Modified oligonucleotides for treating polycystic kidney disease

Abstract

Methods for the treatment of polycystic kidney diseases, including autosomal dominant polycystic kidney disease, using modified oligonucleotides targeting miR-17 are provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 495,576 filed on 12 April 2023 and U.S. Provisional Patent Application No. 63 / 563,881 filed on 11 March 2024, both of which are incorporated herein by reference for all purposes.

[0002] A method for treating polycystic kidney disease using a compound comprising a modified oligonucleotide or a pharmaceutically acceptable salt thereof is provided herein.

[0003] Sequence List This application includes a sequence listing 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 electronic information of this sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Polycystic kidney disease (PKD) is characterized by the accumulation of numerous fluid-filled cysts in the kidneys. These cysts are covered by a single layer of epithelial cells called cystic epithelium. Over time, the cysts increase in size due to increased cell proliferation and active fluid secretion by the cystic epithelium. The enlarged cysts compress the 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 areas of fibrosis, including atrophic tubules. PKD can also cause cysts to develop in the liver and other locations in the body.

[0005] Certain genetic disorders can lead to PKD. The various forms of PKD are distinguished by the mode of inheritance, such as autosomal dominant or autosomal recessive inheritance, organ involvement and presentation of extra-renal phenotypes, the age of onset of end-stage renal disease (e.g., at birth, in childhood or in adulthood, etc.), and the underlying gene mutations associated with the disease. See, for example, Kurschat et al., 2014, Nature Reviews Nephrology, 10:687-699. Summary of the Invention

