratio was shown, and Figure 12B The absolute change in the urinary PC2 / CD133 ratio is shown.

[0122] Figures 13A to 13B The mean change in polycystic ovary syndrome (PCOS) levels from baseline after 3 months of Q2W administration of 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo is shown (mean of all available measurements between day 85 and day 116). Figure 13A The change in the urinary PC1 / CD133 ratio was shown in %, and Figure 13B The percentage change in the urinary PC2 / CD133 ratio was shown.

[0123] Figures 14A to 14B The correlation between polycystic protein levels and pharmacokinetic (PK) parameters was shown. Urinary PC1 and single-dose C max The correlation between them is shown in Figure 14A In the middle. Urinary PC1 and single-dose AUC last The correlation between them is shown in Figure 14B middle.

[0124] Figures 15A to 15CMeasure the height-adjusted total kidney volume (htTKV) at the end of the study compared to baseline for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. Figure 15A ) and total renal cyst volume (TKCV) Figure 15B The change in TKCV was also measured. The correlation between the percentage change in TKCV and the percentage change in htTKV was also measured. Figure 15C ).

[0125] Figures 16A to 16C Measure the total liver volume (TLV) at the end of the study compared to baseline for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. Figure 16A ) and total liver cyst volume (TLCV) Figure 16B The change in TLCV was also measured. The correlation between the absolute change in TLCV and the percentage change in TLV was also measured. Figure 16C ).

[0126] Figures 17A to 17D Measure the change in PC1 (shown as the absolute change in PC1 / CD133, averaged relative to the baseline). D85-D113 The changes in htTKV () Figure 17A Exploratory correlations between ) and changes in PC1 (shown as absolute changes in PC1 / CD133, average) D85-D113 Value) and changes in eGFR ( Figure 17B Exploratory correlation between PC1 and CD133. The change in PC2 was measured (shown as the absolute change in PC1 / CD133, relative to the baseline). D85-D113 The changes in htTKV () Figure 17C Exploratory correlations between ) and changes in PC2 (shown as absolute changes in PC1 / CD133, average) D85-D113 Value) and changes in eGFR ( Figure 17D Exploratory correlations between ).

[0127] Figures 18A to 18C .RG-NG-1015 inhibits miR-17 and delivers efficacy in a dose-responsive manner. Figure 18A The image shows Pkd1 derived from RG-NG-1015 (RGLS8429), PBS, or 20 mg / kg control oligonucleotides administered by injection at various doses. F / RC Cross-section of mouse kidneys. * shows cross-section of kidneys from age-matched wild-type C57BL6 mice (Lakhia et al. 2022 Nat Commun. 2022 Aug 15; 13(1):4765) for reference only. $, Pkd1 in this treatment group F / RCMice were administered the drug only on days (P) 8 and 12 after birth. All other Pkd1 in the study... F / RC Mice were administered the drug at P8, 10, 12, and 15. Various Pkd1 values ​​were observed for RG-NG-1015 (RGLS8429). F / RC Mouse kidney concentrations, calculated as kidney weight / body weight inhibition % (KW / BW), are shown in [data missing]. Figure 18B In the mean, the calculated miR-17 inhibition % for various WT-C57BL6 mouse kidney concentrations of RGLS-4326 and RG-NG-1015 (RGLS8429) is shown in the table. Figure 18C middle. Detailed Implementation

[0128] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless specifically defined, the nomenclature, processes, and techniques used in conjunction with the analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well-known and commonly used in the art. Where a term has multiple definitions herein, the definition in this section shall prevail. Standard techniques are applicable to chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of subjects. Some such techniques and processes can be found, for example, in “Carbohydrate Modifications in Antisense Research”, edited by Sanghvi and Cook, American Chemical Society, Washington DC, 1994; and “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18th edition, 1990; and are incorporated herein by reference for any purpose. Where permitted, unless otherwise stated, all patents, patent applications, published applications and publications, GENBANK sequences, websites, and other published materials mentioned herein are incorporated herein by reference in their entirety. When referencing URLs or other such identifiers or addresses, it should be understood that such identifiers can change, and specific information on the Internet can change, but equivalent information can be found by searching the Internet. Such citations demonstrate the availability and public dissemination of such information.

[0129] Before disclosing and describing the compositions and methods of the present invention, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, unless the context clearly specifies otherwise, the singular forms “a / an” and “described” as used in the specification and appended claims include plural indicators.

[0130] definition

[0131] Polycystic kidney disease (PKD) is a cystic kidney disease characterized by the accumulation of numerous fluid-filled cysts in the kidneys. Multiple cysts form in at least one kidney, often leading to enlargement of the affected kidney and progressive loss of kidney function.

[0132] "Markers of polycystic kidney disease" refers to medical parameters used to assess the severity of polycystic kidney disease, kidney function, and / or the response of a subject with polycystic kidney disease to treatment. Non-limiting examples of markers for polycystic kidney disease include total kidney volume, hypertension, glomerular filtration rate, and kidney pain.

[0133] "Markers of renal function" refers to medical parameters used to assess the renal function of a subject. Non-limiting examples of markers of renal function include glomerular filtration rate, blood urea nitrogen level, and serum creatinine level.

[0134] Autosomal dominant polycystic kidney disease (ADPKD) is a polycystic kidney disease caused by mutations in one or more of the PKD1 and / or PKD2 genes. 85% of ADPKD cases are caused by mutations in the PKD1 gene located on chromosome 16, while the vast majority of other ADPKD cases are caused by mutations in the PKD2 gene located on chromosome 4.

[0135] Autosomal recessive polycystic kidney disease (ARPKD) is a polycystic kidney disease caused by mutations in one or more genes in the PKHD1 gene located on chromosome 6. Up to 50% of newborns with ARPKD die from complications of intrauterine kidney disease, and about one-third of survivors develop end-stage renal disease (ESRD) within 10 years.

[0136] Nephronophthisis, or NPHP, refers to an autosomal recessive cystic kidney disease characterized by corticomedullary cysts, rupture of the tubular basement membrane, and tubulointerstitial nephropathy.

[0137] "Total kidney volume" or "TKV" is a measure of total kidney volume. Total kidney volume can be determined by magnetic resonance imaging (MRI), computed tomography (CT) scans, or ultrasound (US) imaging, and the volume is calculated using standard methods such as the ellipsoidal volume equation (for ultrasound) or by quantitative stereology or boundary tracing (for CT / MRI).

[0138] "Height-adjusted total kidney volume" or "HtTKV" is a measure of the total kidney volume per unit of height. Patients with an HtTKV value ≥600 ml / m² are predicted to develop stage 3 chronic kidney disease within 8 years.

[0139] "Kidney pain" refers to clinically significant kidney pain that requires sick leave, pharmacological treatment (anesthetics or last resort analgesics) or invasive intervention.

[0140] "Worsening hypertension" refers to changes in blood pressure that require the initiation or increase of hypertension treatment.

[0141] "Fibrosis" refers to the formation or development of excessive fibrous connective tissue in an organ or tissue. In some embodiments, fibrosis occurs as a reparative or reactive process. In some embodiments, fibrosis occurs in response to damage or injury. The term "fibrosis" should be understood as the formation or development of excessive fibrous connective tissue in an organ or tissue as a reparative or reactive process, as opposed to the formation of fibrous tissue as a normal component of an organ or tissue.

[0142] "Hematuria" means the presence of red blood cells in the urine.

[0143] Albuminuria refers to the presence of excessive albumin in the urine, and includes, but is not limited to, normoalbuminuria, hypernoralbuminuria, microalbuminuria, and macroalbuminuria. Normally, the glomerular filtration permeability barrier, composed of podocytes, the glomerular basement membrane, and endothelial cells, prevents serum proteins from leaking into the urine. Albuminuria may reflect damage to this barrier. Albuminuria can be calculated from 24-hour urine samples, overnight urine samples, or spot urine samples.

[0144] "Hypernormal albuminuria" refers to elevated albuminuria, characterized by (i) excretion of 15 mg to <30 mg of albumin in the urine every 24 hours and / or (ii) an albumin / creatinine ratio of 1.25 mg / mmol to <2.5 mg / mmol (or 10 mg / g to <20 mg / g) in men and 1.75 mg / mmol to <3.5 mg / mmol (or 15 mg / g to <30 mg / g) in women.

[0145] “Microalbuminuria” refers to elevated albuminuria, characterized by (i) the excretion of 30 mg to 300 mg of albumin in the urine every 24 hours and / or (ii) an albumin / creatinine ratio of 2.5 mg / mmol to <25 mg / mmol (or 20 mg / g to <200 mg / g) in men and 3.5 mg / mmol to <35 mg / mmol (or 30 mg / g to <300 mg / g) in women.

[0146] "Major albuminuria" refers to elevated albuminuria, characterized by the excretion of more than 300 mg of albumin into the urine every 24 hours and / or (ii) an albumin / creatinine ratio of >25 mg / mmol (or >200 mg / g) in men or >35 mg / mmol (or >300 mg / g) in women.

[0147] The "albumin / creatinine ratio" refers to the ratio of urinary albumin (mg / dL) to urinary creatinine (g / dL), expressed in mg / g. In some implementations, the albumin / creatinine ratio can be calculated from a spot urine sample and can be used as an estimate of albumin excretion over a 24-hour period.

[0148] "Glomerular filtration rate" or "GFR" refers to the rate at which filtered fluid passes through the kidneys and is used as an indicator of kidney function in a subject. In some embodiments, a subject's GFR is determined by calculating an estimated glomerular filtration rate. In some embodiments, the inulin method is used to directly measure a subject's GFR.

[0149] "Estimated glomerular filtration rate" or "eGFR" refers to a measure of how well the kidneys filter creatinine and is used to estimate glomerular filtration rate. Because direct measurement of GFR is complex, eGFR is frequently used in clinical practice. Normal results are typically between 90-120 mL / min / 1.73 ml. 2 Within the specified range. A rate below 60 mL / min / 1.73 m for 3 months or longer is not permitted. 2 Levels of this substance may be an indicator of chronic kidney disease. A level below 15 mL / min / 1.73 mcg may indicate this. 2 The level of [something] may be an indicator of kidney failure.

[0150] “Proteinuria” refers to the presence of excessive serum proteins in the urine. Proteinuria is characterized by the excretion of >250 mg of protein in the urine every 24 hours and / or a protein-to-creatinine ratio ≥0.20 mg / mg. Elevated serum proteins associated with proteinuria include, but are not limited to, albumin.

[0151] "Blood urea nitrogen level," or "BUN level," refers to a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea as a waste product of protein digestion during the urea cycle, and urea is removed from the blood by the kidneys. Normal adult blood contains 7 to 21 mg of urea nitrogen per 100 ml (7 mg / dL to 21 mg / dL). Blood urea nitrogen levels are used as an indicator of kidney health. If the kidneys are unable to properly remove urea from the blood, the subject's BUN level will be elevated.

[0152] "Elevation" refers to an increase in a medical parameter that is considered clinically relevant. Healthcare professionals can determine whether an increase is clinically significant.

[0153] "End-stage renal disease (ESRD)" refers to complete or near-complete failure of kidney function.

[0154] "Quality of life" refers to the degree to which a subject's physical, psychological, and social functioning is impaired due to the disease and / or its treatment. Subjects with polycystic kidney disease may experience a reduced quality of life.

[0155] "Impaired kidney function" refers to a decrease in kidney function relative to normal kidney function.

[0156] "Slowing down the progression of..." and "slowing down the progression of..." both refer to reducing the rate at which medical symptoms develop into advanced stages.

[0157] "Delayed dialysis time" means maintaining sufficient kidney function to delay the need for dialysis treatment.

[0158] "Delay time for kidney transplantation" refers to maintaining sufficient kidney function to delay the need for a kidney transplant.

[0159] "Improving life expectancy" means extending the lifespan of a subject by treating one or more symptoms of a disease in the subject.

[0160] "Subject" refers to a human or non-human animal selected for treatment or therapy.

[0161] "Subjects in need" refers to subjects identified as requiring therapy or treatment.

[0162] "Subjects suspected of having..." refers to subjects who exhibit one or more clinical indicators of a disease.

[0163] "MiR-17-related diseases" refers to diseases or conditions regulated by the activity of one or more members of the miR-17 family.

[0164] "Administration" means providing a medicine or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0165] "Parenteral administration" refers to administration via injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

[0166] "Subcutaneous application" means application that is placed directly under the skin.

[0167] "Intravenous administration" means administration through a vein.

[0168] "Concomitant administration" refers to the combined administration of two or more drugs in any manner, where the pharmacological effects of both drugs are simultaneously observed in the patient. Concomitant administration does not require the administration of the two drugs in a single drug composition, in the same dosage form, or through the same route of administration. The effects of the two drugs do not need to appear at the same time. The effects only need to overlap for a period of time, rather than extending together.

[0169] "Duration" refers to the period of time during which the activity or event continues. In some embodiments, the duration of treatment is the period of time during which a dose of the agent or pharmaceutical composition is administered.

[0170] "Therapy" refers to a treatment for a disease. In some implementations, treatment includes, but is not limited to, administering one or more medications to a subject suffering from a disease.

[0171] "Treatment" refers to a specific process of applying one or more indicators to improve at least one of the disease. In some embodiments, the specific process is the administration of one or more agents. In some embodiments, treatment of PKD includes, but is not limited to, reducing total kidney volume, improving renal function, lowering blood pressure, and / or relieving kidney pain.

[0172] "Improvement" means reducing the severity of at least one indicator of a symptom or disease. In some embodiments, improvement includes delaying or slowing the progression of one or more indicators of a symptom or disease. The severity of the indicator can be determined by subjective or objective measurements known to those skilled in the art.

[0173] "At risk of developing..." means a state in which a subject is predisposed to developing a symptom or disease. In some embodiments, a subject at risk of developing a symptom or disease exhibits one or more symptoms of that symptom or disease, but not a sufficient number of symptoms to be diagnosed with that symptom or disease. In some embodiments, a subject at risk of developing a symptom or disease exhibits one or more symptoms of that symptom or disease, but to a lesser extent requires a diagnosis of that symptom or disease.

[0174] "Prevention of the onset of..." means preventing the development of a disease or symptom in a subject who is at risk of developing such a disease or symptom. In some implementations, a subject at risk of developing a disease or symptom receives treatment similar to that received by a subject who already has the disease or symptom.

[0175] "Delaying the onset of..." means delaying the development of a disease or symptom in a subject who is at risk of developing such a disease or symptom. In some implementations, a subject at risk of developing a disease or symptom receives treatment similar to that received by a subject who already has the disease or symptom.

[0176] "Dosage" refers to the specified amount of a drug agent provided in a single administration. In some embodiments, the dosage may be administered in two or more bolus injections, tablets, or injections. For example, in some embodiments, when subcutaneous administration is required, the desired dosage necessitates a volume that is not easily contained in a single injection. In such embodiments, the desired dosage may be achieved using two or more injections. In some embodiments, the dosage may be administered in two or more injections to minimize injection site reactions in an individual. In some embodiments, the dosage is administered as a slow infusion.

[0177] "Dosage unit" refers to the form in which the drug is delivered. In some embodiments, the dosage unit is a vial containing lyophilized oligonucleotides. In some embodiments, the dosage unit is a vial containing reconstituted oligonucleotides.

[0178] "Therapeutic effective dose" refers to the amount of a drug that provides therapeutic benefit to an animal.

[0179] "Pharmaceutical composition" means a mixture of substances suitable for individual administration, including pharmaceutical preparations. For example, a pharmaceutical composition may contain a sterile aqueous solution.

[0180] "Pharmaceutical" means a substance that provides a therapeutic effect when administered to a subject.

[0181] "Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that provides the desired effect.

[0182] "Pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of the compounds provided herein, i.e., a salt that retains the desired biological activity of the compound when administered to a subject and does not have undesirable toxicological effects. Non-limiting exemplary pharmaceutically acceptable salts of the compounds provided herein include sodium and potassium salt forms. Unless otherwise specified, the terms "compound," "oligonucleotide," and "modified oligonucleotide" as used herein include their pharmaceutically acceptable salts.

[0183] "Salt solution" refers to a solution of sodium chloride in water.

[0184] "Improved organ function" refers to a change in organ function toward the normal limit. In some implementations, organ function is assessed by measuring molecules found in the subject's blood or urine. For example, in some implementations, improved kidney function is measured by a decrease in blood urea nitrogen levels, a reduction in proteinuria, a reduction in albuminuria, etc.

[0185] "Acceptable safety profile" refers to a pattern of side effects that is within clinically acceptable limits.

[0186] "Side effects" refers to physiological responses attributable to treatment that are in addition to the desired effect. In some implementations, side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. Such side effects can be detected directly or indirectly. For example, increased serum transaminase levels may indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin may indicate hepatotoxicity or abnormal liver function.

[0187] As used in this article, the term "blood" encompasses whole blood and blood fractions such as serum and plasma.

[0188] "Anti-miR" refers to an oligonucleotide with a nucleobase sequence complementary to that of microRNA. In some implementations, anti-miR is a modified oligonucleotide.

[0189] "Anti-miR-17" refers to an oligonucleotide modified with a nucleobase sequence complementary to one or more miR-17 family members. In some embodiments, anti-miR-17 is fully complementary to one or more miR-17 family members (i.e., 100% complementary). In some embodiments, anti-miR-17 is at least 80%, at least 85%, at least 90%, or at least 95% complementary to one or more miR-17 family members.

[0190] “miR-17” refers to a mature miRNA with the nucleobase sequence 5'-CAAAGUGCUUACAGUGCA GGUAG-3' (SEQ ID NO:1).

[0191] “miR-20a” refers to a mature miRNA with the nucleobase sequence 5'-UAAAGUGCUUAUAGUGCA GGUAG-3' (SEQ ID NO:2).

[0192] “miR-20b” refers to a mature miRNA with the nucleobase sequence 5'-CAAAGUGCUCAUAGUGCA GGUAG-3' (SEQ ID NO:3).

[0193] “miR-93” refers to a mature miRNA with the nucleobase sequence 5'-CAAAGUGCUGUUCGUGCA GGUAG-3' (SEQ ID NO:4).

[0194] “miR-106a” refers to a mature miRNA with the nucleobase sequence 5'-AAAAGUGCUUACAGUGC AGGUAG-3' (SEQ ID NO:5).

[0195] “miR-106b” refers to a mature miRNA with the nucleobase sequence 5'-UAAAGUGCUGACAGUGC AGAU-3' (SEQ ID NO:6).

[0196] The “miR-17 seed sequence” refers to the nucleobase sequence 5'-AAAGUG-3', which is present in every member of the miR-17 family.

[0197] "miR-17 family member" refers to a mature miRNA that has a nucleobase sequence containing the miR-17 seed sequence, and is selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0198] The “miR-17 family” refers to the following group of miRNAs: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b, each having a nucleobase sequence containing the miR-17 seed sequence.

[0199] "Target nucleic acid" refers to an oligomeric compound that is designed to hybridize with a nucleic acid.

[0200] "Targeting" refers to the process of designing and selecting nucleobase sequences that will hybridize with target nucleic acids.

[0201] "Targeted" means having a nucleobase sequence that allows hybridization with the target nucleic acid.

[0202] "Regulation" refers to a disturbance of function, quantity, or activity. In some embodiments, regulation means an increase in function, quantity, or activity. In other embodiments, regulation means a decrease in function, quantity, or activity.

[0203] "Expression" refers to any function and step that translates the encoded information of a gene into the structure that exists and operates within the cell.

[0204] "Nucleobase sequence" refers to the sequence of consecutive nucleobases in an oligomer or nucleic acid, usually listed in a 5' to 3' orientation, and independent of any sugar, bond, and / or nucleobase modifications.

[0205] "Continuous nucleobases" refers to the nucleobases in nucleic acids that are adjacent to each other.

[0206] "Nucleobase complementarity" refers to the ability of two nucleobases to pair non-covalently via hydrogen bonds.

[0207] "Complementarity" means that one nucleic acid can hybridize with another nucleic acid or oligonucleotide. In some implementations, complementarity refers to an oligonucleotide that can hybridize with the target nucleic acid.

[0208] "Complete complementarity" means that each nucleobase of the oligonucleotide is able to pair with a nucleobase at a corresponding position in the target nucleic acid. In some embodiments, the oligonucleotide is completely complementary to the microRNA (also known as 100% complementarity), meaning that each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA. Modified oligonucleotides can be completely complementary to microRNA and have many linked nucleosides shorter than the length of the microRNA. For example, an oligonucleotide with 16 linked nucleosides (where each nucleobase of the oligonucleotide is complementary to a nucleobase at a corresponding position in the microRNA) is completely complementary to the microRNA. In some embodiments, oligonucleotides where each nucleobase is complementary to a nucleobase within a region of the microRNA stem-loop sequence are completely complementary to the microRNA stem-loop sequence.

[0209] "Complementarity percentage" refers to the percentage of nucleobases in an oligonucleotide that are complementary to the same length portion of the target nucleic acid. It is calculated by dividing the number of nucleobases in the oligonucleotide that are complementary to the corresponding nucleobases in the target nucleic acid by the total number of nucleobases in the oligonucleotide.

[0210] "Identity percentage" refers to the number of nucleotides in the first nucleic acid that are identical to those in the corresponding positions in the second nucleic acid, divided by the total number of nucleotides in the first nucleic acid. In some embodiments, both the first and second nucleic acids are microRNAs. In some embodiments, both the first and second nucleic acids are oligonucleotides.

[0211] "Hybridization" refers to the annealing of complementary nucleic acids that occur through nucleobase complementarity.

[0212] "Mismatch" means that the nucleobases of the first nucleic acid cannot be paired with the corresponding nucleobases of the second nucleic acid in a Watson-Crick pairing.

[0213] In the context of nucleobase sequence, “identical” means having the same nucleobase sequence, independent of sugar, bond and / or nucleobase modifications, and independent of the methylation state of any pyrimidine present.

[0214] “MicroRNA” refers to endogenous noncoding RNA with a length between 18 and 25 nucleotides, which is the product of pre-microRNA cleavage by the enzyme Dicer. Examples of mature microRNAs can be found in a microRNA database called miRBase (microrna.sanger.ac.uk / ). In some implementations, microRNA is abbreviated as “miR”.

[0215] "MicroRNA-regulated transcripts" refers to transcripts regulated by microRNAs.

[0216] "Seed matching sequence" refers to a nucleobase sequence that is complementary to the seed sequence and has the same length as the seed sequence.

[0217] "Oligomers" refer to compounds that contain multiple linked monomeric subunits. Oligomeric compounds include oligonucleotides.

[0218] "Oligonucleotide" refers to a compound containing multiple linked nucleosides, each of which may be modified or unmodified independently of the others.

[0219] "Naturally occurring nucleoside bonds" refers to the 3' to 5' phosphodiester bonds between nucleosides.

[0220] "Natural sugars" refers to sugars found in DNA (2'-H) or RNA (2'-OH).

[0221] "Nucleoside internucleotide bond" refers to the covalent bond between adjacent nucleosides.

[0222] "Linked nucleosides" refers to nucleosides linked by covalent bonds.

[0223] "Nucleobase" refers to a heterocyclic moiety that can non-covalently pair with another nucleobase.

[0224] "Nucleoside" refers to a nucleobase that is attached to a sugar.

[0225] "Nucleotide" refers to a nucleoside that has a phosphate group covalently linked to the sugar portion of the nucleoside.

[0226] "A compound comprising a modified oligonucleotide consisting of a plurality of linked nucleosides" means a compound comprising a modified oligonucleotide having a specified number of linked nucleosides. Therefore, the compound may include additional substituents or conjugates. Unless otherwise stated, the modified oligonucleotide does not hybridize with the complementary strand, and the compound does not contain any other nucleosides besides the modified oligonucleotide.

[0227] "Modified oligonucleotides" refer to single-stranded oligonucleotides that have one or more modifications to their ends, sugars, nucleobases, and / or nucleoside bonds relative to their naturally occurring ends. Modified oligonucleotides may contain unmodified nucleosides.

[0228] "Modified nucleoside" refers to a nucleoside that has any variation from naturally occurring nucleosides. Modified nucleosides may have modified sugars and unmodified nucleobases. Modified nucleosides may have modified sugars and modified nucleobases. Modified nucleosides may have natural sugars and modified nucleobases. In some embodiments, the modified nucleoside is a bicyclic nucleoside. In some embodiments, the modified nucleoside is a non-bicyclic nucleoside.

[0229] "Modified nucleoside bonds" refers to any alteration of naturally occurring nucleoside bonds.

[0230] "Thiophosphate nucleoside bond" refers to a bond between nucleosides in which one of the non-bridging atoms is a sulfur atom.

[0231] "Modified sugar portion" means the substitution and / or any alteration of natural sugars.

[0232] "Unmodified nucleobases" refers to naturally occurring heterocyclic bases in RNA or DNA: purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

[0233] "5-methylcytosine" refers to cytosine containing a methyl group attached to the 5-position.

[0234] "Unmethylated cytosine" refers to cytosine without a methyl group attached at the 5-position.

[0235] "Modified nucleobase" means any nucleobase that is not an unmodified nucleobase.

[0236] "Sugar moiety" refers to naturally occurring furanyl groups or modified sugar moieties.

[0237] "Modified sugar portion" refers to the sugar portion that has been replaced or a sugar substitute.

[0238] "2'-O-methyl sugar" or "2'-OMe sugar" refers to a sugar with an O-methyl modification at the 2' position.

[0239] "2'-O-methoxyethyl sugar" or "2'-MOE sugar" refers to a sugar with an O-methoxyethyl modification at the 2' position.

[0240] "2'-F" or "2'-F" refers to sugars that have fluorine modification at the 2' position.

[0241] "Bicyclic sugar moiety" refers to a sugar moiety modified with a 4- to 7-membered ring (including, but not limited to, furanyl glycosyl group), comprising a bridge connecting two atoms of the 4- to 7-membered ring to form a second ring, thereby producing a bicyclic structure. In some embodiments, the 4- to 7-membered ring is a sugar ring. In some embodiments, the 4- to 7-membered ring is a furanyl glycosyl group. In some such embodiments, the bridge connects the 2'-carbon and 4'-carbon of the furanyl glycosyl group. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

[0242] "Locked Nucleic Acid (LNA) Sugar Moment" refers to a substituted sugar moment containing a (CH2)-O bridge between the 4' and 2' furanose ring atoms.

[0243] "ENA sugar moiety" refers to a substituted sugar moiety containing a (CH2)2-O bridge between the 4' and 2' furanose ring atoms.

[0244] "Constrained ethyl (cEt) sugar moiety" means a substituted sugar moiety containing a CH(CH3)-O bridge between the 4' and 2' furanose ring atoms. In some embodiments, the CH(CH3)-O bridge is constrained in the S orientation. In some embodiments, the CH(CH3)-O is constrained in the R orientation.

[0245] "S-cEt sugar moiety" refers to a substituted sugar moiety containing an S-restricted CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0246] "R-cEt sugar moiety" refers to a substituted sugar moiety containing an R-restricted CH(CH3)-O bridge between the 4' and 2' furanose ring atoms.

[0247] "2'-O-methyl nucleoside" refers to a 2'-modified nucleoside with 2'-O-methyl sugar modification.

[0248] "2'-O-methoxyethyl nucleoside" refers to a 2'-modified nucleoside with a 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleoside may contain modified or unmodified nucleobases.

[0249] "2'-Fluoronucleotide" refers to a 2'-modified nucleoside with 2'-fluoro sugar modification. 2'-Fluoronucleotides can contain modified or unmodified nucleobases.

[0250] "Bicyclic nucleoside" refers to a nucleoside with a 2'-modified bicyclic sugar moiety. Bicyclic nucleosides can have modified or unmodified nucleobases.

[0251] "cEt nucleoside" refers to a nucleoside that contains a cEt sugar moiety. cEt nucleosides can contain modified or unmodified nucleobases.

[0252] "S-cEt nucleoside" refers to a nucleoside that contains the S-cEt sugar moiety.

[0253] "R-cEt nucleoside" refers to a nucleoside that contains the R-cEt sugar moiety.

[0254] “β-D-deoxyribonucleoside” refers to naturally occurring DNA nucleosides.

[0255] “β-D-ribonucleoside” refers to naturally occurring RNA nucleosides.

[0256] "LNA nucleoside" refers to a nucleoside that contains the LNA sugar moiety.

[0257] "ENA nucleoside" refers to nucleosides that contain the ENA sugar moiety.

[0258] "Hydrogen bond acceptor" refers to a component that does not provide hydrogen bonds that share hydrogen atoms.

[0259] "Hydrogen bond donor" refers to a bond or molecule that provides a hydrogen atom for a hydrogen bond.

[0260] References to the value or parameter “about” herein include (and describe) embodiments for that value or parameter itself. In some embodiments, the term “about” includes ±10% of the indicated amount. In other embodiments, the term “about” includes ±5% of the indicated amount. In some still embodiments, the term “about” includes ±1% of the indicated amount. Furthermore, the term “about X” includes a description of “X”. Additionally, unless the context clearly indicates otherwise, the singular forms “an” and “described” include plural indicators. Thus, for example, a reference to “compound” includes multiple such compounds.

[0261] Overview

[0262] Polycystic kidney disease (PKD) is a genetic form of kidney disease in which fluid-filled cysts form in the kidneys, leading to renal insufficiency and often end-stage renal disease (ESRD). Some cases of PKD are also characterized by enlarged kidneys. Excessive cyst proliferation is a hallmark pathological feature of PKD. In the management of PKD, the primary goals of treatment are to manage symptoms such as hypertension and infections, maintain kidney function, and prevent or delay the onset of ESRD, which in turn improves the life expectancy of individuals with PKD.

[0263] miR-17 has been identified as a target for the treatment of PKD. The anti-miR-17 compound RGLS4326 was discovered through screening chemically diverse and rationally designed anti-miR-17 oligonucleotide libraries to obtain optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displacing miR-17 from translationally active multimers and de-inhibiting multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, after subcutaneous administration, RGLS4326 attenuates cyst growth in human in vitro autosomal dominant polycystic kidney disease (ADPKD) models and various PKD mouse models. A Phase 1 single-dose escalation (SAD) clinical trial in healthy volunteers (HV) was initiated in December 2017, followed by a Phase 1 multiple-dose escalation (MAD) study in HV in May 2018.

[0264] Following the initiation of the Phase 1b MAD clinical trial, non-clinical toxicology studies revealed CNS-related findings in mice at high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or collapse. RGLS4326 was found to be an antagonist of AMPA receptors (AMPA-R), glutamate receptors and ion channels at excitatory synapses in the central nervous system (CNS) that mediate rapid excitatory neurotransmission and are therefore a key component of all neuronal networks. AMPA receptor antagonism could explain the CNS-mediated findings observed at high doses of RGLS4326 in the non-clinical toxicology model. While such CNS-related findings were not observed in human subjects, avoidance of AMPA receptor antagonism remains preferable. Therefore, a library of anti-miR-17 compounds was screened to identify compounds with comparable physicochemical and pharmacological properties to RGLS4326, and also with more favorable safety profiles (e.g., the ability to avoid AMPA receptor antagonism).

[0265] One such compound, RG-NG-1015, was identified and selected as a candidate therapeutic agent for the treatment of ADPKD.

[0266] RG-NG-1015 and related compounds

[0267] This article provides compounds comprising modified oligonucleotides, wherein the modified oligonucleotides have the following structure 5'-A S G S C M A F C F U F U M U S A S -3', where each cytosine is a nonmethylated cytosine.

[0268] In some implementations, the compound consists of modified oligonucleotides.

[0269] In some implementations, the pharmaceutically acceptable salt is a sodium salt.

[0270] This article provides a modified oligonucleotide named RG-NG-1015, wherein the structure of the modified oligonucleotide is:

[0271]

[0272] This document also provides pharmaceutically acceptable salts of the modified oligonucleotide RG-NG-1015. Therefore, in some embodiments, the modified oligonucleotide has the following structure:

[0273]

[0274] Or a pharmaceutically acceptable salt thereof. A non-limiting exemplary pharmaceutically acceptable salt of RG-NG-1015 has the following structure:

[0275]

[0276] In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide contains fewer cationic counterions (such as Na+) than the number of thiophosphate and / or phosphodiester bonds present per molecule (i.e., some thiophosphate and / or phosphodiester bonds are protonated). + In some embodiments, pharmaceutically acceptable salts of RG-NG-1015 contain fewer than 8 cationic counterions (such as Na+) per molecule of RG-NG-1015. + In other words, in some embodiments, a pharmaceutically acceptable salt of RG-NG-1015 may contain an average of 1, 2, 3, 4, 5, 6 or 7 cationic counterions per molecule of RG-NG-1015, wherein the remaining thiophosphate groups are protonated.

[0277] As used herein, and without specifically referring to a particular pharmaceutically acceptable salt of RG-NG-1015, any dose (whether expressed as, for example, mg / kg, mg or % by weight) should be considered as referring to the amount of RG-NG-1015 in its protonated form (i.e., not as a salt).

[0278] In some embodiments, RG-NG-1015 is formulated for subcutaneous administration. In some such embodiments, RG-NG-1015 is provided in a pre-filled vial containing a sufficient volume to extract 1 mL of 150 mg / mL RG-NG-1015. In some embodiments, RG-NG-1015 is in a solution containing 0.3% saline.

[0279] Treatment methods for polycystic kidney disease

[0280] This article provides a method for inhibiting the activity of one or more members of the miR-17 family in cells, the method comprising contacting cells with a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence.

[0281] This document provides a method for inhibiting the activity of one or more members of the miR-17 family in a subject, the method comprising administering a pharmaceutical composition provided herein to the subject. In some embodiments, the subject suffers from a disease associated with one or more members of the miR-17 family.

[0282] This article provides a method for treating polycystic kidney disease (PKD), comprising administering a compound provided herein to a subject in need, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject has polycystic kidney disease. In some embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and non-autosomal wasting disease (NPHP). In some embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD).

[0283] In some implementations, the subject suffers from a condition characterized by multiple non-renal markers and also has polycystic kidney disease. Such conditions include, for example, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). Therefore, this document provides a method for treating polycystic kidney disease (PKD) comprising administering to a subject a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject suffers from Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). This document provides a method for treating polycystic kidney disease (PKD) comprising administering a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence, wherein the subject is suspected of having Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0284] In some implementations, polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the PKD1 or PKD2 genes. ADPKD is a progressive disease in which cyst formation and kidney enlargement lead to renal insufficiency and eventually end-stage renal disease in 50% of patients aged 60 years. Patients with ADPKD may require lifelong dialysis and / or kidney transplantation. ADPKD is the most common genetic cause of kidney failure. Excessive cyst proliferation is the hallmark pathological feature of ADPKD. In the management of PKD, the primary goal of treatment is to maintain renal function and prevent or delay the onset of end-stage renal disease (ESRD), which in turn improves the life expectancy of PKD patients. The total kidney volume in patients with ADPKD typically increases steadily, and this increase is associated with declining renal function. This article provides a method for treating ADPKD, which involves administering a compound provided herein to a subject who has or is suspected of having ADPKD, the compound containing a nucleobase sequence complementary to the miR-17 seed sequence.

[0285] In some implementations, polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). ARPKD is caused by mutations in the PKHD1 gene and is a cause of chronic kidney disease in children. The typical renal phenotype of ARPKD is enlarged kidneys; however, ARPKD has significant effects on other organs, particularly the liver. Patients with ARPKD progress to end-stage renal disease and require kidney transplantation as young as 15 years of age. This article provides a method for treating ARPKD, which involves administering a compound provided herein to a subject who has or is suspected of having ARPKD, the compound containing a nucleobase sequence complementary to the miR-17 seed sequence.

[0286] In some implementations, polycystic kidney disease is renal wasting disease (NPHP). Renal wasting disease is an autosomal recessive cystic kidney disease and a common cause of ESRD in children. NPHP is characterized by normal or reduced kidney size, cysts concentrated at the corticomedullary junction, and tubulointerstitial fibrosis. Mutations in one of several NPHP genes, such as NPHP1, have been identified in patients with NPHP. This article provides a method for treating NPHP, which involves administering a compound provided herein to a subject who has or is suspected of having NPHP, the compound containing a nucleobase sequence complementary to the miR-17 seed sequence.