[0006] The present disclosure is directed to a method of treating polycystic kidney disease (PKD), optionally autosomal dominant polycystic kidney disease (ADPKD), which comprises administering to a subject in need thereof a therapeutically effective amount of a modified oligonucleotide and a pharmaceutically acceptable salt thereof. Embodiment 1. A method of treating polycystic kidney disease, comprising administering to a subject in need thereof a modified oligonucleotide or a pharmaceutically acceptable salt thereof 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, where the 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', where the nucleoside followed by the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside, the nucleoside followed by the subscript "S" is a S-cEt nucleoside, and each cytosine is an unmethylated cytosine. Embodiment 2. The method according to Embodiment 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. Embodiment 3. The method according to Embodiment 1 or 2, wherein the pharmaceutically acceptable salt is a sodium salt. Embodiment 4. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need of such treatment 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, wherein the modified oligonucleotide has the following structure: [ka] The method comprising a pharmaceutically acceptable salt thereof. Embodiment 5. The method according to Embodiment 4, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg. Embodiment 6. The method according to Embodiment 4 or 5, wherein the pharmaceutically acceptable salt is a sodium salt. Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the modified oligonucleotide is present in a pharmaceutical composition containing a pharmaceutically acceptable diluent. Embodiment 8. The method according to Embodiment 7, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution. Embodiment 9. The method according to Embodiment 8, wherein the sterile aqueous solution is physiological saline. Embodiment 10. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need of such treatment 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, wherein the modified oligonucleotide has the following structure: [ka] The method comprising the above. Embodiment 11. The method according to Embodiment 10, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg. Embodiment 12. The method according to Embodiment 10 or 11, wherein the modified oligonucleotide is present in a pharmaceutical composition containing a pharmaceutically acceptable diluent. Embodiment 13. The method according to Embodiment 12, wherein the pharmaceutically acceptable diluent is an aqueous solution. Embodiment 14. The method according to Embodiment 13, wherein the aqueous solution is physiological saline. Embodiment 15. The method according to any one of Embodiments 1 to 14, wherein the subject has polycystic kidney disease. Embodiment 16. The method according to any one of Embodiments 1 to 15, wherein the subject is diagnosed with polycystic kidney disease using clinical, histopathological, and / or genetic criteria. Embodiment 17. The method according to any one of Embodiments 1 to 16, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). Embodiment 18. The method according to Embodiment 17, wherein the subject has ADPKD Mayo image classification 1C, 1D, or 1E. Embodiment 19. The subject had an estimated glomerular filtration rate (eGFR) of 30-90 mL / min / 1.73 m² prior to administration of the modified oligonucleotide. 2 The method according to any one of embodiments 1 to 18. Embodiment 20. The method according to any one of Embodiments 1 to 19, wherein the subject is determined to have decreased levels of polycystin-1 (PC1) and / or polycystin-2 (PC2) in the subject's kidney, urine, or blood prior to administration of the modified oligonucleotide. Embodiment 21. The method according to any one of Embodiments 1 to 20, wherein the subject has a mutation selected from a mutation in the PKD1 gene or a mutation in the PKD2 gene. Embodiment 22. The method according to any one of Embodiments 1 to 21, wherein the subject is an increase in total kidney volume. Embodiment 23. The method according to any one of Embodiments 1 to 22, wherein the subject has hypertension. Embodiment 24. The method according to any one of Embodiments 1 to 23, wherein the subject has renal dysfunction. Embodiment 25. The method according to any one of Embodiments 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 1 mg / kg. Embodiment 26. The method according to any one of Embodiments 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 2 mg / kg. Embodiment 27. The method according to any one of Embodiments 1 to 24, wherein the method comprises administering the modified oligonucleotide at a dose of 3 mg / kg. Embodiment 28. The method according to any one of Embodiments 1 to 27, wherein the method comprises administering the modified oligonucleotide once every two weeks. Embodiment 29. The method according to any one of Embodiments 1 to 28, wherein the method comprises administering the modified oligonucleotide at least seven times. Embodiment 30. The method according to any one of Embodiments 1 to 29, wherein the modified oligonucleotide is administered subcutaneously. Embodiment 31. The method according to any one of Embodiments 1 to 30, wherein the treatment reduces the total kidney volume in the subject. Embodiment 32. The method according to any one of Embodiments 1 to 31, wherein the treatment slows down the rate of increase in total kidney volume in the subject. Embodiment 33. The method according to Embodiment 31 or 32, wherein the total kidney volume is height-adjusted total kidney volume (htTKV). Embodiment 34. The method according to any one of Embodiments 1 to 33, wherein the treatment slows down the rate of decline of the glomerular filtration rate in the subject. Embodiment 35. The method according to any one of Embodiments 1 to 34, wherein the treatment increases the glomerular filtration rate in the subject. Embodiment 36. The method according to Embodiment 34 or 35, wherein the glomerular filtration rate is the estimated glomerular filtration rate. Embodiment 37. The method according to any one of Embodiments 1 to 36, wherein the treatment inhibits or slows the increase in cyst growth in the kidney and / or liver of the subject. Embodiment 38. The method according to Embodiment 37, wherein the treatment inhibits or slows the increase in total cyst volume, number, and / or size distribution. Embodiment 39. By the above treatment, a) The rate of decrease in creatinine clearance in the subject is improved or slowed down. b) The rate of increase in the albumin:creatinine ratio in the subject is reduced or slowed down. c) The rate of increase in blood urea nitrogen (BUN) levels in the subject is reduced or slowed down. d) The rate of increase in serum creatinine (SCr) levels in the subject is reduced or slowed down. e) The amount of polycystin-1 (PC1) in the urine of the subject increases. f) The amount of polycystin-2 (PC2) in the urine of the subject increases. g) The rate of increase of neutrophil gelatinase-binding lipocalin (NGAL) protein in the urine of the subject is reduced or slowed, and / or h) The method according to any one of Embodiments 1 to 38, wherein the rate of increase of kidney injury molecule 1 (KIM-1) protein in the urine of the subject is reduced or slowed down. Embodiment 40. By administering the above, a) The rate of increase of monocyte chemotactic protein 1 (MCP-1) in the urine of the subject is reduced or slowed down. b) The rate of increase of beta-2 microglobulin (B2M) in the urine of the subject is reduced or slowed down. c) The rate of increase of complement degradation products C3a and / or Bb in the plasma of the subject is reduced or slowed down. d) The rate of increase of serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the subject is reduced or slowed down. e) The rate of increase of serum copeptin (CT-proAVP) in the subject is reduced or slowed down. f) The rate of increase of serum N-acetyl-1-methylhistidine in the subject is reduced or slowed, and / or g) The method according to any one of Embodiments 1 to 39, wherein the rate of increase of acute-phase protein in the subject is reduced or slowed down. Embodiment 41. The method according to any one of Embodiments 1 to 40, wherein CNS failure in the subject is reduced to almost none. Embodiment 42. The method according to Embodiment 41, wherein the treatment results in little to no change in the Scale for the Assessment and Rating of Ataxia (SARA) test score for the subject. Embodiment 43. a) Measure height-adjusted total kidney volume (HtTKV) in the subject. b) Measuring polycystin-1 (PC1) in the urine of the subject, c) Measure polycystin-2 (PC2) in the urine of the subject, d) Measuring the blood urea nitrogen (BUN) level in the subject, e) Measuring serum creatinine (SCr) levels in the subject, f) Measuring creatinine clearance in the subject, g) Measure the urinary albumin:creatinine ratio (UACR) in the subject. h) Measure the estimated glomerular filtration rate (eGFR) in the subject. i) Measure the neutrophil gelatinase-binding lipocalin (NGAL) protein in the urine of the subject. j) Measure the kidney injury molecule 1 (KIM-1) protein in the urine of the subject. k) Measure monocyte chemotactic protein 1 (MCP-1) in the urine of the subject. l) Measure beta-2 microglobulin (B2M) in the urine of the subject. m) Measure the serum insulin-like growth factor-binding protein acid unstable subunit (IGFALS) in the subject. n) Measure serum copeptin (CT-proAVP) in the subject. o) Measure serum N-acetyl-1-methylhistidine in the subject, p) Measure the complement degradation products C3a and / or Bb in the plasma of the subject. q) To measure the total cyst volume, number, and / or size distribution in the subject, and / or r) The method according to any one of Embodiments 1 to 42, comprising measuring the SARA test score for the subject. Embodiment 44. The method according to any one of Embodiments 1 to 43, wherein the subject is a human subject. Embodiment 45. The method according to any one of Embodiments 1 to 44, having an acceptable safety and tolerability profile. Embodiment 46. A modified oligonucleotide or a pharmaceutically acceptable salt thereof for use in the treatment of polycystic kidney disease, wherein the modified oligonucleotide has structure 5'-A S G S C M A F C F U F U M U S A S It has -3', Nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, nucleosides followed by the subscript "S" are S-cEt nucleosides, and each cytosine is an unmethylated cytosine. The modified oligonucleotide for use, or a pharmaceutically acceptable salt thereof, is administered in 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. Embodiment 47. The modified oligonucleotide for use according to Embodiment 46, wherein the modified oligonucleotide is administered at a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg. Embodiment 48. A modified oligonucleotide for use according to Embodiment 46 or 47, wherein the pharmaceutically acceptable salt is a sodium salt. Embodiment 49. The modified oligonucleotide for use according to any one of Embodiments 46 to 48, wherein the modified oligonucleotide is present in a pharmaceutical composition containing sterile physiological saline. Embodiment 50. A modified oligonucleotide for use according to any one of Embodiments 46 to 49, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). Embodiment 51. The modified oligonucleotide for use according to any one of Embodiments 46 to 50, wherein the modified oligonucleotide is administered once every two weeks. Embodiment 52. A modified oligonucleotide for use according to any one of Embodiments 46 to 51, wherein the modified oligonucleotide is administered at least seven times. Embodiment 53. Use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical for the treatment of polycystic kidney disease, wherein the modified oligonucleotide has structure 5'-A S G S C M A F C F U F U M U S A S It has -3', Nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, nucleosides followed by the subscript "S" are S-cEt nucleosides, and each cytosine is an unmethylated cytosine. The use wherein the modified oligonucleotide is formulated for administration in 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. Embodiment 54. The use according to Embodiment 53, wherein the modified oligonucleotide is administered in a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg. Embodiment 55. The use according to Embodiment 53 or 54, wherein the pharmaceutically acceptable salt is a sodium salt. Embodiment 56. The use according to any one of Embodiments 53 to 55, wherein the modified oligonucleotide is present in a pharmaceutical composition containing sterile physiological saline. Embodiment 57. The use according to any one of Embodiments 53 to 56, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). Embodiment 58. The use according to any one of Embodiments 53 to 57, wherein the modified oligonucleotide is administered at least once every two weeks. Embodiment 59. The use according to any one of Embodiments 53 to 58, wherein the modified oligonucleotide is administered at least seven times. [Brief explanation of the drawing]