[0287] In some embodiments, the subject with polycystic kidney disease also has Joubert syndrome and related conditions (JSRD). JSRD encompasses a wide range of hallmark features, including brain, retinal, and skeletal abnormalities. Some subjects with JSRD also have polycystic kidney disease in addition to the hallmark features of JSRD. Therefore, this document provides a method for treating polycystic kidney disease in a subject with JSRD, comprising administering to the subject a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having JSRD.

[0288] In some embodiments, the subject with polycystic kidney disease also has Meckel syndrome (MKS). MKS is a condition characterized by severe signs and symptoms in many parts of the body, including the central nervous system, skeletal system, liver, kidneys, and heart. A common feature of MKS is the presence of numerous fluid-filled cysts in the kidneys and kidney enlargement. Therefore, this document provides a method for treating MKS, comprising administering a compound provided herein to a subject with MKS, the compound containing a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having MKS.

[0289] In some embodiments, the subject with polycystic kidney disease also has Bardet-Biedl syndrome (BBS). BBS is a condition that affects many parts of the body, including the eyes, heart, kidneys, liver, and digestive system. A hallmark feature of BBS is the presence of kidney cysts. Therefore, this document provides a method for treating polycystic kidney disease in a subject with BBS, comprising administering to the subject a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence. In some embodiments, the subject is suspected of having BBS.

[0290] In some implementations, the subject has been diagnosed with PKD prior to administration of the compound containing the modified oligonucleotide. The diagnosis of PKD can be achieved by assessing parameters including, but not limited to, the subject's family history, clinical characteristics (including, but not limited to, hypertension, albuminuria, hematuria, and impaired GFR), renal imaging studies (including, but not limited to, MRI, ultrasound, and CT scans), and / or histological analysis.

[0291] In some implementations, subjects with ADPKD are classified into categories 1C, 1D, or 1E based on Mayo imaging classification of ADPKD. In some implementations, subjects with ADPKD are defined as those with an estimated glomerular filtration rate (eGFR) between 30 and 90 mL / min / 1.73 m³ / min. 2 The subjects between.

[0292] In some embodiments, a method for treating ADPKD is provided, comprising administering to a subject in need about 0.5-5 mg / kg, about 0.5-4.5 mg / kg, about 0.5-4 mg / kg, about 0.5-3.5 mg / kg, about 0.5-3 mg / kg, about 1-5 mg / kg, about 1-4.5 mg / kg, about 1-4 mg / kg, about 1-3.5 mg / kg, or about 1-3 mg / kg of a compound containing an oligonucleotide modified as discussed herein, such as RG-NG-1015, or a pharmaceutically acceptable salt thereof, such as a sodium salt of RG-NG-1015. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at doses of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at doses of about 1 mg / kg, about 2 mg / kg, or about 3 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at doses of 1 mg / kg, 2 mg / kg, or 3 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at doses of about 1 mg / kg or 1 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a dose of about 2 mg / kg or 2 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a dose of about 3 mg / kg or 3 mg / kg. In some embodiments, the compound comprising the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered subcutaneously.

[0293] In some embodiments, a compound containing a modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered every 2 weeks (14 days). In some embodiments, the compound containing a modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at least once, at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, or more. In some embodiments, tolvaptan is not administered to the subject within 28 days prior to administration of a compound containing a modified oligonucleotide as discussed herein or a pharmaceutically acceptable salt thereof.

[0294] In some embodiments, PKD diagnosis includes screening for mutations in one or more of the PKD1 or PKD2 genes. In some embodiments, ARPKD diagnosis includes screening for mutations in the PKHP1 gene. In some embodiments, NPHP diagnosis includes screening for mutations in one or more of the NPHP1, NPHP2, NPHP3, NPHP4, NPHP5, NPHP6, NPHP7, NPHP8, or NPHP9 genes. In some embodiments, JSRD diagnosis includes screening for mutations in the NPHP1, NPHP6, AHI1, MKS3, or RPGRIP1L genes. In some embodiments, MKS diagnosis includes screening for mutations in the NPHP6, MKS3, RPGRIP1L, NPHP3, CC2D2A, BBS2, BBS4, BBS6, or MKS1 genes. In some implementations, BBS diagnosis includes screening for mutations in the BBS2, BBS4, BBS6, MKS1, BBS1, BBS3, BBS5, BBS7, BBS8, BBS9, BBS10, BBS11, or BBS12 genes.

[0295] In some implementations, the subject has an increased total kidney volume. In some implementations, the total kidney volume is the height-adjusted total kidney volume (HtTKV). In some implementations, the subject has hypertension. In some implementations, the subject has impaired kidney function. In some implementations, the subject requires improved kidney function. In some implementations, the subject is identified as having impaired kidney function.

[0296] In some embodiments, the levels of one or more miR-17 family members are increased in the kidneys of a subject with PKD. In some embodiments, prior to administration, the subject is identified as having increased levels of one or more miR-17 family members in the kidneys. The levels of miR-17 family members can be measured from kidney biopsy material. In some embodiments, prior to administration, the subject is identified as having increased levels of one or more miR-17 family members in the urine or blood of the subject. In some embodiments, prior to administration, the subject is identified as having decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine of the subject. In some embodiments, prior to administration, the subject is identified as having decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine of the subject. In some embodiments, prior to administration, the subject is identified as having decreased levels of polycystic protein-1 (PC1) and / or polycystic protein-2 (PC2) in the urine of the subject.

[0297] In some embodiments, prior to administration, the presence of elevated levels of neutrophil gelatinase-associated lipocalin (NGAL) and / or kidney injury molecule-1 (KIM-1) in the subject's urine is determined. In some embodiments, prior to administration, the presence of elevated levels of neutrophil gelatinase-associated lipocalin (NGAL) or kidney injury molecule-1 (KIM-1) in the subject's urine is determined.

[0298] In any of the implementations provided herein, subjects may undergo certain tests before, during, and / or after administration to diagnose polycystic kidney disease, for example, to determine the cause of polycystic kidney disease, assess the severity of polycystic kidney disease, and / or determine the subject's response to treatment. Such tests can assess biomarkers of polycystic kidney disease. Some of these tests (such as glomerular filtration rate (GFR) and blood urea nitrogen (BUN) levels) are also indicators of renal function. Biomarkers for polycystic ovary syndrome (PCOS) include, but are not limited to: measurement of total kidney volume and height-adjusted total kidney volume (htTKV); measurement of hypertension; assessment of renal pain; measurement of fibrosis; measurement of polycystic protein-1 (PC1) in urine; measurement of polycystic protein-2 (PC2) in urine; measurement of blood urea nitrogen (BUN) levels; measurement of serum creatinine (SCr) levels; measurement of creatinine clearance; measurement of albuminuria; measurement of albumin:creatinine ratio; measurement of glomerular filtration rate (GFR) and estimated GFR (eGFR); measurement of hematuria; measurement of NGAL protein in urine; and / or measurement of KIM-1 protein in urine. Unless otherwise stated herein, blood urea nitrogen (BUN) levels, serum creatinine (SCr) levels, creatinine clearance, albuminuria, albumin:creatinine ratio, glomerular filtration rate (GFR), and hematuria refer to measurements in the subject's blood (such as whole blood or serum).

[0299] In some implementations, subjects may also undergo additional tests before, during, and / or after administration, such as measurements of monocyte chemoattractant protein-1 (MCP-1) and / or β-2 microglobulin (B2M) in the subject's urine; measurements of insulin-like growth factor binding protein acid unstable subunits (IGFALS), CT-proAVP, and / or N-acetyl-1-methylhistidine in the subject's serum; measurements of complement cleavage products C3a and / or Bb in the subject's plasma; and / or measurements of the total cyst volume, number, and / or size distribution of the subject's cysts.

[0300] Biomarkers for polycystic kidney disease are determined through laboratory testing. Reference ranges for individual biomarkers may vary from laboratory to laboratory. Variations may be due to, for example, differences in the specific assays used. Therefore, the upper and lower limits of the normal distribution of a biomarker within a population (also referred to as the upper limit of normal (ULN) and lower limit of normal (LLN) respectively) can vary from laboratory to laboratory. For any given biomarker, a healthcare professional can determine which levels outside the normal distribution are clinically relevant and / or indicative of disease. For example, a healthcare professional can determine glomerular filtration rate, which can indicate a rate of decline in kidney function in a subject with polycystic kidney disease.

[0301] In some embodiments, administration of the compounds provided herein results in one or more clinically beneficial outcomes. In some embodiments, administration improves renal function in a subject. In some embodiments, administration slows the rate of decline in renal function in a subject. In some embodiments, administration reduces total kidney volume in a subject. In some embodiments, administration slows the rate of increase in total kidney volume in a subject. In some embodiments, administration reduces height-adjusted total kidney volume (HtTKV). In some embodiments, administration slows the rate of increase in HtTKV.

[0302] In some embodiments, administration increases polycystic protein-1 (PC1) in the urine of the subject. In some embodiments, administration increases polycystic protein-2 (PC2) in the urine of the subject. In some embodiments, administration increases both polycystic protein-1 (PC1) and polycystic protein-2 (PC2) in the urine of the subject.

[0303] In some embodiments, the administration inhibits cyst growth (total cyst volume, number, and / or size distribution) in the subject. In some embodiments, the administration slows the rate of increase in cyst growth (total cyst volume, number, and / or size distribution) in the subject. In some embodiments, the cyst is located in the subject's kidney. In some embodiments, the cyst is located in an organ other than the kidney, such as the liver.

[0304] In some embodiments, the administration reduces kidney pain in the subject. In some embodiments, the administration slows the increase in kidney pain in the subject. In some embodiments, the administration delays the onset of kidney pain in the subject.

[0305] In some implementations, the administration lowers the subject's blood pressure. In some implementations, the administration slows the worsening of the subject's blood pressure. In some implementations, the administration delays the onset of the subject's blood pressure.

[0306] In some embodiments, administration reduces fibrosis in the kidneys of the subject. In some embodiments, administration slows the progression of fibrosis in the kidneys of the subject.

[0307] In some embodiments, administration delays the onset of end-stage renal disease in the subject. In some embodiments, administration delays the duration of dialysis in the subject. In some embodiments, administration delays the time to kidney transplantation in the subject. In some embodiments, administration improves the life expectancy of the subject.

[0308] In some embodiments, the administration reduces albuminuria in the subject. In some embodiments, the administration slows the worsening of albuminuria in the subject. In some embodiments, the administration delays the onset of albuminuria in the subject. In some embodiments, the administration reduces hematuria in the subject. In some embodiments, the administration slows the worsening of hematuria in the subject. In some embodiments, the administration delays the onset of hematuria in the subject. In some embodiments, the administration reduces or slows the rate of increase in blood urea nitrogen (BUN) levels in the subject. In some embodiments, the administration reduces or slows the rate of increase in serum creatinine (SCr) levels in the subject. In some embodiments, the administration improves or slows the rate of decrease in creatinine clearance in the subject. In some embodiments, the administration reduces or slows the rate of increase in the urinary albumin:creatinine ratio in the subject.

[0309] In some embodiments, the administration improves the subject's glomerular filtration rate (GFR). In some embodiments, the administration slows the rate of decline in the subject's GFR. In some embodiments, the GFR is an estimated GFR (eGFR). In some embodiments, the GFR is a measured GFR (mGFR).

[0310] In some embodiments, administration reduces or slows the rate of increase of neutrophil gelatinase-associated lipocalin (NGAL) protein in the subject's urine. In some embodiments, administration reduces or slows the rate of increase of kidney injury molecule-1 (KIM-1) protein in the subject's urine.

[0311] In some embodiments, administration reduces or slows the rate of increase of monocyte chemoattractant protein-1 (MCP-1) in the subject's urine. In some embodiments, administration reduces or slows the rate of increase of β-2 microglobulin (B2M) in the subject's urine.

[0312] In some implementations, administration reduces or slows the rate of increase in complement cleavage products C3a and / or Bb in the subject's plasma.

[0313] In some implementations, administration reduces or slows the rate of increase in acute-phase proteins (i.e., Alb, fibrinogen, and / or hypersensitive C-reactive protein).

[0314] In some embodiments, administration reduces or slows the rate of increase of insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the subject's serum. In some embodiments, administration reduces or slows the rate of increase of CT-proAVP in the subject's serum. In some embodiments, administration reduces or slows the rate of increase of N-acetyl-1-methylhistidine in the subject's serum.

[0315] In any of the implementations provided herein, the subject may undergo certain tests to assess the subject's disease severity. Such tests include, but are not limited to, measuring the subject's total kidney volume; measuring the subject's htTKV; measuring the subject's hypertension; measuring the subject's renal pain; measuring the subject's fibrosis in the kidneys; measuring the subject's blood urea nitrogen (BUN) level; measuring the subject's serum creatinine (SCr) level; measuring the subject's creatinine clearance in the blood; measuring the subject's albuminuria; measuring the subject's albumin:creatinine ratio; measuring the subject's glomerular filtration rate (GFR), wherein the glomerular filtration rate is estimated or measured; and measuring the subject's urine. Polycystin-1 (PC1) and / or polycystin-2 (PC2) in urine; neutrophil gelatinase-associated lipocalin (NGAL) protein in urine; and / or kidney injury molecule-1 (KIM-1) protein in urine; MCP-1 and / or B2M in urine; IGFALS, CT-proAVP and / or N-acetyl-1-methylhistidine in serum; total cyst volume, number and / or size distribution.

[0316] In some embodiments, administration of a compound comprising an oligonucleotide modified as discussed herein, or a pharmaceutically acceptable salt thereof, results in little or no CNS damage in subjects. In some embodiments, CNS damage is measured by an ataxia assessment and rating scale (SARA) test. SARA is a tool for assessing ataxia. In some embodiments, the SARA test is performed on the subject prior to administration. In some embodiments, administration results in little or no change in the subject's SARA test score compared to pre-treatment levels.

[0317] In some implementations, subjects may undergo certain tests to assess the pharmacokinetics of compounds containing oligonucleotides modified as discussed herein, or pharmaceutically acceptable salts thereof, before, during, and / or after administration. Pharmacokinetic analyses are performed to measure and compare one or more of the following parameters: maximum observed concentration (C0). max ), the time to reach the maximum observed concentration (T) max ), up to 24 hours after administration, the area under the concentration-time curve (AUC) 0-24 ), until the final quantifiable concentration is reached, the area under the concentration-time curve (AUC) 0-t The area under the concentration-time curve (AUC) within the dosing interval. tau The area under the concentration-time curve extrapolated to infinity (AUC) inf ), half-life (t) 1 / 2 Apparent clearance rate (CL / F), apparent distribution volume (V) z / F), the fraction of its unmodified compounds excreted in urine (fe) and / or the total amount of its unmodified compounds excreted in urine (Ae).

[0318] In some implementations, during and / or after administration of a compound containing an oligonucleotide modified as discussed herein or a pharmaceutically acceptable salt thereof, the subject may undergo certain tests to assess the development of antidrug antibodies (ADA) in the subject's plasma.

[0319] In some implementations, subjects with polycystic kidney disease experience a reduced quality of life. For example, subjects with polycystic kidney disease may experience kidney pain, which can reduce their quality of life. In some implementations, administration improves the subject's quality of life.

[0320] In any of the embodiments provided herein, the subjects are human subjects. In some embodiments, the human subjects are adults. In some embodiments, adults are at least 21 years old. In some embodiments, the human subjects are pediatric subjects, i.e., subjects are under 21 years old. The pediatric population may be defined by a regulatory agency. In some embodiments, the human subjects are adolescents. In some embodiments, adolescents are at least 12 years old and under 21 years old. In some embodiments, the human subjects are children. In some embodiments, children are at least two years old and under 12 years old. In some embodiments, the human subjects are infants. In some embodiments, infants are at least one month old and under two years old. In some embodiments, the subjects are newborns. In some embodiments, newborns are under one month old. In some embodiments, the subjects are between 18 and 70 years old.

[0321] Any compound described herein may be used in a therapy. Any compound provided herein may be used to treat polycystic kidney disease. In some embodiments, the polycystic kidney disease is autosomal dominant polycystic kidney disease. In some embodiments, the polycystic kidney disease is autosomal recessive polycystic kidney disease. In some embodiments, the polycystic kidney disease is renal wasting disease. In some embodiments, the subject has Joubert syndrome and related conditions (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0322] Any modified oligonucleotides described in this article may be used in therapy. Any modified oligonucleotides provided in this article may be used to treat polycystic kidney disease.

[0323] Any compound provided herein may be used to prepare a drug. Any compound provided herein may be used to prepare a drug for the treatment of polycystic kidney disease.

[0324] Any modified oligonucleotides provided in this article can be used to prepare drugs. Any modified oligonucleotides provided in this article can be used to prepare drugs for the treatment of polycystic kidney disease.

[0325] Any of the pharmaceutical compositions described herein may be used to treat polycystic kidney disease.

[0326] In some implementations, the treatment method has acceptable safety and tolerability characteristics. In some implementations, the treatment method is generally safe and well-tolerated.

[0327] Some other treatments

[0328] Treatment of polycystic kidney disease or any of the conditions listed herein may include more than one therapy. Therefore, in some embodiments, this document provides a method for treating a subject who has or is suspected of having polycystic kidney disease, the method comprising administering at least one therapy in addition to administering a compound provided herein, the compound comprising a nucleobase sequence complementary to the miR-17 seed sequence.

[0329] In some embodiments, at least one additional therapy comprises a pharmaceutical agent. In some embodiments, the pharmaceutical agent is an antihypertensive agent. The antihypertensive agent is used to control the subject's blood pressure.

[0330] In some embodiments, the agent is a vasopressin receptor 2 antagonist. In some embodiments, the vasopressin receptor 2 antagonist is tolvaptan.

[0331] In some embodiments, the agent includes an angiotensin II receptor blocker (ARB). In some embodiments, the angiotensin II receptor blocker is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, or eprosartan.