[0007] [Figure 1] Purine nucleic acid base structure. [Figure 2A] Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on (2A) kidney-to-body weight ratio, (2B) blood urea nitrogen (BUN) levels, and (2C) blood creatinine levels. [Figure 2B] Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on (2A) kidney-to-body weight ratio, (2B) blood urea nitrogen (BUN) levels, and (2C) blood creatinine levels. [Figure 2C]Efficacy of RG-NG-1015 in the Pkd1-F / RC model of PKD. Effect of treatment on (2A) kidney-to-body weight ratio, (2B) blood urea nitrogen (BUN) levels, and (2C) blood creatinine levels. [Figure 3] Maximum tolerated dose (MTD) studies and comparative dose evaluations for RG-NG-1001, RGLS4326, and RG-NG-1017. Male C57BL / 6J mice aged 6-7 weeks were administered 4 μL of single intracerebroventricular (ICV) injections of various dose levels of RG-NG-1001 and RGLS4326 (anti-miR-17 oligonucleotides that inhibit AMPA-R) and RG-NG-1017 (anti-miR-17 oligonucleotides that do not inhibit AMPA-R), and monitored for 7 days. Mouse mortality is indicated for the three different compounds at different doses. [Figure 4] A-F. This shows the evaluation of the activity of RG-NG-1015 and RGLS4326 against the luciferase sensor activity of miR-17(A), miR-20a(B), miR-93(C), and miR106(a)(D) in vitro in HeLa cells. This also shows the evaluation of the activity of RG-NG-1015 and RGLS4326 against luciferase sensors containing the full-length 3' untranslated region (UTR) of the miR-17 direct target genes PKD1(E) and PKD2(F). [Figure 5] A-D. Pharmacokinetic and target involvement (measured by miPSA) of RGLS4326 and RG-NG-1015 after a single subcutaneous administration in C57BL6 mice were measured. Plasma concentration (A), tissue concentration (B), renal target binding (C), and hepatic target binding (D) are shown. [Figure 6]A-E. The effects of RG-NG-1015 in combination with tolvaptan on a Pcy / DBA mouse model of PKD were measured at various dosages and regimens. The drug schedule is shown in A, and the key for graphs C-E is shown in B. Kidney weight / body weight (6C), cystic area (%) (6D), and urinary Ngal / Cr (6E) are shown. Error bars represent standard deviation. Compared to the Pcy vehicle treatment group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.001, (ns)p>0.05; one-way ANOVA Bonferroni multiple comparison study. Compared to the tolvaptan monotherapy group, #p<0.05, ##p<0.01, ###p<0.001, ####p<0.001, (ns)p>0.05; one-way ANOVA Sadik multiple comparison study. Compared to the group treated with dose-matched RG-NG-1015 alone, $p<0.05, $$p<0.01, $$$p<0.001, $$$$p<0.001, (ns)p>0.05; one-way ANOVA Sadik multiple comparison trial. [Figure 7A] Urinary polycystin 1 (PC1; Figure 7A) and polycystin 2 (PC2; Figure 7C) levels (expressed as the ratio of PC1 / CD133 or PC2 / CD133) were measured in healthy individuals and patients with chronic kidney disease and ADPKD. 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 (chronic obstructive pulmonary disease) with CKD stages 2-4; ADPKD: Autosomal dominant polycystic kidney disease (Mayo classification based on htTKV and age); CD133: Prominin 1 (shown to co-localize 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). *One-way ANOVA compared with HV using p-value and Dunnett's correction. Urinary PC1 (Figure 7B) and PC2 (Figure 7D) levels (shown as PC1 / CD133 or PC2 / CD133 ratios) were measured over placebo and RG-NG-1015 (RGLS8429) baseline and mean D85-D113 values ​​at 1 mg / kg and 2 mg / kg. [Figure 7B] Urinary polycystin 1 (PC1; Figure 7A) and polycystin 2 (PC2; Figure 7C) levels (expressed as the ratio of PC1 / CD133 or PC2 / CD133) were measured in healthy individuals and patients with chronic kidney disease and ADPKD. 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 (chronic obstructive pulmonary disease) with CKD stages 2-4; ADPKD: Autosomal dominant polycystic kidney disease (Mayo classification based on htTKV and age); CD133: Prominin 1 (shown to co-localize 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). *One-way ANOVA compared with HV using p-value and Dunnett's correction. Urinary PC1 (Figure 7B) and PC2 (Figure 7D) levels (shown as PC1 / CD133 or PC2 / CD133 ratios) were measured over placebo and RG-NG-1015 (RGLS8429) baseline and mean D85-D113 values ​​at 1 mg / kg and 2 mg / kg. [Figure 7C]Urinary polycystin 1 (PC1; Figure 7A) and polycystin 2 (PC2; Figure 7C) levels (expressed as the ratio of PC1 / CD133 or PC2 / CD133) were measured in healthy individuals and patients with chronic kidney disease and ADPKD. 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 (chronic obstructive pulmonary disease) with CKD stages 2-4; ADPKD: Autosomal dominant polycystic kidney disease (Mayo classification based on htTKV and age); CD133: Prominin 1 (shown to co-localize 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). *One-way ANOVA compared with HV using p-value and Dunnett's correction. Urinary PC1 (Figure 7B) and PC2 (Figure 7D) levels (shown as PC1 / CD133 or PC2 / CD133 ratios) were measured over placebo and RG-NG-1015 (RGLS8429) baseline and mean D85-D113 values ​​at 1 mg / kg and 2 mg / kg. [Figure 7D]Urinary polycystin 1 (PC1; Figure 7A) and polycystin 2 (PC2; Figure 7C) levels (expressed as the ratio of PC1 / CD133 or PC2 / CD133) were measured in healthy individuals and patients with chronic kidney disease and ADPKD. 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 (chronic obstructive pulmonary disease) with CKD stages 2-4; ADPKD: Autosomal dominant polycystic kidney disease (Mayo classification based on htTKV and age); CD133: Prominin 1 (shown to co-localize 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). *One-way ANOVA compared with HV using p-value and Dunnett's correction. Urinary PC1 (Figure 7B) and PC2 (Figure 7D) levels (shown as PC1 / CD133 or PC2 / CD133 ratios) were measured over placebo and RG-NG-1015 (RGLS8429) baseline and mean D85-D113 values ​​at 1 mg / kg and 2 mg / kg. [Figure 8A] The absolute change in the urinary PC1 / CD133 ratio from baseline was measured for 2 mg / kg RG-NG-1015 (RGLS8429) (Figure 8A), 1 mg / kg RG-NG-1015 (Figure 8B), and placebo (Figure 8C). [Figure 8B] The absolute change in the urinary PC1 / CD133 ratio from baseline was measured for 2 mg / kg RG-NG-1015 (RGLS8429) (Figure 8A), 1 mg / kg RG-NG-1015 (Figure 8B), and placebo (Figure 8C). [Figure 8C] The absolute change in the urinary PC1 / CD133 ratio from baseline was measured for 2 mg / kg RG-NG-1015 (RGLS8429) (Figure 8A), 1 mg / kg RG-NG-1015 (Figure 8B), and placebo (Figure 8C). [Figure 9A]The absolute change in the urinary PC2 / CD133 ratio from individual baseline was measured for 2 mg / kg RG-NG-1015 (Figure 9A), 1 mg / kg RG-NG-1015 (Figure 9B), and placebo (Figure 9C). [Figure 9B] The absolute change in the urinary PC2 / CD133 ratio from individual baseline was measured for 2 mg / kg RG-NG-1015 (Figure 9A), 1 mg / kg RG-NG-1015 (Figure 9B), and placebo (Figure 9C). [Figure 9C] The absolute change in the urinary PC2 / CD133 ratio from individual baseline was measured for 2 mg / kg RG-NG-1015 (Figure 9A), 1 mg / kg RG-NG-1015 (Figure 9B), and placebo (Figure 9C). [Figure 10] A-B. Exploratory regression analysis of the absolute change in urinary PC1 / CD133 (A) and PC2 / CD133 (B) ratios from baseline was performed for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. Filled circles represent RG-NG-1015 treatment (1 mg / kg Q2W × 7), and filled squares represent RG-NG-1015 treatment (2 mg / kg Q2W × 7). White circles represent placebo (cohort 1+2). For RG-NG-1015 (1 mg / kg): #, Change in PC1 from baseline; statistical significance by Wilcoxon paired signed-rank test based on PC1 / CD133 ratio at days 85 and 86. #, Change in PC2 from baseline; statistical significance by Wilcoxon paired signed-rank test based on PC2 / CD133 ratio at day 113. For RG-NG-1015 (2 mg / kg): # Change from baseline in PC1; statistical significance by Wilcoxon paired signed-rank test based on PC1 / CD133 ratio at days 57, 86, 99, and 113. # Change from baseline in PC2; statistical significance by Wilcoxon paired signed-rank test based on PC2 / CD133 ratio at day 57. Exploratory regression analysis using nonlinear regression (quadratic polynomial). For the purpose of curve fitting, one subject was excluded in which PC1 / CD133=3.85 and PC2 / CD133=0.033 increased absolutely at day 113. [Figure 11] A-B. The percentage change in urinary PC1 / CD133 (A) and PC2 / CD133 (B) ratios from baseline was measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo, and is shown in group mean plots. Urinary polycystin levels were measured every two weeks for 113 days, before, during, and 28 days after the final (and 7th) dose of 1 mg / kg and 2 mg / kg RG-NG-1015. Urinary PC1 levels are shown in A, and urinary PC2 levels are shown in B. The filled circles represent RG-NG-1015 treatment (1 mg / kg Q2W × 7) (total N=9 subjects, Mayo class 1C / 1D / 1E=5 / 3 / 1). The white circles represent the placebo cohort (cohort 1+2) (Q2W x 7) (total N=6 subjects, Mayo class 1C / 1D / 1E = 1 / 4 / 1). The filled squares represent the RG-NG-1015 treatment (2 mg / kg Q2W x 7) (total N=11 subjects, Mayo class 1C / 1D / 1E = 5 / 4 / 1). [Figure 12] A and B. Mean change in polycystin levels after 3 months of Q2W treatment compared to baseline (mean of all available measurements from day 85 to day 116) for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. A shows the absolute change in urinary PC1 / CD133 ratio, and B shows the absolute change in urinary PC2 / CD133 ratio. [Figure 13] A and B. Mean change in polycystin levels after 3 months of Q2W treatment compared to baseline (mean of all available measurements from day 85 to day 116) for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. A shows the % change in urinary PC1 / CD133 ratio, and B shows the % change in urinary PC2 / CD133 ratio. [Figure 14] A and B. These show the correlation between polycystin levels and pharmacokinetic (PK) parameters. The correlation between urinary PC1 and a single dose of Cmax is shown in A. The correlation between urinary PC1 and a single dose of AUClast is shown in B. [Figure 15A]Changes in height-adjusted total kidney volume (htTKV) (Figure 15A) and total renal cystic volume (TKCV) (Figure 15B) at the end of the study compared to baseline were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the percentage change in TKCV and the percentage change in htTKV was also measured (Figure 15C). [Figure 15B] Changes in height-adjusted total kidney volume (htTKV) (Figure 15A) and total renal cystic volume (TKCV) (Figure 15B) at the end of the study compared to baseline were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the percentage change in TKCV and the percentage change in htTKV was also measured (Figure 15C). [Figure 15C] Changes in height-adjusted total kidney volume (htTKV) (Figure 15A) and total renal cystic volume (TKCV) (Figure 15B) at the end of the study compared to baseline were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the percentage change in TKCV and the percentage change in htTKV was also measured (Figure 15C). [Figure 16A] Changes in total liver volume (TLV) at the end of the study compared to baseline (Figure 16A) and total liver cyst volume (TLCV) (Figure 16B) were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the absolute change in TLCV and the percentage change in TLV was also measured (Figure 16C). [Figure 16B] Changes in total liver volume (TLV) at the end of the study compared to baseline (Figure 16A) and total liver cyst volume (TLCV) (Figure 16B) were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the absolute change in TLCV and the percentage change in TLV was also measured (Figure 16C). [Figure 16C]Changes in total liver volume (TLV) at the end of the study compared to baseline (Figure 16A) and total liver cyst volume (TLCV) (Figure 16B) were measured for 2 mg / kg RG-NG-1015, 1 mg / kg RG-NG-1015, and placebo. The correlation between the absolute change in TLCV and the percentage change in TLV was also measured (Figure 16C). [Figure 17A] Exploratory correlations were measured between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17A), and between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17B). Exploratory correlations were also measured between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17C), and between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17D). [Figure 17B] Exploratory correlations were measured between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17A), and between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17B). Exploratory correlations were also measured between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17C), and between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17D). [Figure 17C]Exploratory correlations were measured between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17A), and between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17B). Exploratory correlations were also measured between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17C), and between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17D). [Figure 17D] Exploratory correlations were measured between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17A), and between changes in PC1 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17B). Exploratory correlations were also measured between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 from baseline) and changes in htTKV (Figure 17C), and between changes in PC2 (shown as absolute changes in PC1 / CD133, mean D85-D113 values) and changes in eGFR (Figure 17D). [Figure 18A]RG-NG-1015 inhibits miR-17, conferring efficacy in a dose-response manner. Figure 18A shows cross-sectional images of kidneys from Pkd1F / RC mice administered with various doses of RG-NG-1015 (RGLS8429), PBS, or a control oligonucleotide at 20 mg / kg. *, Cross-sectional images of kidneys from age-matched wild-type C57BL6 mice (Lakhia et al. 2022 Nat Commun. 2022 Aug 15;13(1):4765) are shown for reference only. $, Pkd1F / RC mice in this treatment group were administered only on postnatal (P) days 8 and 12. All other Pkd1F / RC mice in the study were administered on P8, 10, 12, and 15. Figure 18B shows the calculated inhibition percentages of kidney weight / body weight (KW / BW) for various kidney concentrations of RG-NG-1015 (RGLS8429) in Pkd1F / RC mice. Figure 18C shows the calculated inhibition percentages of miR-17 for various kidney concentrations of RGLS-4326 and RG-NG-1015 (RGLS8429) in WT-C57BL6 mice. [Figure 18B] RG-NG-1015 inhibits miR-17, conferring efficacy in a dose-response manner. Figure 18A shows cross-sectional images of kidneys from Pkd1F / RC mice administered with various doses of RG-NG-1015 (RGLS8429), PBS, or a control oligonucleotide at 20 mg / kg. *, Cross-sectional images of kidneys from age-matched wild-type C57BL6 mice (Lakhia et al. 2022 Nat Commun. 2022 Aug 15;13(1):4765) are shown for reference only. $, Pkd1F / RC mice in this treatment group were administered only on postnatal (P) days 8 and 12. All other Pkd1F / RC mice in the study were administered on P8, 10, 12, and 15. Figure 18B shows the calculated inhibition percentages of kidney weight / body weight (KW / BW) for various kidney concentrations of RG-NG-1015 (RGLS8429) in Pkd1F / RC mice. Figure 18C shows the calculated inhibition percentages of miR-17 for various kidney concentrations of RGLS-4326 and RG-NG-1015 (RGLS8429) in WT-C57BL6 mice. [Figure 18C]RG-NG-1015 inhibits miR-17, conferring efficacy in a dose-response manner. Figure 18A shows cross-sectional images of kidneys from Pkd1F / RC mice administered with various doses of RG-NG-1015 (RGLS8429), PBS, or a control oligonucleotide at 20 mg / kg. *, Cross-sectional images of kidneys from age-matched wild-type C57BL6 mice (Lakhia et al. 2022 Nat Commun. 2022 Aug 15;13(1):4765) are shown for reference only. $, Pkd1F / RC mice in this treatment group were administered only on postnatal (P) days 8 and 12. All other Pkd1F / RC mice in the study were administered on P8, 10, 12, and 15. Figure 18B shows the calculated inhibition percentages of kidney weight / body weight (KW / BW) for various kidney concentrations of RG-NG-1015 (RGLS8429) in Pkd1F / RC mice. Figure 18C shows the calculated inhibition percentages of miR-17 for various kidney concentrations of RGLS-4326 and RG-NG-1015 (RGLS8429) in WT-C57BL6 mice. [Modes for carrying out the invention]