[0332] In some embodiments, the agent includes an angiotensin II converting enzyme (ACE) inhibitor. In some embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, or ramipril.

[0333] In some implementations, the agent is a diuretic. In some implementations, the agent is a calcium channel blocker.

[0334] In some embodiments, the agent is a glucocorticoid synthase inhibitor. In some embodiments, the glucocorticoid synthase inhibitor is venglustat.

[0335] In some embodiments, the agent is an antihyperglycemic agent. In some embodiments, the antihyperglycemic agent is biguanide. In some embodiments, the biguanide is metformin.

[0336] In some embodiments, the agent is a kinase inhibitor. In some embodiments, the kinase inhibitor is bosutinib or KD019.

[0337] In some implementations, the agent is an adrenergic receptor antagonist.

[0338] In some embodiments, the agent is an aldosterone receptor antagonist. In some embodiments, the aldosterone receptor antagonist is spironolactone. In some embodiments, spironolactone is administered at a dose of 10 mg to 35 mg daily. In some embodiments, spironolactone is administered at a dose of 25 mg daily.

[0339] In some embodiments, the agent is a mammalian target of a rapamycin (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

[0340] In some embodiments, the agent is a hormone analogue. In some embodiments, the hormone analogue is somatostatin or adrenocorticotropic hormone.

[0341] In some embodiments, the agent is an antifibrotic agent. In some embodiments, the antifibrotic agent is a modified oligonucleotide complementary to miR-21.

[0342] In some implementations, an additional treatment is dialysis. In some implementations, an additional treatment is kidney transplantation.

[0343] In some embodiments, the pharmaceutical agent includes an anti-inflammatory agent. In some embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In some embodiments, the steroidal anti-inflammatory agent is a corticosteroid. In some embodiments, the corticosteroid is prednisone. In some embodiments, the anti-inflammatory agent is a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, the NSAID is ibuprofen, a COX-1 inhibitor, or a COX-2 inhibitor.

[0344] In some implementations, the agent is an agent that blocks one or more responses to fibrosis signals.

[0345] In some implementations, additional therapies may include agents that enhance the body’s immune system, including low-dose cyclophosphamide, thymosin, vitamins and nutritional supplements (e.g., antioxidants, including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea), and vaccines, such as immunostimulatory complexes (ISCOM), which are multimer-presented vaccine formulations containing a combination of antigens and adjuvants.

[0346] In some embodiments, alternative therapies are selected to treat or improve side effects of one or more of the pharmaceutical compositions provided herein. Such side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, elevated serum transaminase levels may indicate hepatotoxicity or abnormal liver function. For example, elevated bilirubin may indicate hepatotoxicity or abnormal liver function.

[0347] Certain microRNA nucleobase sequences

[0348] The miR-17 family includes miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b. Each member of the miR-17 family has a nucleotide sequence containing the 5'-AAAGUG-3' nucleobase sequence or the miR-17 seed sequence, which is the nucleotide sequence at positions 2 to 7 of SEQ ID NO:1. Additionally, each member of the miR-17 family shares some nucleotide sequence identity outside the seed region. Therefore, in addition to miR-17, oligonucleotides modified with a nucleotide sequence complementary to the miR-17 seed sequence can target other microRNAs in the miR-17 family.

[0349] In some implementations, the modified oligonucleotide contains the nucleobase sequence 5'-AGCACUUUA-3'.

[0350] In some embodiments, each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. In some embodiments, at least one cytosine is unmethylated cytosine. In some embodiments, each cytosine is unmethylated cytosine. In some embodiments, at least one cytosine is 5-methylcytosine. In some embodiments, each cytosine is 5-methylcytosine.

[0351] In some embodiments, the number of linked nucleotides in the modified oligonucleotide is less than the length of its target microRNA. A modified oligonucleotide with linked nucleotides shorter than the length of the target microRNA (where each nucleotide of the modified oligonucleotide is complementary to a nucleotide at a corresponding position in the target microRNA) is considered a modified oligonucleotide with a nucleotide sequence that is perfectly complementary (also known as 100% complementary) to a region of the target microRNA sequence. For example, a modified oligonucleotide consisting of nine linked nucleotides is perfectly complementary to miR-17, where each nucleotide is complementary to a corresponding position in miR-17.

[0352] In some embodiments, the modified oligonucleotide has one mismatched nucleotide sequence relative to the target microRNA's nucleotide sequence. In some embodiments, the modified oligonucleotide has two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some such embodiments, the modified oligonucleotide has no more than two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some such embodiments, the mismatched nucleotides are consecutive. In some such embodiments, the mismatched nucleotides are not consecutive.

[0353] Although the sequence listing appended to this application identifies each nucleobase sequence as “RNA” or “DNA” as needed, in practice, these sequences can be modified with combinations of chemical modifications specified herein. Those skilled in the art will readily understand that the names such as “RNA” or “DNA” used to describe the modified oligonucleotides in the sequence listing are, to some extent, arbitrary. For example, a modified oligonucleotide containing a nucleoside with a 2'-O-methoxyethyl sugar moiety and a thymine base can be described in the sequence listing as a DNA residue, even if the nucleoside is modified and not a native DNA nucleoside.

[0354] Therefore, the nucleic acid sequences provided in the sequence listing are intended to cover nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to such nucleic acids having modified nucleotides. As a further example and not a limitation, the modified oligonucleotides in the sequence listing having the nucleotide sequence “ATCGATCG” cover any oligonucleotide having such a nucleotide sequence, whether modified or unmodified, including but not limited to compounds containing RNA bases, such as compounds having the sequence “AUCGAUCG”, and compounds having some DNA bases and some RNA bases, such as “AUCGATCG”, and oligonucleotides having other modified bases, such as “AT…”. me "CGAUCG", among which me C indicates 5-methylcytosine.

[0355] certain modifications

[0356] In some embodiments, the oligonucleotides provided herein may contain one or more modifications to nucleotide bases, sugars, and / or nucleoside bonds, and are therefore modified oligonucleotides. Modified nucleotide bases, sugars, and / or nucleoside bonds can be selected instead of unmodified forms due to desired properties such as, for example, enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases.

[0357] In some implementations, the modified oligonucleotide comprises one or more modified nucleosides.

[0358] In some embodiments, the modified nucleoside is a sugar-modified nucleoside. In some such embodiments, the sugar-modified nucleoside may also comprise a native or modified heterocyclic base moiety and / or be linked to another nucleoside via a native or modified internucleotide bond and / or may include further modifications independent of the sugar modification. In some embodiments, the sugar-modified nucleoside is a 2'-modified nucleoside, wherein the sugar ring is modified at the 2' carbon by native ribose or 2'-deoxyribose.

[0359] In some embodiments, the 2'-modified nucleoside has a bicyclic sugar moiety. In some such embodiments, the bicyclic sugar moiety is an α-configured D sugar. In some such embodiments, the bicyclic sugar moiety is a β-configured D sugar. In some such embodiments, the bicyclic sugar moiety is an α-configured L sugar. In some such embodiments, the bicyclic sugar moiety is a β-configured L sugar.

[0360] Nucleosides containing such bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. In some embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy(4'-CH2-O-2')BNA; (B) β-D-methyleneoxy(4'-CH2-O-2')BNA; (C) ethyloxy(4'-(CH2)2-O-2')BNA; (D) aminooxy(4'-CH2-ON(R)-2')BNA; (E) oxyamino(4'-CH2-N(R)-O-2')BNA; and (F) methyl(methyleneoxy)(4'-C H(CH3)-O-2')BNA (also known as restricted ethyl or cEt); (G) methylene-thio(4'-CH2-S-2')BNA; (H) methylene-amino(4'-CH2-N(R)-2')BNA; (I) methylcarbocyclic(4'-CH2-CH(CH3)-2')BNA; (J) c-MOE(4'-CH(CH2-OMe)-O-2')BNA; and (K) propylenecarbocyclic(4'-(CH2)3-2')BNA, as shown below.

[0361]

[0362] Where Bx is the nucleobase moiety and R is independently H, a protecting group, or C1-C. 12 alkyl.

[0363] In some embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from F, OCF3, O-CH3 (also known as "2'-OMe"), OCH2CH2OCH3 (also known as "2'-O-methoxyethyl" or "2'-MOE"), 2'-O(CH2)2SCH3, O-(CH2)2-ON(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3.

[0364] In some embodiments, the 2'-modified nucleoside contains a 2'-substituent selected from F, O-CH3, and OCH2CH2OCH3.

[0365] In some embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In some embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. The 4'-thio-modified nucleoside has β-D-ribonucleoside, wherein 4'-O is replaced by 4'-S. The 4'-thio-2'-modified nucleoside is a 4'-thio-modified nucleoside in which 2'-OH is replaced by a 2'-substituent. Suitable 2'-substituents include 2'-OCH3, 2'-OCH2CH2OCH3, and 2'-F.

[0366] In some embodiments, the modified oligonucleotide comprises one or more inter-nucleoside modifications. In some such embodiments, each inter-nucleoside bond of the modified oligonucleotide is a modified inter-nucleoside bond. In some embodiments, the modified inter-nucleoside bond comprises a phosphorus atom.

[0367] In some embodiments, the modified oligonucleotide contains at least one phosphate-thioester nucleoside bond. In some embodiments, each nucleoside bond of the modified oligonucleotide is a phosphate-thioester nucleoside bond.

[0368] In some embodiments, the modified oligonucleotide comprises one or more modified nucleobases. In some embodiments, the modified nucleobase is selected from 5-hydroxymethylcytosine, 7-deazoguanine, and 7-deazoadenine. In some embodiments, the modified nucleobase is selected from 7-deazo-adenine, 7-deazoguanosine, 2-aminopyridine, and 2-pyridone. In some embodiments, the modified nucleobase is selected from 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0369] In some embodiments, the modified nucleobase comprises a polycyclic heterocycle. In some embodiments, the modified nucleobase comprises a tricyclic heterocycle. In some embodiments, the modified nucleobase comprises a phenoxazine derivative. In some embodiments, the phenoxazine may be further modified to form a nucleobase known in the art as a G-clamp.

[0370] In some embodiments, the modified oligonucleotide is conjugated to one or more moieties that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. In some such embodiments, the moieties are cholesterol moieties. In some embodiments, the moieties are lipid moieties. Other moieties used for conjugation include carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folic acid, phenanthridine, anthraquinones, acridine, fluorescein, rhodamine, coumarin, and dyes. In some embodiments, the carbohydrate moieties are N-acetyl-D-galactosamine (GalNac). In some embodiments, the conjugation group is directly linked to the oligonucleotide. In some embodiments, the conjugating group is linked to the modified oligonucleotide via a linker selected from amino, azide, hydroxy, carboxylic acid, thiol, unsaturated group (e.g., double or triple bond), 8-amino-3,6-dioxanoic acid (ADO), 4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl groups. In some such embodiments, the substituent is selected from hydroxyl, amino, alkoxy, azide, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl groups.

[0371] In some such embodiments, the compound comprises a modified oligonucleotide having one or more stabilizing groups attached to one or both ends of the modified oligonucleotide to enhance properties such as, for example, nuclease stability. The stabilizing groups contain a cap structure. These end modifications protect the modified oligonucleotide from exonuclease degradation and can aid in intracellular delivery and / or localization. The cap may be present at the 5' end (5'-cap), at the 3' end (3'-cap), or at both ends. Cap structures include, for example, a reverse deoxy-base cap.

[0372] Certain pharmaceutical compositions

[0373] This document provides pharmaceutical compositions comprising oligonucleotides or compounds modified herein and pharmaceutically acceptable diluents. In some embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In some embodiments, the aqueous solution is a saline solution. As used herein, a pharmaceutically acceptable diluent should be understood as a sterile diluent. Suitable routes of administration include, but are not limited to, intravenous and subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. In some embodiments, administration is oral.

[0374] In some embodiments, the pharmaceutical composition is administered in the form of dosage units. For example, in some embodiments, the dosage units are in the form of tablets, capsules, or bolus injections.

[0375] In some embodiments, the pharmaceutical agent is a modified oligonucleotide that has been prepared in a suitable diluent, adjusted to pH 7.0–9.0 with acid or base during preparation, and then lyophilized under aseptic conditions. The lyophilized modified oligonucleotide is then reconstituted with a suitable diluent (e.g., an aqueous solution such as water) or a physiologically compatible buffer (e.g., saline solution, Hanks' solution, or Ringer's solution). The reconstituted product is administered as a subcutaneous injection or intravenous infusion. The lyophilized pharmaceutical product can be packaged in a 2 mL Type I clear glass vial (ammonium sulfate treated), plugged with a bromobutyl rubber closure and sealed with an aluminum outer seal.

[0376] In some embodiments, the pharmaceutical compositions provided herein may additionally contain other auxiliary components conventionally present in pharmaceutical compositions, at levels defined in the art for use. Thus, for example, the composition may contain additional compatible pharmaceutically active materials, such as, for example, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents.

[0377] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials that can be used to physically formulate various dosage forms of the compositions provided herein, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickeners, and stabilizers; such additional materials also include, but are not limited to, excipients such as alcohols, polyethylene glycol, gelatin, lactose, amylase, magnesium stearate, talc, silica, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone. In various embodiments, the addition of such materials should not unduly interfere with the bioactivity of the components of the compositions provided herein. The formulation may be sterilized and, if desired, mixed with adjuvants such as lubricants, preservatives, stabilizers, humectants, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, which do not adversely interact with the oligonucleotides of the formulation. Some injectable pharmaceutical compositions are suspensions, solutions, or emulsions in oily or aqueous media and may contain formulations such as suspending agents, stabilizers, and / or dispersants. Solvents suitable for injectable pharmaceutical compositions include, but are not limited to, lipophilic solvents and fatty oils (such as sesame oil), synthetic fatty acid esters (such as ethyl oleate or triglycerides), and liposomes. Aqueous injectable suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical preparation to allow for the preparation of highly concentrated solutions.

[0378] The lipid moiety has been used in nucleic acid therapy in various methods. In one method, nucleic acids are introduced into a pre-formed liposome or lipid complex made from a mixture of cationic and neutral lipids. In another method, a DNA complex with monocationic or polycationic lipids is formed in the absence of neutral lipids. In some embodiments, the lipid moiety is selected to increase the distribution of the agent to specific cells or tissues. In some embodiments, the lipid moiety is selected to increase the distribution of the agent to adipose tissue. In some embodiments, the lipid moiety is selected to increase the distribution of the agent to muscle tissue.

[0379] In some embodiments, the pharmaceutical compositions provided herein comprise a polyamine compound or a lipid moiety complexed with a nucleic acid. In some embodiments, such formulations comprise one or more compounds, each individually having a structure defined by formula (Z), or a pharmaceutically acceptable salt thereof.

[0380]

[0381] Each X a and X b It is C independently each time it appears. 1-6 Alkylene; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least about 80% of the at least n+2 R portions in the formula (Z) compound molecule in the formulation are not H; m is 1, 2, 3, or 4; Y is O, NR. 2 Or S; R 1 It is alkyl, alkenyl, or ynyl; each of which is optionally substituted by one or more substituents; and R 2 It is H, alkyl, alkenyl, or alkynyl; each of which is optionally substituted, and each of which is optionally substituted by one or more substituents; provided that, if n = 0, at least n+3 R moieties are not H. Such formulations are described in PCT Publication WO / 2008 / 042973, which is incorporated herein by reference in its entirety as a lipid formulation. Certain other formulations are described in Akinc et al., Nature Biotechnology 26, 561-569 (May 1, 2008), which is incorporated herein by reference in its entirety as a lipid formulation.

[0382] In some embodiments, the pharmaceutical compositions provided herein are prepared using known techniques, including but not limited to mixing, dissolving, granulating, preparing sugar-coated pellets, grinding, emulsifying, encapsulating, embedding, or tableting processes.

[0383] In some embodiments, the pharmaceutical compositions provided herein are solids (e.g., powders, tablets, and / or capsules). In some such embodiments, solid pharmaceutical compositions comprising one or more oligonucleotides are prepared using ingredients known in the art, including but not limited to starch, sugars, diluents, granulating agents, lubricants, binders, and disintegrants.

[0384] In some embodiments, the pharmaceutical compositions provided herein are formulated as reservoir preparations. Some of these reservoir preparations typically have a longer duration of action than non-reservoir preparations. In some embodiments, such preparations are administered via implantation (e.g., subcutaneously or intramuscularly) or via intramuscular injection. In some embodiments, reservoir preparations are prepared using suitable polymers or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or as slightly soluble derivatives, such as slightly soluble salts.

[0385] In some embodiments, the pharmaceutical compositions provided herein comprise a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Some delivery systems are used to prepare certain pharmaceutical compositions, including those comprising hydrophobic compounds. In some embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0386] In some embodiments, the pharmaceutical compositions provided herein comprise one or more tissue-specific delivery molecules designed to deliver one or more of the pharmaceutical agents provided herein to a specific tissue or cell type. For example, in some embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0387] In some embodiments, the pharmaceutical compositions provided herein comprise a sustained-release system. A non-limiting example of such a sustained-release system is a semi-permeable matrix of a solid hydrophobic polymer. In some embodiments, depending on their chemical properties, a sustained-release system can release the agent over time periods of hours, days, weeks, or months.

[0388] Some injectable pharmaceutical compositions are presented in unit dosage forms, such as in ampoules or in multi-dose containers.

[0389] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a modified oligonucleotide. In some embodiments, the therapeutically effective amount is sufficient to prevent, alleviate, or improve symptoms of the disease or prolong the survival of a subject undergoing treatment.

[0390] In some embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In some embodiments, when administered in vivo, the prodrug is chemically converted to a biologically, pharmaceutically, or therapeutically more active form of the oligonucleotide. In some embodiments, prodrugs are useful because they are easier to administer than their corresponding active forms. For example, in some cases, prodrugs may have higher bioavailability than their corresponding active forms (e.g., by oral administration). In some cases, prodrugs may have improved solubility compared to their corresponding active forms. In some embodiments, prodrugs are less water-soluble than their corresponding active forms. In some cases, such prodrugs have superior ability to be transported across cell membranes, where water solubility is detrimental to migration. In some embodiments, the prodrug is an ester. In some of these embodiments, the ester is metabolically hydrolyzed to a carboxylic acid upon administration. In some cases, the compound containing the carboxylic acid is the corresponding active form. In some embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In some of these embodiments, the peptide is cleaved upon administration to form the corresponding active form.

[0391] In some embodiments, prodrugs are produced by modifying the pharmaceutically active compound so that the active compound will be regenerated after administration in vivo. Prodrugs can be engineered to alter the metabolic stability or transport characteristics of a drug, mask side effects or toxicities, improve the flavor of a drug, or change other characteristics or properties of a drug. Once the pharmaceutically active compound is known, those skilled in the art can engineer prodrugs of the compound by understanding pharmacodynamic processes and in vivo drug metabolism (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392).

[0392] Other routes of administration include, but are not limited to, oral, rectal, mucosal, intestinal, enteric, local, suppository, inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In some embodiments, intrathecal administration is used to achieve local rather than systemic exposure. For example, the pharmaceutical composition can be injected directly into the area of ​​desired effect (e.g., into the kidney).

[0393] Some kits

[0394] A kit is also provided. In some embodiments, the kit comprises one or more compounds containing oligonucleotides with modifications disclosed herein. In some embodiments, the kit can be used to administer the compound to a subject.

[0395] In some embodiments, the kit contains a pharmaceutical composition ready for administration. In some embodiments, the pharmaceutical composition is contained in a vial. Multiple vials (such as 10) may be contained, for example, in a dispensing pack. In some embodiments, the vials are manufactured to be accessible with a syringe. The kit may also contain instructions for using the compound.

[0396] In some embodiments, the kit contains a pharmaceutical composition present in a pre-filled syringe (such as a single-dose syringe with, for example, a 27-gauge 1 / 2-inch needle with a needle guard) rather than in a vial. Multiple pre-filled syringes (such as 10) may be present in, for example, a dispensing pack. The kit may also contain instructions for administering a compound comprising an oligonucleotide with modifications disclosed herein.

[0397] In some implementations, the kit contains modified oligonucleotides as lyophilized pharmaceutical products provided herein and pharmaceutically acceptable diluents. The lyophilized pharmaceutical product is reconstituted in a pharmaceutically acceptable diluent before administration to a subject.

[0398] In some implementations, in addition to compounds containing the modified oligonucleotides disclosed herein, the kit may also contain one or more of the following: syringes, alcohol swabs, cotton balls, and / or gauze pads.

[0399] Some experimental models

[0400] In some implementations, methods are provided for using and / or testing the modified oligonucleotides provided herein in experimental models. Those skilled in the art can select and modify protocols for such experimental models to evaluate the agents provided herein.

[0401] Typically, the modified oligonucleotides are first tested in cultured cells. Suitable cell types include those associated with the cell types from which in vivo delivery of the modified oligonucleotides is desired. For example, suitable cell types for studying the methods described herein include primary or cultured cells.

[0402] In some embodiments, the extent to which modified oligonucleotides interfere with the activity of one or more miR-17 family members is assessed in cultured cells. In some embodiments, inhibition of microRNA activity can be assessed by measuring the levels of one or more predicted or verified microRNA-regulated transcripts. Inhibition of microRNA activity may lead to an increase in miR-17 family member-regulated transcripts and / or proteins encoded by miR-17 family member-regulated transcripts (i.e., the miR-17 family member-regulated transcripts are derepressed). Furthermore, in some embodiments, certain phenotypic outcomes can be measured.

[0403] Those skilled in the art can utilize several animal models to study one or more miR-17 family members in human disease models. Models of polycystic kidney disease include, but are not limited to, models with mutations and / or deletions in Pkd1 and / or Pkd2; and models containing mutations in other genes. Non-limiting exemplary models of PKD containing mutations and / or deletions in Pkd1 and / or Pkd2 include subtype models, such as models containing missense mutations in Pkd1 and models with reduced or unstable expression of Pkd2; inducible conditional knockout models; and conditional knockout models. Non-limiting exemplary PKD models containing mutations in genes other than Pkd1 and Pkd2 include models with mutations in Pkhd1, Nek8, Kif3a, and / or Nphp3. PKD models are reviewed in, for example, Shibazaki et al., Human Mol. Genet., 2008; 17(11):1505-1516; Happe and Peters, Nat Rev Nephrol., 2014; 10(10):587-601; Patel et al., PNAS, 2013; 110(26):10765-10770.

[0404] Some quantitative determinations

[0405] In some embodiments, microRNA levels are quantified in cells or tissues, either in vitro or in vivo. In some embodiments, changes in microRNA levels are measured by microarray analysis. In some embodiments, changes in microRNA levels are measured by one of several commercially available PCR assays, such as... MicroRNA assay (Applied Biosystems).

[0406] The regulation of microRNA activity by anti-miRs or microRNA mimics can be assessed through mRNA microarray mapping. Seed sequences of microRNAs regulated (increased or decreased) by anti-miRs or microRNA mimics are searched to compare the regulation of mRNAs that are microRNA targets with that of mRNAs that are not. In this way, the interaction between anti-miRs and their target microRNAs, or between microRNA mimics and their targets, can be evaluated. In the case of anti-miRs, mRNAs with increased expression levels are screened against seed-matched mRNA sequences containing microRNAs complementary to the anti-miR.

[0407] The regulation of microRNA activity by antimiR compounds can be assessed by measuring the levels of microRNA messenger RNA targets, or by measuring the levels of the messenger RNA itself or the proteins transcribed from it. Antisense repression of microRNAs typically leads to an increase in the levels of microRNA messenger RNA and / or the proteins of their targets; that is, antimiR treatment results in the derepression of one or more target messenger RNAs.

[0408] Example

[0409] The following examples are provided to illustrate some embodiments of the invention more fully. However, they should not in any way be construed as limiting the broad scope of the invention.

[0410] Those skilled in the art will readily employ the basic principles of this discovery to design various compounds without departing from the spirit of the invention.

[0411] Example 1: The role of miR-17 in PKD

[0412] Members of the miR-17 family of the miR-17-92 microRNA cluster are upregulated in a mouse model of PKD. Genetic deletion of the miR-17-92 cluster in the PKD mouse model reduced renal cyst growth, improved renal function, and prolonged survival (Patel et al., PNAS, 2013; 110(26):10765-10770). The miR-17-92 cluster contains six different microRNAs, each with a distinct sequence: miR-17, miR-18a, miR-19a, miR-19-b-1, and miR-92a-1.

[0413] The miR-17-92 cluster comprises two microRNAs: miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Each member of this family shares seed sequence identity, as well as varying degrees of sequence identity outside the seed region. Other members of the miR-17 family are miR-20b, miR-93, miR-106a, and miR-106b. miR-20b and miR-106a are located in the miR-106a-363 ​​cluster on human chromosome X, and miR-93 and miR-106b are located in the miR-106b-25 cluster on human chromosome 7. The sequences of the miR-17 family members are shown in Table 1.

[0414] Table 1: miR-17 family of microRNAs

[0415]

[0416] The anti-miR-17 compound RGLS4326 was discovered through screening chemically diverse and rationally designed anti-miR-17 oligonucleotide libraries to obtain optimal pharmaceutical properties. RGLS4326 preferentially distributes to kidney and collecting duct-derived cysts, displacing miR-17 from translationally active multimers and de-inhibiting multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, after subcutaneous administration, RGLS4326 attenuates cyst growth in human ADPKD models and various PKD mouse models. A Phase 1 single-dose escalation (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a Phase 1 multiple-dose escalation (MAD) clinical trial in healthy volunteers in May 2018. A Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) was initiated in October 2020.

[0417] Following the initiation of the Phase 1 MAD clinical trial, non-clinical toxicology studies revealed central nervous system (CNS)-related findings at high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or collapse. 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 potential 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 nM–600 nM (0.9 μg / mL–1.8 μg / mL) based on patch-clamp activity. AMPA receptors are ion channels at excitatory synapses in the CNS that mediate rapid excitatory neurotransmission and are therefore a key component of all neuronal networks. This interaction with AMPA receptors could explain the CNS-mediated findings observed in a non-clinical toxicology model at high doses of RGLS4326.

[0418] Example 2: Screening of anti-miR-17 compounds with reduced AMPA receptor binding

[0419] RGLS4326 has the following sequence and chemical modification pattern: A S G S C M A F C F U F U M U S G SIn this model, the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, and the nucleoside followed by the subscript "S" is an S-cEt nucleoside. Each cytosine is unmethylated, and all bonds are phosphate thioester bonds. Chemical modifications and length variants of RGLS4326 were designed and screened to identify compounds that retain the potency and pharmacokinetic characteristics of RGLS4326 and exhibit reduced binding to the AMPA receptor (AMPA-R).

[0420] Compared to RGLS4326, a library of compounds with different chemical modifications, nucleobase sequences, and lengths was designed.

[0421] Table 2: Anti-miR-17 Library

[0422]

[0423]

[0424] The activity of antimiR-17 compounds was evaluated in a radioligand binding assay, which was performed in the presence of increased concentrations of the antimiR-17 compounds. 3 [H]AMPA ligand binds to AMPA-R present on the synaptic membrane of the rat brain. AntimiR-17 compounds with affinity for AMPA-R will bind to [ 3 H]AMPA ligands bind and compete with them for binding.

[0425] The determination was performed according to previously published methods (Honore et al., J Neurochem., 1982, 38(1):173-178; Olsen et al., Brain Res., 1987, 402(2):243-254). A 5.0 nM ligand [ 3 [H]AMPA, 1.0 mM of the nonspecific ligand L-glutamate, and μM concentrations of antimiR compounds were incubated with synaptic membranes prepared from the cerebral cortex of Wistar rats for 90 min. The compounds shown in Table 2 were tested in three experiments. AntimiR compounds targeting microRNAs other than miR-17 were used as control compounds (RG5124 targets miR-33a; RG5365 targets let-7a; RG8093 targets miR-214). RGLS4326 and RG-NG-1001 were also tested in each experiment because they were shown to bind to and inhibit AMPA-R activity. 3 The amount of H]AMPA ligands was quantified by radioligand binding and is shown in Tables 3, 4, and 5. As the data show, the compounds exhibit varying abilities to inhibit the binding of radiolabeled ligands to AMPA-R.

[0426] Table 3: Inhibition of ligand binding to AMPA-R, Experiment #1

[0427]

[0428] Table 4: Inhibition of ligand binding to AMPA-R, Experiment #2

[0429]

[0430] Table 5: Inhibition of ligand binding to AMPA-R, Experiment #3

[0431]

[0432] To assess the functional antagonism of antimiR-17 oligonucleotides to AMPA-R, certain oligonucleotides were tested using a manual whole-cell patch-clamp technique that records membrane currents as a measure of AMPA-R activity.

[0433] Manual whole-cell patch-clamp studies were conducted by Metrion Biosciences (Cambridge, UK). Whole-cell voltage-clamp experiments were performed at room temperature (18°C–21°C) using Patchmaster software (HEKA Elektronik) and an EPC10 patch-clamp amplifier. Glass patch pipettes were made of borosilicate glass capillaries (Harvard Apparatus) with resistances between 1.4 MΩ and 2.5 MΩ. Membrane currents were recorded using whole-cell patch-clamp techniques. GluA1 / GluA4EZ cells were clamped at a holding potential of -80 mV and subjected to membrane currents induced by 10 μM (s)-AMPA delivered using the VC38 perfusion system (ALA Scientific Instruments). Minimum current amplitude values ​​were measured each time 10 μM (s)-AMPA was applied. The fractional change in current amplitude produced by each concentration of the compound was calculated relative to the control current (pre-compound) and expressed as a percentage change (inhibition %) per cell. The compounds tested are shown in Table 6. RGLS4326 was tested in a separate study from all other compounds listed in Table 6.

[0434] As shown in Table 6, compared to RGLS4326, based on human... Manual whole-cell patch-clamp studies in GluA1 / GluA4EZ cells showed that compounds RG-NG-1015, RG-NG-1016, and RG-NG-1017 exhibited reduced functional antagonism against AMPA-R.

[0435] Table 6: Functional antagonistic effects of AMPA-R in whole-cell patch-clamp studies

[0436]

[0437] Example 3: The relationship between nucleobase characteristics and AMPA-R binding

[0438] As demonstrated by AMPA-R binding and whole-cell patch-clamp studies, guanosine, present at the 3'-terminus of the anti-miR-17 oligonucleotide at the site complementary to the first nucleotide of miR-17, influences the functional antagonism of AMPA-R. Like guanosine, adenosine is a purine; however, adenosine does not inhibit AMPA-R. Guanosine and adenosine are similar in several properties except for hydrogen bonding; therefore, differences in hydrogen bonds at positions 1, 2, and 6 of the purine bases were assessed. The tested purine nucleobases are shown in... Figure 1 And in Table 7. In the “Purine Location” column of Table 7, “A” indicates the location of the purine as a hydrogen acceptor, and “D” indicates the location of the purine as a hydrogen donor. In the “Purine Location” column of Table 7, “N” indicates a neutral location that is neither a hydrogen acceptor nor a donor. Different 2'-sugar moieties on the purine nucleotide bases were also tested to assess the effect of the 2'-sugar moieties’ chemical properties on the ability of the purine nucleotide bases to inhibit AMPA-R.

[0439] Table 7: AntimiR-17 compounds with different nucleobase and sugar moiety chemical properties

[0440]

[0441]

[0442] Compounds were tested in the radioligand binding assay described herein to determine the interaction between the antimiR-17 compound and […]. 3 The ability of H]AMPA ligands to bind and compete with them. As shown in Table 8, a correlation was observed between the inhibition of ligand binding to +AMPA-R and the presence of a hydrogen bond acceptor at the purine position #6 of the 3'-terminus of the oligonucleotide. For example, compounds with guanosine or inosine at the 3'-terminus result in inhibition of ligand binding to AMPA-R. Compounds with a 3'-terminal nucleotide containing a hydrogen bond acceptor at the purine position #6 (e.g., RG-NG-1037 and RG-NG-1039) are unlikely to inhibit ligand binding to AMPA-R.

[0443] Table 8: Inhibition of ligand binding to AMPA-R

[0444]

[0445] Example 4: AntimiR-17 compounds with reduced AMPA-R binding and inhibition did not show CNS toxicity in high-dose studies.

[0446] 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 escalating doses (100 mg / kg, 450 mg / kg, and 2000 mg / kg). As shown in Table 9, while escalating doses of RG-NG-1001 and RGLS4326 resulted in ataxia, somnolence, and, in the case of RGLS4326, loss of consciousness at the highest dose, no CNS toxicity was observed with RG-NG-1015, RG-NG-1016, or RG-NG-1017.

[0447] Table 9: Anti-miR-17 compounds and CNS-related findings

[0448]

[0449] Example 5: Maximum Tolerable Dose (MTD) Studies and Comparative Dose Assessments of Different Compounds

[0450] Data from the following studies further support the idea that AMPA-R antagonism is the cause of CNS toxicity and mortality observed in previous toxicity studies of RGLS4326.

[0451] Study 1: Maximum Tolerated Dose (MTD) Study and Comparative Dose Assessment of RG-NG-1017, RGLS4326, and RG-NG-1001

[0452] Compounds (RG-NG-1017, RGLS4326, RG-NG-1001) were evaluated in a preliminary maximum tolerated dose (MTD) study (discussed below). RG-NG-1017, RGLS4326, and RG-NG-1001 were initially evaluated at four dose levels, respectively. RG-NG-1017 was evaluated as a non-AMPA-R-binding compound, compared to AMPA-R-bound RGLS4326 and RG-NG-1001. Male C57Bl / 6J mice (Jackson Laboratories) (6–7 weeks old) were used in this study. Mice were randomized to the treatment group, and the study was blinded. Animals were acclimatized for at least 5 days and housed on a 12-hour light / dark cycle (7:00 AM light). No more than four mice were housed per cage in a ventilated cage system. The diet consisted of standard rodent food and free access to water.

[0453] MTD Preliminary Study

[0454] The following parameters were used in this study:

[0455] 1. Route of administration: Intraventricular (ICV) administration of RG-NG-1017, RG-NG-1001, and RGLS4326.

[0456] 2. Dosage volume: 4 μL

[0457] 3. Formulation: Free of media, Ca 2+ and Mg 2+ dPBS

[0458] 4. Dosage frequency: once

[0459] 5. Study duration: 8 days

[0460] 6. Number of groups: 3

[0461] 7. Number of animals per group: (2-4 animals per group)

[0462] 8. Total number of animals: 54

[0463] For ICV administration, mice were anesthetized and positioned for injection. The skin above the skull was incised, and a small hole was drilled in the skull above the target using a micro-drill. Stereotactic coordinates were anterior-posterior (AP), -0.4 mm; medial-lateral (ML), + / -1.0–1.5 mm; dorsoventral (DV), 3.0 mm from the anterior fontanelle, for injection into the right and left ventricles (Hironaka et al., 2015). 4 μl was injected unilaterally into the animal's right ventricle. The compound was injected over 1–2 minutes, and the needle was left in place for 0.5–1 minute before removal. The incision was sutured with sutures, wound clips, or VetBond.

[0464] Animals were monitored for 7 days following ICV treatment (day 0), during which daily health checks, weight, and mortality were recorded. On day 7, the brain and kidneys were collected, fixed (in 10% formalin), and stored for histological examination.

[0465] Results from the MTD study are shown in Table 10 and Figure 3As shown in the figure. All animal deaths reported occurred within the first 5–8 hours after ICV injection. Mice injected with 2.5 μg RG4326 reported some immediate signs of respiratory distress and were provided with heating pads. RG-NG-1017 (a non-AMPA-R binding compound) was well tolerated at high doses, and the MTD of this compound was not determined (0 deaths in the 600 μg, 100 μg, or 50 μg groups; 1 death in the 300 μg group). For RG4326 and RG-NG-1001 (e.g., 600 μg, 300 μg, 100 μg), 100% mortality was observed at high doses; furthermore, for both AMPA-R binding compounds, 100% mortality was observed at 50 μg and 25 μg. The MTD of RG-NG-1001 was not reached in this study and is expected to be below 2.5 μg. ICV predicts the MTD of RG4326 to be approximately 2.5 < 5.0 μg. According to reports, all the animals fully recovered by the second day of observation.