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. Unless otherwise specified, the nomenclature, procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. If there are multiple definitions for a term herein, the definition in this section shall prevail. Standard techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and for the treatment of patients. Specific such techniques and procedures can be found, for example, in "Carbohydrate Modifications in Antisense Research," edited by Sanghvi and Cook, American Chemical Society, Washington DC, 1994, and "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990, which are incorporated herein by reference for any purpose. Where permitted, all patents, patent applications, published applications and publications, GENBANK sequences, websites, and other publicly available materials referenced throughout this disclosure are incorporated by reference in their entirety unless otherwise specified. Where URLs or other such identifiers or addresses are referenced, it is understood that such identifiers may change, and certain information on the Internet may change, but equivalent information can be found by searching the Internet. References to them demonstrate the availability and public dissemination of such information.

[0009] Before disclosing and describing the compositions and methods of the present invention, it should be understood that the technical terms used herein are intended solely to describe specific embodiments and are not intended to be limiting. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural nouns unless otherwise explicitly indicated by the context.

[0010] definition Polycystic kidney disease, or 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 and often lead to enlargement of the affected kidney(s) and progressive loss of kidney function.

[0011] "Markers of polycystic kidney disease" refers to medical parameters used to assess the severity of polycystic kidney disease, renal function, and / or response to treatment in subjects with polycystic kidney disease. Non-exclusive examples of markers of polycystic kidney disease include total kidney volume, hypertension, glomerular filtration rate, and renal pain.

[0012] "Kidney function markers" refer to medical parameters used to assess kidney function in a subject. Non-exclusive examples of kidney function markers include glomerular filtration rate, blood urea nitrogen levels, and serum creatinine levels.

[0013] Autosomal dominant polycystic kidney disease, or ADPKD, is a polycystic kidney disease caused by one or more gene mutations in the PKD1 and / or PKD2 genes. 85% of ADPKD cases are caused by mutations in PKD1, located on chromosome 16, while the majority of the remaining ADPKD cases are caused by mutations in PKD2, located on chromosome 4.

[0014] Autosomal recessive polycystic kidney disease, or ARPKD, is a polycystic kidney disorder caused by one or more gene mutations in the PKHD1 gene located on chromosome 6. Up to 50% of newborns with ARPKD die from complications of intrauterine kidney disease, and about one-third of survivors develop end-stage renal disease (ESRD) within 10 years.

[0015] "Nephronophthiriasis" or "NPHP" refers to an autosomal recessive cystic kidney disease characterized by corticomedullary cysts, tubular basement membrane disruption, and tubulointerstitial nephropathy.

[0016] "Total kidney volume" or "TKV" is a measure of total kidney volume. Total kidney volume may be determined by magnetic resonance imaging (MRI), computed tomography (CT) scan, or ultrasound (US) imaging, and the volume may be calculated by standard methodologies such as the ellipsoidal volume equation (for ultrasound), or determined by quantitative stereology or boundary tracing (for CT / MRI).

[0017] Height-adjusted total kidney volume (HtTKV) is a measure of total kidney volume per unit height. Patients with an HtTKV value of 600 ml / m² or higher are predicted to develop stage 3 chronic kidney disease within 8 years.

[0018] "Kidney pain" refers to clinically significant kidney pain requiring medical leave, pharmacological treatment (narcotic or last-resort analgesics), or invasive intervention.

[0019] "Worsening of hypertension" refers to a change in blood pressure that requires the initiation or increase of hypertension treatment.

[0020] "Fibrosis" refers to the formation or development of excessive fibrous connective tissue in an organ or tissue. In certain embodiments, fibrosis occurs as a reparative or reactive process. In certain embodiments, fibrosis occurs in response to injury or damage. 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 the organ or tissue.

[0021] "Hematuria" refers to the presence of red blood cells in the urine.

[0022] Albuminuria refers to the presence of excess albumin in the urine and includes, but is not limited to, normal albuminuria, hypernormal albuminuria, microalbuminuria, and overt albuminuria. Normally, the glomerular filtration barrier, composed of podocytes, glomerular basement membrane, and endothelial cells, prevents serum proteins from leaking into the urine. Albuminuria may reflect damage to the glomerular filtration barrier. Albuminuria can be calculated from a 24-hour urine sample, an overnight urine sample, or a spot urine sample.