[0466] Table 10: Summary Results from the 7-Day MTD Study

[0467]

[0468] Maximum tolerated dose (MTD) study of RGLS4326

[0469] A second MTD study of RGL4326 was conducted via ICV to evaluate the dosage selection of the compound for evaluation in a disease model (Table 11). In this study, different mouse strains (Swiss:Rjorl male mice, 5 weeks old, derived from Janvier) were evaluated. Mice were anesthetized with isoflurane (5% for induction and 2% for maintenance, at 100% O2) and administered 5 mg / kg sc carbofen. They were then placed in a stereotactic frame. A midline sagittal incision was made in the scalp, and a hole was drilled in the skull above the left ventricle. A stainless steel cannula (0.51 mm outer diameter) was stereotactically placed in the left ventricle at the following coordinates: +0.5 mm posterior to the anterior fontanelle, L ± 0.7 mm, V = -2.7 mm. After a 2-minute delay allowing brain tissue to slide through the cannula, 4 μL of a solution containing 0.625 mg / mL RG4326 was slowly infused over 2 minutes. After infusion, the cannula was left in place for another 5 minutes to prevent backflow of the solution along the cannula's path. Mice were given 5 mg / kg carbofenac subcutaneously at 24 and 48 hours post-surgery. Mice were monitored for 3–7 days post-surgery (starting 24 hours after ICV administration), and their weight was measured daily to assess their health. For mice monitored within 7 days, their weight was measured on days 1 and 7 post-surgery to assess their health.

[0470] Table 11: Design of the MTD study for RGLS4326

[0471]

[0472] In Study 1, six mice were injected with 4 μL of a 0.625 mg / mL solution (total 2.5 μg per ICV injection; Table 10). At the end of anesthesia, the mice were kept in a lateral recumbent position. They were quiet for the first few hours post-surgery, with some periods of scratching. No toxic effects were observed in the six mice administered the solution at 24, 48, or 72 hours. In Study 2, four mice were injected with four different doses of RGLS4326 (0.75 mg / mL, 1.0 mg / mL, 1.25 mg / mL, and 1.875 mg / mL, in 4 μL volumes). One mouse receiving the highest dose (1.875 mg / mL, or 7.5 μg / mouse) was found dead approximately 24 hours after the ICV injection. All other mice remained healthy until the end of the preliminary study (7 days post-administration).

[0473] The combined results of Studies 1 and 2 indicate that RG4326 was generally well tolerated in the tested subjects, but only at significantly lower doses than RG-NG-1017 (see [link to study 1]). Figure 3 ).

[0474] Table 12 summarizes the MTD data of RGLS4326 in the mouse models of Studies 1 and 2. Based on these results from Study 2, the predicted MTD of RGLS4326 in the Swiss:Rjorl mouse strain is approximately 4 μg.

[0475] Table 12: 7-day survival data from MTD studies 1 and 2

[0476]

[0477] In summary, the compounds RG-NG-1017, RGLS4326, and RG-NG-1001 were evaluated in two MTD studies, demonstrating a significant difference in tolerability between the non-AMPA-R bound compound (RG-NG-1017) and the AMPA-R bound compounds (RGLS4326, RG-NG-1001) (see [link to MTD study]). Figure 3Although one death occurred at an ICV dose of 300 μg, the MTD of RG-NG-1017 was not determined because no deaths occurred at the higher test dose of 600 μg. Furthermore, no effect on mortality was observed at doses of 100 μg and 50 μg with RG-NG-1017. By comparison, the AMPA-R combination compounds RGLS4326 and RG-NG-1001 had a significant effect on mortality, with no surviving animals in the test dose range of 25 μg to 600 μg. A trend toward improved survival was observed at lower doses of RGLS4326 (10 μg), with a survival rate of 50% in animals treated with RGLS4326 at 5 μg and 100% at 2.5 μg. Similarly, with RG-NG-1001 (which showed stronger AMPA-R binding compared to RGLS4326), 100% mortality was evident at a low dose of 5 μg, with a trend toward improved survival at 2.5 μg. The results from Study 1 regarding RGLS43426 were further confirmed in a second MTD study using different mouse strains (Study 2). This study found that there may be modest differences in RGLS4326 tolerance between strains; in Study 1 using C57 / Bl / 6J, the observed survival only affected mice at the highest dose of 7.5 μg compared to 5 μg. However, these results still support the view that the MTD of AMPA-R-bound RGLS4326 occurs between approximately 2.5 μg and 5–7.5 μg (depending on the strain), compared to the significantly higher MTD (at least >40-fold or higher) of non-AMPA-R-bound RG-NG-10017. Figure 3 ).

[0478] Example 6: In vitro and in vivo efficacy of anti-miR-17 compounds

[0479] The in vitro potency of certain compounds was evaluated using a miR-17 luciferase sensor assay. This assay employed a luciferase reporter vector for miR-17, containing two fully complementary miR-17 binding sites tandemly 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 inhibited luciferase signaling. HeLa cells were then treated alone with anti-miR-17 oligonucleotides at concentrations of 0.045 nM, 0.137 nM, 0.412 nM, 1.23 nM, 3.70 nM, 11.1 nM, 33.3 nM, 100 nM, and 300 nM. Luciferase activity was measured at the end of the 18-24 hour transfection period. RG5124 was included as a control compound. As shown in Table 13, these compounds exhibited similar EC50 activity compared to RGLS4326 in vitro. 50It inhibits miR-17 function and de-represses miR-17 luciferase reporter gene activity.

[0480] Table 13: Inhibition of miR-17 in luciferase assay

[0481]

[0482]

[0483] As shown in Figure 4, RG-NG-1015 showed a similar EC50 in HeLa cells compared to RGLS4326 in vitro. 50 Values ​​inhibit miR-17 as well as miR-20a, miR-106a and miR-93.

[0484] Compared to RGLS4326 in vitro, RG-NG-1015 also exhibits similar ECG activity. 50 The value is to repress the full-length 3' untranslated region (UTR) of the miR-17 direct target genes PKD1 and PKD2.

[0485] The activity of certain compounds was assessed using a mouse miR-17 pharmacodynamic signature (miR-17PD-Sig), which consists of the expression of 18 unique miR-17 target genes normalized by six reference housekeeping genes to provide an unbiased and comprehensive assessment of miR-17 activity. The mouse miR-17PD-Sig score is the calculated mean of the individual log2 fold change (normalized by the six housekeeping genes) of the 18 genes compared to a simulated transfection (Lee et al., Nat. Commun., 2019, 10, 4148).

[0486] As shown in Table 13, compared with in vitro RGLS4326, the tested oligonucleotides showed similar EC values. 50 The values ​​inhibited miR-17 function and depressed the expression of multiple direct miR-17 target genes in normal and PKD kidney cell lines (mouse and human) (as measured by miR-17 PD-signature). No PD-Sig (77.2, indicated by "*") was generated for RGLS4326 in mIMCD3 cells in this experiment; values ​​in Table 14 are reported by Lee et al., Nat. Commun., 2019, 10, 4148. Blank cells in the table indicate compounds not tested in the specific cell lines.

[0487] Table 14: miR-17PD-Sig in normal and PKD cell lines

[0488]

[0489] In vivo potency was assessed using the microRNA multimer translocation assay (miPSA). This assay was used to determine the extent to which compounds directly bind to miR-17 targets in the kidneys of normal and PKD mice. miPSA relies on the principle that active miRNAs bind to their mRNA targets in translationally active high molecular weight (HMW) multimers, while inhibited miRNAs are present in low MW (LMW) multimers. Treatment with anti-miRs causes microRNAs to transition from HMW multimers to LMW multimers. Therefore, miPSA provides a direct measurement of microRNA target binding via complementary anti-miRs (Androsavich et al., Nucleic Acids Research, 2015, 44:e13).

[0490] Wild-type mice were administered single doses of 0.3 mg / kg, 3 mg / kg, or 30 mg / kg. Kidney tissue was collected seven days later for miPSA. The mean replacement fractions 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 replaced miR-17 from translationally active multimers (as measured by miPSA) in normal mouse kidneys.

[0491] Table 15: miPSA Permutation Score

[0492]

[0493] In addition, as shown in Table 16 and Figures 5A to 5D As shown, RGLS4326 and RG-NG-1015 exhibited similar pharmacokinetic and target binding characteristics (as measured by miPSA) after a single subcutaneous administration in C57BL6 mice.

[0494] Table 16: Pharmacokinetic and Target Binding Characteristics

[0495]

[0496] Example 7: Efficacy of RG-NG-1015 in an experimental model of ADPKD

[0497] 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 containing a germline subtype Pkd1 mutation (the mouse equivalent of the human PKD1-R3277C (RC mutation)) in one allele and loxP sites flanking exons 2 and 4 of Pkd1 in the other allele. KspCre-mediated recombination was used to delete fluxed Pkd1 exons, generating a compound mutant mouse with a tubule-specific somatic null mutation in one allele and a germline subtype mutation in the other. This is an aggressive but long-lived model of ADPKD (Hajarnis et al., Nat. Commun., 2017, 8, 14395).

[0498] On days 8, 10, 12, and 15 of age, sex-matched Pkd1-F / RC mice were administered subcutaneous injections of RGLS4326 (n=8; 4 males and 4 females per treatment group), RG5124 (n=8), RG-NG-1015 (n=8), or PBS (n=8). Mice were sacrificed at 18 days of age, and kidney weight, body weight, cyst index, serum creatinine level, and blood urea nitrogen (BUN) level were measured. BUN level is a marker of renal function. Higher BUN levels are associated with poorer renal function; therefore, a decrease in BUN level is an indicator of reduced renal injury and functional improvement. Statistical significance was calculated using a one-way ANOVA with Dunnett multiple correction.

[0499] 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 ratio of kidney weight to body weight (KW / BW ratio) was significantly lower in Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015 than in Pkd1-F / RC mice treated with PBS. Figure 2A Compared with mice treated with PBS, mean BUN levels were significantly lower in Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively. Figure 2B Compared to mice treated with PBS, the mean serum creatinine level in Pkd1-F / RC mice was lower in mice treated with RGLS4326 and RG-NG-1015, respectively; however, the decrease was not statistically significant. Figure 2CTreatment with the control oligonucleotide RG5124 did not reduce the kidney weight to body weight ratio, serum creatinine, or serum BUN, thus demonstrating that the results observed with RGLS4329 and RG-NG-1015 were specific for the inhibition of miR-17.

[0500] Table 17: Efficacy of RG-NG-1015 in a mouse model of ADPKD

[0501]

[0502] The efficacy of RG-NG-1015 was also evaluated, both alone and in combination with tolvaptan, in the Pcy / DBA mouse model of PKD. Pcy / DBA mice exhibit slowly progressive PKD caused by missense mutations in the Nphp3 gene, a cause of adolescent renal wasting disease in humans (akahashi et al., J Am Soc Nephrol 1991, 1:980–989; Olbrich et al., NatGenet 2003, 34:455–459). In Pcy mice, cysts originate from the distal tubules, and the entire nephron segment is diffusely occupied by disease-promoting cysts by 30 weeks of age, often associated with ESRD (Nagao et al., Exp Anim 2012, 61:477–488). In particular, male Pcy / DBA mice have been used to characterize the pharmacological properties of many investigational products for the treatment of ADPKD, including tolvaptan and the first-generation anti-miR-17RGLS4326 (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 initiating treatment at approximately 5 weeks of age and continuing until 15–30 weeks of age.

[0503] like Figure 6A and Figure 6BAs outlined herein, five groups of male Pcy / DBA mice (n=13 / treatment group) were subcutaneously treated every two weeks (Q2W) with PBS or RG-NG-1015 at 25 mg / kg, 5 mg / kg, 1 mg / kg, or 0.2 mg / kg. Two groups of male Pcy / DBA mice (n=13 / group) were also treated with RG-NG-1015 at 50 mg / kg every four weeks (Q4W) or at 12.5 mg / kg weekly (QW). Four additional groups of male Pcy / DBA mice (n=13 / group) were subcutaneously treated with PBS or RG-NG-1015 at 25 mg / kg, 5 mg / kg, or 1 mg / kg Q2W in any combination with 0.3% (w / w) tolvaptan. One 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 randomly assigned to the treatment group at 5 weeks of age and began treatment for 17 weeks at 6 weeks of age, and were sacrificed 7 days after the final treatment. Kidney weight, body weight, renal cyst index, and urinary Ngal / Cr ratio (Ngal / Cr) were measured. Urinary Ngal / Cr is a biomarker of kidney injury.

[0504] like Figures 6C to 6E As shown in Tables 18 to 20, RG-NG-1015 was effective in the Pcy / DBA mouse model of PKD at various doses and regimens, and provided an additive or synergistic effect when used in combination with tolvaptan. Specifically, RG-NG-1015 treatment significantly reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index in Pcy / DBA mice in a dose-dependent manner (Tables 18 and 20). Figures 6C to 6E Furthermore, in Pcy / DBA mice, treatment with RG-NG-1015 at similar total doses (212.5 mg–250 mg per mouse during the study period) but with different dosing regimens (including QW, Q2W, and Q4W) reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index to similar levels (Table 19). Figures 6C to 6E 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 and tolvaptan further reduced mean KW / BW, urinary Ngal / Cr and renal cyst index (Table 20). Figures 6C to 6E As indicated by the Bliss additive analysis, the observed effects of the drug combinations on KW / BW, urinary Ngal / Cr, and renal cyst index were synergistic, primarily additive, and less than additive, respectively (Table 20).

[0505]

[0506]

[0507] Example 8: Metabolites of RG-NG-1015

[0508] In vitro and in vivo studies were conducted to investigate the metabolism of RG-NG-1015. For both in vitro and in vivo samples, tissue samples were homogenized in lysis buffer on ice, and RG-NG-1015 and / or metabolites were isolated from plasma, tissue homogenates, or urine via liquid-liquid extraction and solid-phase extraction steps. Calibration samples containing known amounts of RG-NG-1015 were extracted in parallel with test tissue homogenates, plasma, or urine samples. The molecular weights (MW) of RG-NG-1015 and potential metabolites were calculated based on MS signals and compared with theoretical values.

[0509] 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 at 5 μM in these matrices with kidney and liver homogenates (equivalent to 307 μg / g tissue) or serum samples (equivalent to 15.3 μg / mL) at 37 °C for 24 h. RG-NG-1015 and metabolites were then extracted and analyzed by HPLC-TOF.

[0510] In vivo metabolism was assessed in the liver and kidneys after a single dose of RG-NG-1015 was administered to CD-1 mice, and in plasma, tissues, and urine after single and / or repeated administrations to monkeys. CD-1 mice received a single SC dose of RG-NG-1015 at 2000 mg / kg, and monkeys received up to five weekly SC doses of RG-NG-1015 at 15 mg / kg, 75 mg / kg, or 150 mg / kg. RG-NG-1015 and its metabolites were then extracted and analyzed by HPLC-TOF.

[0511] RG-NG-1015 is sequentially hydrolyzed from the 3' and 5' ends to produce 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 in that they sequentially remove the terminal nucleotides and terminate with hydroxyl groups at the 3' and 5' ends. No 5' terminal short polymers (from N-5 to N-8) or 3' terminal short polymers (from N-6 to N-8) were observed.

[0512] Table 21: Sequences, precise mass, m / z, and charge state of RG-NG-1015 and its potential metabolites

[0513]

[0514] Example 9: Clinical study evaluating the safety, tolerability, pharmacodynamics, and pharmacokinetics of RG-NG-1015 in patients with autosomal dominant polycystic kidney disease.

[0515] A. Overview of Research Design

[0516] This was a phase Ib, double-blind, placebo-controlled, multiple escalation dose (MAD) study in which RG-NG-1015 or placebo was administered subcutaneously (SC) to approximately 36 subjects diagnosed with Mayo imaging classification 1C, 1D, or 1E autosomal dominant polycystic kidney disease (ADPKD) (based on MRI obtained during screening or a previous MRI with a documented Mayo classification obtained within 5 years of screening). Subjects were required to sign an informed consent form (ICF) and were assessed according to inclusion / exclusion criteria during the screening period. Patients meeting all inclusion / exclusion criteria were centrally randomized 3:1 to receive RG-NG-1015 or placebo x7 doses via subcutaneous (SC) injection every two weeks (Q2W).

[0517] Cohort 1 (12 subjects): 1 mg / kg RG-NG-1015 or placebo

[0518] Cohort 2 (12 subjects): 2 mg / kg RG-NG-1015 or placebo

[0519] Cohort 3 (12 subjects): 3 mg / kg RG-NG-1015 or placebo.

[0520] The investigational drug is administered via SC injection at the clinic by the investigator or another qualified and trained research center staff member, and is subject to at least 4 hours of safety monitoring by research center staff.

[0521] Participants were involved in the study for a total of 141 days. The study consisted of a 28-day screening period (day -28 to day -1), followed by an 85-day treatment period (day 1 to day 86), and then a 28-day follow-up period (day 92 to day 113). During the treatment period, outpatient visits were conducted on days 1 and / or 2 (day 1, dose 1), day 15 (dose 2), day 29 (dose 3), day 43 (dose 4), day 57 (dose 5), day 71 (dose 6), and day 85 and / or day 86 (day 85, dose 7). During the follow-up period, outpatient visits were conducted on days 92, 99, and 113 (the end of the study visits).

[0522] B. Subject population

[0523] This study consisted of three consecutive cohorts, with 12 subjects in each cohort being centrally randomized in a 3:1 ratio to receive RG-NG-1015 1 mg / kg, 2 mg / kg, or 3 mg / kg or placebo x7 via SC injection every two weeks (Q2W) (a total of 36 subjects).

[0524] Inclusion criteria

[0525] Participants must meet all of the following inclusion criteria to participate in the study:

[0526] 1) The signatory of the informed consent form must be between 18 and 70 years old (inclusive);

[0527] 2) Diagnosis of ADPKD (based on Mayo imaging classification 1C, 1D or 1E, based on magnetic resonance imaging [MRI] obtained during screening, or a previous MRI with a recorded Mayo classification obtained within 5 years of screening);

[0528] 3) Estimated glomerular filtration rate (eGFR) is between 30-90 mL / min / 1.73 M. 2 between;

[0529] 3) Body Mass Index (BMI) between 18 and 35 kg / m² 2 ;

[0530] 4) If the subject has hypertension, the antihypertensive regimen must be stable for at least 28 days before randomization, and the blood pressure must be adequately controlled before randomization;

[0531] 5) Screen for the following hematological and clinical chemistry results:

[0532] a) Platelet count is within the normal range

[0533] b) Total bilirubin and direct bilirubin <1.5x the upper limit of normal (ULN), unless the bilirubin elevation is associated with a known benign condition (e.g., Gilbert's syndrome).

[0534] c) Alanine aminotransferase (ALT) < 1.5x ULN,

[0535] d) Aspartate aminotransferase (AST) <1.5x ULN,

[0536] e) Alkaline phosphatase (ALP) < 1.5 x ULN,

[0537] f) γ-glutamyltransferase (GGT) <1.5x ULN.

[0538] 6) You must understand and agree to the research process as explained in the Informed Consent Form (ICF), and be willing and able to comply with the protocol.

[0539] 7) Female subjects of reproductive potential must not breastfeed and must not plan to become pregnant within 28 days of the last dose of the study drug. Heterosexual female subjects of reproductive potential must agree to use one of the following highly effective methods of contraception (i.e., with a failure rate of <1% when used consistently and correctly) from screening to 28 days of the last dose of the study drug:

[0540] a) Had an intrauterine device (IUD) or intrauterine system (IUS) in place for at least 3 months prior to randomization.

[0541] b. The partner has undergone vasectomy. A partner's vasectomy is considered highly effective only if the partner is the only sexual partner of the female participant of reproductive potential and underwent vasectomy 6 months or more prior to randomization.

[0542] c. At least 3 months prior to randomization, use stable hormonal contraception (approved oral, transdermal, or long-acting regimens) that is associated with ovulation suppression.

[0543] 8) Female subjects who are not of reproductive potential must undergo one of the following sterilization procedures at least 6 months prior to the first dose of the study drug:

[0544] a. Hysterectomy

[0545] b. Bilateral oophorectomy

[0546] c. Bilateral fallopian tube obstruction

[0547] d. Bilateral salpingectomy

[0548] Or, the patient has not menstruated for at least one year after menopause before the first dose of the study drug.

[0549] 9) Any non-vasectomized heterosexual male participant must consent to the use of spermicide-containing condoms. (For heterosexual men who have undergone vasectomy, no restrictions apply if they had the procedure 6 months or more prior to the start of the study. Heterosexual men who had vasectomy less than 6 months prior to the start of the study must adhere to the same restrictions as heterosexual men who have not undergone vasectomy.)

[0550] 10) Male and female participants must agree not to donate sperm or preserve eggs (ovum cells) for 1 to 28 days after the last dose of the study drug.

[0551] 11) You must agree not to donate blood within 28 days prior to randomization, or from 7 days after randomization until the end-of-study visit (EOS).

[0552] Exclusion criteria

[0553] Participants who met any of the following criteria were excluded from the study:

[0554] 1) Administer tolvaptan 28 days prior to randomization;

[0555] 2) The subject is mentally incapacitated or has significant emotional problems;

[0556] 3) Any medical condition or social environment that the researcher believes may make it impossible for the subject to complete the study or to comply with the study process and requirements, or may pose a risk to the safety of the subject;

[0557] 4) A history of alcohol poisoning or drug abuse within the two years prior to screening;

[0558] 5) Active infection of the urinary tract (e.g., kidneys, bladder, etc.);

[0559] 6) Known infection with hepatitis B, hepatitis C, or human immunodeficiency virus (HIV);

[0560] 7) Has only one kidney or is a kidney transplant recipient;

[0561] 8) History of malignant tumors, excluding squamous or basal cell carcinoma and skin cancer that have been successfully treated;

[0562] 9) A history of significant clinical response to oligonucleotide compounds, as believed by researchers;

[0563] 10) Tattoos or scars at or near the SC injection site, or any other condition that the investigator believes may interfere with the examination of the injection site;

[0564] 11) Participation in another clinical trial and / or exposure to any investigational drug or therapy approved for investigational use within 28 days or 5 half-lives (whichever is longer) of the study drug administration. The 28-day or 5-half-life window is calculated from the last administration date in the previous study to day 1 of the current study.

[0565] C. Pharmaceutical Products

[0566] RG-NG-1015 is provided in a 2 mL clear glass vial containing sufficient volume to extract a labeled volume of 1 mL of RG-NG-1015 at 150 mg / mL in 0.3% saline. The placebo injection is provided in a 2 mL clear glass vial containing sufficient volume to extract a labeled volume of 1 mL of riboflavin at 1.5 μg / mL in 0.9% sodium chloride. Both the RG-NG-1015 and placebo solutions are clear to pale yellow.

[0567] D. Application

[0568] RG-NG-1015 and placebo were administered as a bolus injection into the anterior abdominal wall via subcutaneous (SC) injection, following standard care procedures for this site. The injection was rotated to different quadrants of the abdomen on each dosing day. The study drug was administered by qualified and trained research center staff. Because the volume of the study drug varies considerably at dose levels, the following guidelines must be followed: the maximum volume per injection must not exceed 2 mL (e.g., a 6 mL dose requires three 2 mL injections in the same quadrant of the abdomen).

[0569] E. Endpoint

[0570] The main objective and endpoint of this study are:

[0571]

[0572] The secondary objectives and endpoints of this study are:

[0573]

[0574]

[0575] The exploratory goals and endpoints of this study are:

[0576]

[0577] F. Evaluation

[0578] Clinical and safety assessments at pre-specified time points during screening and the study include:

[0579] • Demographics, medical history, and concomitant medications,

[0580] • Height and weight measurement

[0581] • Vital signs (body temperature, sitting systolic and diastolic blood pressure, heart rate, and respiratory rate),

[0582] • Physical examination (comprehensive and limited),

[0583] • SARA assessment to detect potential CNS damage caused by the investigational drug.

[0584] • Safety laboratory tests (hematological complete blood count, chemometome, urinalysis, coagulation and lipid profile),

[0585] ·12-lead ECG,

[0586] • ADPKD gene testing

[0587] • Plasma sample testing for C3a and Bb complement,

[0588] • Testing of anti-drug antibodies in plasma samples

[0589] • Urine biomarker testing (PC1, PC2, NGAL, KIM-1)

[0590] • Kidney function tests (eGFR calculation and UACR, SCr, BUN tests),

[0591] ○ During the screening period, eGFR was calculated using the 2021 CKD-EPI creatinine-cystatin C age-sex equation (Inker, 2021).

[0592] • Plasma and urine pharmacokinetic tests

[0593] ○ Plasma and urine concentrations compared to time data were used to derive the following PK parameters: C max T max AUC 0-24 AUC inf (where it is computable) AUC tau t 1 / 2 CL / F, V z / F, fe, and Ae, and appropriate additional PK parameters),

[0594] Plasma PK samples taken on days 1 and 85 prior to administration should be obtained within 60 minutes prior to administration. Plasma samples taken on days 1 and 2, and days 85 and 86 post-administration should be obtained within the following timeframes: 2 hours, 4 hours, 6 hours and 8 hours ± 15 minutes, 12 hours ± 30 minutes and 24 hours ± 60 minutes.

[0595] Pre-dose PK samples were collected on days 15, 43, and 71. Samples were collected before and 4 hours ± 15 minutes after dosing on days 29 and 57. PK samples were collected during EOS visits on days 99 and 113.

[0596] ○ On day 1 (0-24 hours) and day 71 (0-24 hours), 24-hour urine collection for pharmacokinetic analysis should begin immediately after drug administration. Subjects should urinate immediately before administration (before starting 24-hour urine collection). The last urine collected in the 24-hour period is 24 hours after administration.

[0597] • MRI to determine the change in htTKV relative to baseline.

[0598] • Exploratory renal biomarker testing in residual urine (MCP-1 and B2M) and residual serum (e.g., IGFALS, CT-proAVP, N-acetyl-1-methylhistidine, etc.); and exploratory image-based biomarkers (e.g., total cyst volume, number, and / or size distribution, etc.).

[0599] • Acute phase response (albumin, fibrinogen, and high-sensitivity C-reactive protein) tests in plasma samples.

[0600] G. Data Analysis / Statistical Methods

[0601] Results will be summarized and analyzed in tables based on RG-NG-1015 and placebo dose levels (pooled across all cohorts). Descriptive statistics for dose levels and treatment groups will be listed by visit.

[0602] The following analysis will be conducted:

[0603] Safety analysis: Safety data (frequency of TEAEs (treatment-associated adverse events) and SAEs (serious adverse events), safety laboratory tests, vital signs, ECG, and SARA test scores) were descriptively summarized as appropriate by dose level and treatment group. Analysis of safety laboratory tests, vital signs, and ECG included summary statistics over time and descriptive summaries of changes relative to baseline. For each category and total score, descriptive summaries of changes in SARA test scores relative to baseline were provided.

[0604] The SARA test has eight categories, with cumulative scores ranging from 0 (no ataxia) to 40 (most severe ataxia). Each category is assessed and scored accordingly when completing the outcome measurement. The score ranges for the eight items are as follows:

[0605] 1. Gait (0-8 points)

[0606] 2. Standing posture (0-6 points)

[0607] 3. Sitting posture (0-4 points)

[0608] 4. Speech impairment (0-6 points)

[0609] 5. Finger chasing (0-4 points)

[0610] 6. Finger-to-nose test (0-4 points)

[0611] 7. Rapid alternating hand movements (0-4 points)

[0612] 8. Heel-tibial gliding (0-4 points).

[0613] Once each of the eight categories has been assessed, the total will be calculated to determine the severity of the ataxia.

[0614] Pharmacodynamic (Biomarker) Analysis: Characterizing urinary biomarker (PC1, PC2, NGAL, KIM-1) response. Changes in urinary biomarkers (i.e., PC1, PC2, NGAL, KIM-1) over time were compared to baseline (screening and day 1 samples), and comparisons were made using analysis of covariance and adjusted for baseline biomarker values ​​and treatment groups, at each RG-NG-1015 dose level and between placebo (placebo pooled across cohorts 1–3). Data collected on days 29, 57, 85, 86, 92, 99, and 113 were analyzed, but primary comparisons were performed at day 86 and day 113. The figure will be used to assess biomarker (PC1, PC2, NGAL, KIM-1) and htTKV compared to plasma exposure (e.g., AUC). tau C max C min The relationship between dose level and change relative to baseline;

[0615] MRI analysis: Descriptive statistics will be used to summarize the absolute change and percentage change of htTKV relative to baseline. Analysis of covariance will be used, adjusting for baseline htTKV values, baseline Mayo classification (1C, 1D, or 1E), and treatment group. Changes in htTKV values ​​relative to baseline will be compared between treatment groups at each dose level, and among all subjects receiving RG-NG-1015 compared to placebo (placebo combined across cohorts 1–3).

[0616] Renal function analysis: Descriptive analysis and summary of changes in eGRF (calculated using creatinine and cystatin-C using the CKD-EPI equation and regardless of race), UACR, SCr, and BUN relative to baseline;

[0617] Pharmacokinetic Analysis: The laboratories analyzing the PK samples will be unblinded; therefore, PK analysis will only be performed on subjects who received RG-NG-1015. Plasma and urine concentrations compared to time data were used to derive the following PK parameters: C max t max AUC 0-24 AUC inf (where it is computable) AUC tau t 1 / 2 CL / F, V z / F, fe, and Ae. Descriptive statistics of plasma concentrations were summarized by time point and cohort. Using non-compartmental methods, plasma and urine concentrations were compared to time-coordinated data to derive the following PK parameters: Cmax, Tmax, AUC0-24, AUCinf (where calculable), AUCtau, t1 / 2, CL / F, Vz / F, fe, and Ae. Where data allowed, C...max AUC 0-24 AUC inf and AUC tau The efficacy model explores dosage ratios;

[0618] ADA analysis: The incidence and titer of ADA in subjects who experienced it at any time during the study period are listed by cohort. The effect of ADA on PK parameters will be assessed by subgroup analysis (e.g., calculating PK parameters in subjects with and without ADA);

[0619] Exploratory analyses: Descriptive statistics of exploratory renal biomarkers in urine (MCP-1 and B2M) and serum (e.g., IGFALS, CT-proAVP, N-acetyl-1-methylhistidine, etc.) listed by visit; and exploratory image-based biomarkers (e.g., total cyst volume, number, and / or size distribution, etc.). Explore the relationship between ADPKD gene mutations (e.g., PKD1 or PKD2 mutations, truncated or missense mutations, etc.) and biomarker responses (e.g., PC1 and PC2 levels).

[0620] Interim Analysis: After participants in each cohort completed study drug administration and EOS visits, the sponsor analyzed unblinded safety, biomarkers, renal function, and PK data.

[0621] CRO medical monitors continuously review safety data. Sponsor and CRO medical monitors will conduct a blinded safety review of adverse events (AEs) and safety laboratory test results monthly to monitor safety throughout the study. A cohort dose escalation meeting will be held at least 4 weeks after the last subject in the enrolled cohort receives their first dose (Day 1). The sponsor will review available safety data to make dose escalation decisions for Cohort 2, and repeat the process to make dose escalation decisions for Cohort 3.

[0622] H. Results

[0623] Treatment with RG-NG-1015 achieved one or more of the primary endpoint, secondary endpoints, and / or exploratory endpoints, with acceptable safety and tolerability.

[0624] Table 22 provides the baseline characteristics of queue 1 and queue 2:

[0625]

[0626] The enrolled population represented a significant disease burden due to decreased kidney size and eGFR.

[0627] The study showed that RG-NG-1015 (RGLS8429) administered every two weeks for 12 weeks at doses of 1 mg / kg and 2 mg / kg was well tolerated, with no significant safety findings.

[0628] Table 23 provides the adverse events observed for queue 1 and queue 2:

[0629]

[0630] Repeated dosing every other week did not result in the accumulation of RG-NG-1015 in plasma or urine. Patients administered 1 mg / kg of RG-NG-1015 had almost twice the AUC plasma exposure of healthy volunteers, consistent with a reduction of approximately 35% in renal excretion. AUC plasma exposure increased at 2 mg / kg relative to 1 mg / kg.

[0631] Polycystic protein (PC) measurement in urine :

[0632] Measurements of PC1 and PC2 in urinary exosomes have demonstrated a significant difference between healthy subjects and patients with ADPKD, and are negatively correlated with disease severity. See also Figure 7A and Figure 7B .

[0633] Multiple analytical methods were used to assess urinary PC levels:

[0634] • Absolute changes in urinary PC1 and PC2 levels relative to baseline, including the best-fit regression model over the duration of treatment (see [reference]). Figures 8A to 8C , Figures 9A to 9C and Figures 10A to 10B );

[0635] • Percentage change in urinary PC1 and PC2 levels relative to baseline (see...) Figure 11A and Figure 11B );

[0636] • Mean changes in urinary PC1 and PC2 levels relative to baseline after three months of RG-NG-1015 administration (see [link to RG-NG-1015 administration]) Figures 12A to 12B and Figures 13A to 13B ).

[0637] Studies showed that treatment with 1 mg / kg and 2 mg / kg RG-NG-1015 (RGLS8429) resulted in increased absolute and % changes in urinary polycystic protein (PC1 and PC2) levels relative to baseline. See also Figures 8A to 8C , Figures 9A to 9C , Figures 10A to 10B and Figures 11A to 11B .like Figure 11AAs shown, for RG-NG-1015 at 1 mg / kg, a statistically significant increase in urinary PC1 relative to baseline (mean of three separate samples taken before treatment) was observed at 12 weeks of administration (36% and 41% on days 85 and 86, respectively). Figure 11B As shown, for RG-NG-1015 at 1 mg / kg, an increase in urinary PC2 relative to baseline was also observed at 12 weeks; however, the change did not reach statistical significance (see statistical analysis). Figure 10A and Figure 10B The polycystic protein pattern was consistent with tissue PK characteristics observed in preclinical studies. The study also showed increased absolute changes in PC1 and PC2 levels with treatment at 2 mg / kg RG-NG-1015 compared to 1 mg / kg RG-NG-1015. See also Figures 8A to 8C , Figures 9A to 9C and Figures 10A to 10B .exist Figure 10A For example, with RG-NG-1015 at 2 mg / kg, a statistically significant increase in urinary PC1 relative to baseline was observed on days 57, 86, 99, and 113 after administration. Figure 10B In the study, for RG-NG-1015 at 2 mg / kg, a statistically significant increase in urinary PC2 relative to baseline was observed on day 57.

[0638] The study also showed that for the absolute changes in PC1 and PC2 ( Figures 12A to 12B ) and the percentage changes in PC1 and PC2 ( Figures 13A to 13B After 3 months of treatment with 1 mg / kg and 2 mg / kg RG-NG-1015 (RGLS8429) (Q2W), mean polycystic protein levels increased. 2 mg / kg RG-NG-1015 showed a greater increase in mean polycystic protein levels compared to 1 mg / kg RG-NG-1015.

[0639] Urinary measurements from PC1 and PC2 indicated that RG-NG-1015 (RGLS8429) at 2 mg / kg had greater bioactivity compared to placebo, which was most pronounced three months after administration. A mechanical dose-response was also observed at the 2 mg / kg dose level based on urinary polycystic protein analysis.