[0023] "Elevated normal albuminuria" refers to elevated albuminuria characterized by (i) urinary excretion of 15 to <30 mg of albumin per 24 hours and / or (ii) an albumin / creatinine ratio of 1.25 to <2.5 mg / mmol (or 10 to <20 mg / g) in males, or 1.75 to <3.5 mg / mmol (or 15 to <30 mg / g) in females.

[0024] "Microalbuminuria" refers to elevated albuminuria characterized by (i) the excretion of 30-300 mg of albumin in the urine per 24 hours, and / or (ii) an albumin / creatinine ratio of 2.5 to <25 mg / mmol (or 20 to <200 mg / g) in males, or 3.5 to <35 mg / mmol (or 30 to <300 mg / g) in females.

[0025] "Overt albuminuria" means elevated albuminuria characterized by the excretion of more than 300 mg of albumin in the urine per 24 hours, and / or (ii) an albumin / creatinine ratio of >25 mg / mmol (or >200 mg / g) in males and >35 mg / mmol (or >300 mg / g) in females.

[0026] The "albumin / creatinine ratio" refers to the ratio of urinary albumin (mg / dL) to urinary creatinine (g / dL) and is expressed as mg / g. In certain embodiments, the albumin / creatinine ratio may be calculated from a spot urine sample and may be used as an estimate of albumin excretion over 24 hours.

[0027] "Glomerular filtration rate" or "GFR" refers to the flow rate of filtered fluid through the kidney and is used as an indicator of renal function in a subject. In certain embodiments, the subject's GFR is determined by calculating the estimated glomerular filtration rate. In certain embodiments, the subject's GFR is measured directly in the subject using the inulin method.

[0028] "Estimated glomerular filtration rate" or "eGFR" is 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. A normal result is 90-120 mL / min / 1.73m². 2 It may be within the range of 60 mL / min / 1.73 m³ over 3 or more days. 2 Levels below 15 mL / min / 1.73 m³ may be an indicator of chronic kidney disease. 2 Levels below this may be an indicator of renal failure.

[0029] "Proteinuria" means the presence of excess serum protein in the urine. Proteinuria can be characterized by the excretion of more than 250 mg of protein in the urine per 24 hours, and / or a urinary protein-to-creatinine ratio of 0.20 mg / mg or higher. Serum proteins elevated in association with proteinuria include, but are not limited to, albumin.

[0030] Blood urea nitrogen level, or "BUN level," is a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea in the urea cycle as a waste product of protein digestion, and urea is removed from the blood by the kidneys. Normal human adult blood contains 7–21 mg of urea nitrogen per 100 ml (7–21 mg / dL) of blood. Measuring blood urea nitrogen levels is used as an indicator of kidney health. If the kidneys are unable to properly remove urea from the blood, the BUN level will be elevated.

[0031] "Increase" refers to an increase in a medical parameter that is considered clinically relevant. Healthcare professionals can determine whether the increase is clinically significant.

[0032] "End-stage renal disease (ESRD)" refers to complete or near-complete failure of kidney function.

[0033] "Quality of life" refers to the degree to which an individual's physical, psychological, and social functions are impaired by the disease and / or treatment of the disease. Quality of life may be reduced in individuals with polycystic kidney disease.

[0034] "Renal dysfunction" refers to a decline in kidney function compared to normal kidney function.

[0035] "To slow down the deterioration" means to reduce the rate at which a medical condition progresses toward a progressive state.

[0036] "Delay time to dialysis" means maintaining sufficient kidney function so that the need for dialysis treatment is delayed.

[0037] "Delay time until kidney transplant" means maintaining sufficient kidney function so that the need for a kidney transplant is delayed.

[0038] "Improving life expectancy" means extending the lifespan of a subject by treating one or more symptoms of a disease.

[0039] "Subject" means a human or non-human animal selected for treatment or therapy.

[0040] "Those who need it" refers to those who have been identified as needing therapy or treatment.

[0041] "Subjects suspected of having the disease" refers to subjects exhibiting one or more clinical indicators of the disease.

[0042] "miR-17-related diseases" refers to diseases or conditions regulated by the activity of one or more miR-17 family members.

[0043] "Administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administration by a medical professional and self-administration.

[0044] "Pareral administration" refers to administration via infusion or injection. Parenteral administration methods include, but are not limited to, subcutaneous, intravenous, and intramuscular administration.

[0045] "Subcutaneous administration" means administration directly beneath the skin.

[0046] "Intravenous administration" means administering the drug into a vein.

[0047] "Simultaneous administration" refers to the co-administration of two or more drugs in any manner in which their pharmacological effects appear simultaneously in a patient. Simultaneous administration does not require that both drugs be administered in a single pharmaceutical composition, in the same dosage form, or via the same route of administration. The effects of both drugs do not need to appear simultaneously. The effects only need to overlap for a certain period of time and do not need to have the same extent.

[0048] "Duration" refers to the period during which an activity or event persists. In a particular embodiment, the treatment period is the period during which a certain dose of a pharmaceutical agent or pharmaceutical composition is administered.

[0049] "Therapy" refers to a method of treating a disease. In certain embodiments, therapy includes, but is not limited to, administering one or more pharmaceutical agents to a subject with a disease.

[0050] "To treat" means to apply one or more specific procedures used to alleviate at least one indicator of the disease. In certain embodiments, a specific procedure is to administer one or more medicinal agents. In certain embodiments, treatment of PKD includes, but is not limited to, a reduction in total kidney volume, an improvement in renal function, a reduction in hypertension, and / or a reduction in renal pain.

[0051] "To alleviate" means a reduction in the severity of at least one indicator of a condition or disease. In certain embodiments, alleviation includes delaying or slowing the progression of one or more indicators of a condition or disease. The severity of an indicator may be determined by a subjective or objective scale known to those skilled in the art.