[0640] This study also demonstrated that polycystic protein is a valid pharmacodynamic biomarker (i.e., for dose range) because urinary polycystic protein exhibits appropriate PK / PD correlation to serve as a pharmacodynamic biomarker for ADPKD. See also Figure 14A and Figure 14B .like Figures 14A to 14BAs shown, a novel dose response was observed at 0.3 mg / kg and 1 mg / kg RGLS4326 (NCT04536688; archived data) and 1 mg / kg RG-NG-1015 (RGLS8429). When the RGLS4326 and RG-NG-1015 datasets were combined, the two pK parameters (C1 and C2) were measured at PC1. max and AUC last A positive correlation was observed between them. Furthermore, similar pharmacodynamic responses were observed between RGLS 4326 and RG-NG-1015 at a dose level of 1 mg / kg.

[0641] Kidney function parameters and kidney MRI measurements :

[0642] Based on published longitudinal studies, the kidneys of patients with ADPKD grow by about 6% per year, so the kidney volume is expected to increase by about 1% to 2% over 12 weeks.

[0643] At the end of the study, MRI was used to assess its impact on novel imaging biomarkers characterizing cystic structures. Exploratory results from the MRI image analysis are shown in... Figures 15A to 15C and Figures 16A to 16C middle.

[0644] Figures 15A to 15B The changes in height-adjusted total kidney volume (htTKV) and total renal cyst volume (TKCV) are shown in subjects receiving 1 mg / kg RG-NG-1015, 2 mg / kg RG-NG-1015, and placebo. Figure 15C The correlation between changes in TKCV and htTKV is shown.

[0645] Table 24 shows the mean % htTKV changes in the 2 mg / kg group, the 1 mg / kg group, and the placebo group:

[0646] %HtTKV 2mpk 1mpk Pb - All average value -0.84 +1.79 +0.52 SD 3.11 5.90 3.13

[0647] Table 25 shows the mean %TKCV changes in the 2 mg / kg group, the 1 mg / kg group, and the placebo group:

[0648] %TKCV 2mpk 1mpk Pb - All average value -0.53 +4.36 +0.47 SD 3.90 8.21 1.94

[0649] Figure 16A and Figure 16B The changes in total liver volume (TLV) and total liver cyst volume (TLCV) are shown in subjects who received 1 mg / kg RG-NG-1015, 2 mg / kg RG-NG-1015, and placebo. Figure 16C The correlation between changes in TLCV and changes in TLV is shown.

[0650] Table 26 shows the mean %TLV changes in the 2 mg / kg group, the 1 mg / kg group, and the placebo group:

[0651] %TLV 2mpk 1mpk Pb - All average value -1.03 -2.99 +5.03 SD 7.91 7.65 5.61

[0652] Table 27 shows the mean absolute TLCV changes in the 2 mg / kg group and the placebo group:

[0653] Absolute TLCV (mL) 2mpk Pb average value -1.72 +0.71 SD 14.95 1.966

[0654] Figure 17A An exploratory correlation was shown between changes in PC1 and changes in HtTKV. Table 28 shows a simple linear regression for placebo, 1 mg / kg, and 2 mg / kg RG-NG-1015:

[0655] Simple linear regression <![CDATA[R 2 ]]> P-value placebo 0.0092 0.857 RGLS8429 (1 mg / kg) 0.0211 0.709 RGLS8429 (2mg / kg) 0.0719 0.425

[0656] Figure 17B An exploratory correlation was shown between changes in PC1 and changes in eGFR. Table 29 shows a simple linear regression for placebo, 1 mg / kg, and 2 mg / kg RG-NG-1015:

[0657] Simple linear regression <![CDATA[R 2 ]]> P-value placebo 0.0021 0.930 RGLS8429 (1 mg / kg) 0.2418 0.179 RGLS8429 (2mg / kg) 0.4027 0.036

[0658] Figure 17C An exploratory correlation was shown between changes in PC2 and changes in HtTKV. Table 30 shows a simple linear regression for placebo, 1 mg / kg, and 2 mg / kg RG-NG-1015:

[0659] Simple linear regression <![CDATA[R 2 ]]> P-value placebo 0.0728 0.605 RGLS8429 (1 mg / kg) 0.0639 0.570 RGLS8429 (2mg / kg) 0.0373 0.512

[0660] Figure 17D An exploratory correlation was shown between changes in PC2 and changes in eGFR. Table 31 shows a simple linear regression for placebo, 1 mg / kg, and 2 mg / kg RG-NG-1015:

[0661]

[0662]

[0663] For cohort 1, renal function parameters did not show significant changes during the short-term 12-week dosing period. Baseline measurements were consistent with the ADPKD Mayo classification phase. No significant changes in renal function indices (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM) were observed within 12 weeks.

[0664] For cohort 2, the exploratory results of MRI image analysis are as follows:

[0665] The mean change in htTKV was -0.84% ​​in the 2 mg / kg group and +0.52% in the placebo group.

[0666] • 4 / 11 subjects who received 2 mg / kg showed a decrease in htTKV >2%, a decrease in TKCV, and an increase in urinary PC1 and PC2;

[0667] The change in TKCV is related to the change in htTKV;

[0668] • A reduction in liver volume and liver cyst volume was observed in some patients treated with RG-NG-1015;

[0669] • No significant changes in renal function parameters (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM 1) were observed within 12 weeks, based on the expected changes from short-term treatment and a small number of subjects.

[0670] Key points of some cases are illustrated below:

[0671] Subject 1: Highest increase in PC1 and PC2*

[0672] ○ 47 male gynecologists were diagnosed in 2006.

[0673] ○ Baseline eGFR 66 mL / min and htTKV 941 mL / min

[0674] ○D113 MRI: htTKV decreased by 4.96%; TKCV decreased by 4.34%;

[0675] Subject 2: Second highest increase in PC1*

[0676] ○ 44 women diagnosed with gynecology in 2019

[0677] ○ Baseline eGFR 65 mL / min and htTKV 1253 mL / min

[0678] ○D113 MRI: htTKV decreased by 6.28%; TKCV decreased by 6.93%;

[0679] Subject 3: Second highest increase in PC2*

[0680] ○ 29 male gynecologists were diagnosed in 2020.

[0681] ○ Baseline eGFR 88 mL / min and htTKV 1162 mL / min

[0682] ○D113 MRI: htTKV decreased by 4.22%; TKCV decreased by 2.73%.

[0683] • In cohort 2, among the four active subjects with a htTKV decrease of >2%, both PC1 and PC2 were increased in all four subjects.

[0684] *The average percentage change in polycystic protein (PC) between day 85 and day 113.

[0685] The results of cohort 2 showed that, compared with placebo, patients with ADPKD had numerical improvements in total kidney and liver volume and cyst volume.

[0686] Modeling of RG-NG-1015 target engagement research data

[0687] The efficacy of RG-NG-1015 (RGLS8429) was evaluated in a KspCre;Pkd1F / RC (Pkd1-F / RC) mouse model. Pkd1-F / RC mice were administered various doses of RG-NG-1015, 20 mg / kg of control oligonucleotide, or PBS (N = 8–13 per group) subcutaneously on days 8, 10, 12, and 15 postnatally. A separate group of Pkd1-F / RC mice was administered 20 mg / kg of RG-NG-1015 subcutaneously on days 8 and 12 postnatally. Mice were sacrificed at 18 days of age. The left kidney was perfused with cold PBS and 4% PFA prior to collection. All other mouse kidneys were collected, fixed in 10% formalin, dehydrated, and embedded in paraffin using standard protocols. Samples were sectioned at 5 μm and stained with hematoxylin and eosin.

[0688] like Figures 18A to 18B As shown, RGLS8429 demonstrated efficacy in reducing the kidney weight to body weight ratio (KW / BW) in a dose-response manner. Figure 18B Left: Individual KW / BW grouped by dose level and regimen; Right: Individual calculated percentage of KW / BW inhibition (CPI) plotted against individual kidney concentrations for RG-NG-1015. The estimated kidney concentration corresponding to 50% inhibition of KW / BW is shown (i.e., IC50 value). Following administration of RGLS8429, both kidney size and cyst number decreased in a dose-responsive manner. See also Figure 18A .

[0689] Wild-type C57BL6 mice received a single SC dose of RGLS8429 or RGLS4326 at doses of 0.003 mg / kg, 0.03 mg / kg, 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 10 mg / kg, 30 mg / kg, or 300 mg / kg. Mice were sacrificed 7 days after administration. Kidney samples were harvested and target binding (displacement of miR-17 from the high molecular weight polymer) was measured by miPSA assay (Androsavich, Nucleic Acids Res. 44, e13 (2016)). Individually calculated percentage of inhibition (CPI) of target binding in mouse kidneys was plotted against individual kidney concentrations of RGLS8429. The estimated kidney concentration corresponding to 80% inhibition of miR-17, i.e., the IC80 value, is shown. See also Figure 18C The predicted AUC was approximately 12,494 h*ug / g, and was used as a benchmark for renal exposure driving maximal target binding (approximately 80% inhibition of miR-17).

[0690] Based on recent results from cohorts 1 and 2 (RGLS8429-02), the predicted renal exposures of RG-NG-1015 in ADPKD patients at doses of 1 mg / kg (cohort 1) and 2 mg / kg (cohort 2) are approximately 6,052 h*ug / g and 8,900 h*ug / g, respectively (lower than the predicted baseline renal exposure of approximately 12,494 h*ug / g). Based on these results, a dose of 3 mg / kg (cohort 3) is expected to result in predicted renal exposures close to or slightly higher than approximately 12,494 h*ug / g.

[0691] Plasma-tissue modeling showed that 1 mg / kg was less than half the dose-response curve. Data suggest that at higher doses, a greater urinary polycystic protein response may be observed.

[0692] Based on extensive nonclinical analysis and PK / PD modeling, renal exposure associated with peak miR-17 target binding is expected to reach >2.4 mg / kg in humans.

Claims

1. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide or a pharmaceutically acceptable salt thereof to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg. The modified oligonucleotide described therein has a 5'-A structure. S G S C M A F C F U F U M U S A S -3', The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine is a non-methylated cytosine.

2. The method of claim 1, wherein the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a dose of 1 mg / kg, 2 mg / kg or 3 mg / kg.

3. The method of claim 1 or 2, wherein the pharmaceutically acceptable salt is a sodium salt.

4. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg, wherein the modified oligonucleotide has the following structure: Or its pharmaceutically acceptable salt.

5. The method of claim 4, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

6. The method of claim 4 or 5, wherein the pharmaceutically acceptable salt is a sodium salt.

7. The method of any one of claims 1 to 6, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

8. The method of claim 7, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

9. The method of claim 8, wherein the sterile aqueous solution is a saline solution.

10. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need at a dose of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg, wherein the modified oligonucleotide has the following structure:

11. The method of claim 10, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

12. The method of claim 10 or 11, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

13. The method of claim 12, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution.

14. The method of claim 13, wherein the sterile aqueous solution is a saline solution.

15. The method of any one of claims 1 to 14, wherein the subject suffers from polycystic kidney disease.

16. The method of any one of claims 1 to 15, wherein the subject has been diagnosed with polycystic kidney disease using clinical, histopathological, and / or genetic criteria.

17. The method of any one of claims 1 to 16, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

18. The method of claim 17, wherein the subject has a Mayo imaging classification of ADPKD 1C, 1D, or 1E.

19. The method of any one of claims 1 to 18, wherein prior to administration of the modified oligonucleotide, the subject's estimated glomerular filtration rate (eGFR) is between 30 and 90 mL / min / 1.73 m 2 between.

20. The method of any one of claims 1 to 19, wherein prior to administering the modified oligonucleotide, the subject is determined to have reduced polycystin-1 (PC1) and / or polycystin-2 (PC2) levels in the subject's kidneys, urine, or blood.

21. The method of any one of claims 1 to 20, wherein the subject has a mutation selected from a mutation in the PKD1 gene or a mutation in the PKD2 gene.

22. The method of any one of claims 1 to 21, wherein the subject has an increased total kidney volume.

23. The method of any one of claims 1 to 22, wherein the subject suffers from hypertension.

24. The method of any one of claims 1 to 23, wherein the subject has impaired renal function.

25. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 1 mg / kg.

26. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 2 mg / kg.

27. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 3 mg / kg.

28. The method of any one of claims 1 to 27, wherein the method comprises applying the modified oligonucleotide once every two weeks.

29. The method of any one of claims 1 to 28, wherein the method comprises applying the modified oligonucleotide at least 7 times.

30. The method of any one of claims 1 to 29, wherein the modified oligonucleotide is administered subcutaneously.

31. The method of any one of claims 1 to 30, wherein the treatment reduces the total kidney volume of the subject.

32. The method of any one of claims 1 to 31, wherein the treatment slows the rate of increase in the total kidney volume of the subject.

33. The method of claim 31 or 32, wherein the total kidney volume is the total kidney volume adjusted for height (htTKV).

34. The method of any one of claims 1 to 33, wherein the treatment slows the rate of decline in the glomerular filtration rate of the subject.

35. The method of any one of claims 1 to 34, wherein the treatment increases the glomerular filtration rate of the subject.

36. The method of claim 34 or 35, wherein the glomerular filtration rate is an estimated glomerular filtration rate.

37. The method of any one of claims 1 to 36, wherein the treatment inhibits or slows the increase in cyst growth in the kidneys and / or liver of the subject.

38. The method of claim 37, wherein the treatment inhibits or slows the increase in total cyst volume, number, and / or size distribution.

39. The method of any one of claims 1 to 38, wherein the treatment: a) Improve or slow the rate of decrease in the subject's creatinine clearance; b) Reduce or slow the rate of increase in the albumin:creatinine ratio of the subject; c) Reduce or slow the rate of increase in the subject's blood urinary nitrogen (BUN) level; d) Reduce or slow the rate of increase in the subject's serum creatinine (SCr) level; e) Increase polycystic protein-1 (PC1) in the urine of the subjects; f) Increase polycystic protein-2 (PC2) in the urine of the subjects; g) Reduce or slow the rate of increase of neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subjects; and / or h) Reduce or slow the rate of increase of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects.

40. The method of any one of claims 1 to 39, wherein the application: a) Reduce or slow the rate of increase of monocyte chemoattractant protein-1 (MCP-1) in the urine of the subjects; b) Reduce or slow the rate of increase of β-2 microglobulin (B2M) in the urine of the subjects; c) Reduce or slow the rate of increase of complement cleavage products C3a and / or Bb in the plasma of the subject; d) Reduce or slow the rate of increase in serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the subjects; e) Reduce or slow the rate of increase of serum and pro-AVP in the subjects; f) Reduce or slow the rate of increase in serum N-acetyl-1-methylhistidine in the subjects; and / or g) Reduce or slow the rate of increase in acute-phase proteins in the subject.

41. The method of any one of claims 1 to 40, wherein the treatment results in little or no CNS damage in the subject.

42. The method of claim 41, wherein the treatment results in minimal or no change in the subject's ataxia assessment and rating scale (SARA) test score.

43. The method according to any one of claims 1 to 42, comprising: a) Measure the height-adjusted total kidney volume (HtTKV) of the subject; b) Measure polycystic protein-1 (PC1) in the urine of the subjects; c) Measure polycystic protein-2 (PC2) in the urine of the subjects; d) Measure the blood urea nitrogen (BUN) level of the subjects; e) Measure the serum creatinine (SCr) level of the subjects; f) Measure the creatinine clearance rate of the subjects; g) Measure the urine albumin:creatinine ratio (UACR) of the subject; h) Measure the estimated glomerular filtration rate (eGFR) of the subject; i) Measure the level of neutrophil gelatinase-associated lipocalin (NGAL) protein in the urine of the subjects; j) Measure the amount of kidney injury molecule-1 (KIM-1) protein in the urine of the subjects; k) Measure the level of monocyte chemoattractant protein-1 (MCP-1) in the urine of the subjects; l) Measure β-2 microglobulin (B2M) in the urine of the subjects; m) Measure the serum insulin-like growth factor-binding protein acid unstable subunit (IGFALS) of the subjects; n) Measure the serum and peptide (CT-proAVP) of the subjects; o) Measure the serum N-acetyl-1-methylhistidine of the subject; p) Measure the complement cleavage products C3a and / or Bb in the plasma of the subjects; and / or q) Measure the total cyst volume, number, and / or size distribution of the subjects; and / or r) Measure the subject's SARA test score.

44. The method of any one of claims 1 to 43, wherein the subject is a human subject.

45. The method according to any one of claims 1 to 44, having acceptable safety and tolerability characteristics.

46. ​​A modified oligonucleotide or a pharmaceutically acceptable salt thereof for use in the treatment of polycystic kidney disease, wherein said modified oligonucleotide has a 5'-A structure. S G S C M A F C F U F U M U S A S -3', The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine in it is a non-methylated cytosine. The modified oligonucleotide is administered at doses of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg.

47. The modified oligonucleotide for use as described in claim 46, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

48. The modified oligonucleotide for use as described in claim 46 or 47, wherein the pharmaceutically acceptable salt is a sodium salt.

49. The modified oligonucleotide for use as described in any one of claims 46 to 48, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

50. The modified oligonucleotide for use as described in any one of claims 46 to 49, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

51. The modified oligonucleotide for use as described in any one of claims 46 to 50, wherein the modified oligonucleotide is applied once every two weeks.

52. The modified oligonucleotide for use as described in any one of claims 46 to 51, wherein the modified oligonucleotide is applied at least seven times.

53. Use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of polycystic kidney disease, wherein said modified oligonucleotide has the structure 5'-A S G S C M A F C F U F U M U S A S -3', The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is S-cEt nucleoside, and each cytosine in it is a non-methylated cytosine. The modified oligonucleotides are formulated for administration at doses of 0.5-5 mg / kg, 0.5-4.5 mg / kg, 0.5-4 mg / kg, 0.5-3.5 mg / kg, 0.5-3 mg / kg, 1-5 mg / kg, 1-4.5 mg / kg, 1-4 mg / kg, 1-3.5 mg / kg, or 1-3 mg / kg.

54. The use as described in claim 53, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

55. The use as described in claim 53 or 54, wherein the pharmaceutically acceptable salt is a sodium salt.

56. The use according to any one of claims 53 to 55, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

57. The use as claimed in any one of claims 53 to 56, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

58. The use as described in any one of claims 53 to 57, wherein the modified oligonucleotide is applied at least once every two weeks.

59. The use as described in any one of claims 53 to 58, wherein the modified oligonucleotide is applied at least seven times.

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