[0052] "At risk of developing" means that the subject has a predisposition to developing a condition or disease. In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but not enough symptoms to warrant a diagnosis of the condition or disease. In certain embodiments, a subject at risk of developing a condition or disease exhibits one or more symptoms of the condition or disease, but to a low degree to which a diagnosis of the condition or disease is necessary.

[0053] "Preventing onset" means preventing the onset of a condition or disease in subjects at risk of developing the condition or disease. In certain embodiments, subjects at risk of developing the condition or disease receive treatment similar to that received by subjects who already have the condition or disease.

[0054] "Delaying onset" means delaying the onset of a condition or disease in subjects at risk of developing the disease or condition. In certain embodiments, subjects at risk of developing the disease or condition receive treatment similar to that received by subjects who already have the disease or condition.

[0055] "Dose" refers to a specific amount of a pharmaceutical agent provided in a single dose. In certain embodiments, the dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments where subcutaneous administration is desired, the desired dose requires a volume that cannot be readily addressed by a single injection. In such embodiments, the desired dose may be achieved using two or more injections. In certain embodiments, the dose may be administered in two or more injections to minimize injection site reactions in the individual. In certain embodiments, the dose is administered as a slow infusion.

[0056] "Dosage unit" refers to the form in which the pharmaceutical agent is provided. In certain embodiments, the dosage unit is a vial containing lyophilized oligonucleotides. In certain embodiments, the dosage unit is a vial containing reconstituted oligonucleotides.

[0057] "Therapeutic dose" refers to the amount of a drug that provides a therapeutic benefit to an animal.

[0058] "Pharmaceutical composition" means a mixture of substances suitable for administration to an individual, including a pharmaceutical agent. For example, a pharmaceutical composition may include a sterile aqueous solution.

[0059] A "pharmaceutical agent" refers to a substance that, when administered to a target, provides a therapeutic effect.

[0060] "Active pharmaceutical ingredient" refers to a substance in a pharmaceutical composition that provides the desired effect.

[0061] "Pharmacologically acceptable salt" means a physiologically and pharmaceutically acceptable salt of a compound provided herein, i.e., a salt that retains the desired biological activity of the compound and does not have undesirable toxicological effects when administered to a subject. Non-limiting exemplary pharmaceutically acceptable salts of the compounds provided herein include sodium and potassium salt forms. As used herein, the terms "compound," "oligonucleotide," and "modified oligonucleotide" include their pharmaceutically acceptable salts unless otherwise specified.

[0062] "Physiological saline solution" refers to an aqueous solution of sodium chloride.

[0063] "Improvement of organ function" refers to a change in organ function towards the normal range. In certain embodiments, organ function is assessed by measuring molecules found in the subject's blood or urine. In certain embodiments, improvement in renal function is measured by a decrease in blood urea nitrogen, a decrease in proteinuria, a decrease in albuminuria, etc.

[0064] "Acceptable safety profile" refers to a pattern of adverse events that falls within clinically acceptable limits.

[0065] "Side effects" refer to physiological responses resulting from treatment other than the desired effects. In certain embodiments, side effects may 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 may be detected directly or indirectly. For example, increased levels of aminotransferase in serum may indicate hepatotoxicity or abnormal liver function. For example, increased bilirubin may indicate hepatotoxicity or abnormal liver function.

[0066] As used herein, the term "blood" includes whole blood and blood fractions, such as serum and plasma.

[0067] "Anti-miR" refers to an oligonucleotide having a nucleic acid base sequence complementary to microRNA. In certain embodiments, anti-miR is a modified oligonucleotide.

[0068] "Anti-miR-17" means a modified oligonucleotide having a nucleic acid base sequence complementary to one or more miR-17 family members. In certain embodiments, anti-miR-17 is fully complementary (i.e., 100% complementary) to one or more miR-17 family members. In certain embodiments, anti-miR-17 is at least 80%, at least 85%, at least 90%, or at least 95% complementary to one or more miR-17 family members.

[0069] "miR-17" refers to a mature miRNA with the nucleic acid base sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (Sequence ID 1).

[0070] "miR-20a" refers to a mature miRNA with the nucleic acid base sequence 5'-UAAAGUGCUUAUAGUGCAGGUAG-3' (Sequence ID 2).

[0071] "miR-20b" refers to a mature miRNA with the nucleic acid base sequence 5'-CAAAGUGCUCAUAGUGCAGGUAG-3' (Sequence ID 3).

[0072] "miR-93" refers to a mature miRNA with the nucleic acid base sequence 5'-CAAAGUGCUGUUCGUGCAGGUAG-3' (SEQ ID NO: 4).

[0073] "miR-106a" refers to a mature miRNA with the nucleic acid base sequence 5'-AAAAGUGCUUACAGUGCAGGUAG-3' (Sequence ID 5).

[0074] "miR-106b" refers to a mature miRNA with the nucleic acid base sequence 5'-UAAAGUGCUGACAGUGCAGAU-3' (SEQ ID NO: 6).

[0075] The "miR-17 seed sequence" refers to the nucleic acid base sequence 5'-AAAGUG-3,' present in each member of the miR-17 family.

[0076] "miR-17 family members" refers to mature miRNAs that have a nucleic acid sequence containing the miR-17 seed sequence and are selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0077] The "miR-17 family" refers to the following group of miRNAs, each having a nucleic acid sequence containing the miR-17 seed sequence: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

[0078] "Target nucleic acids" refer to nucleic acids that are designed to hybridize with oligomeric compounds.

[0079] "Targeting" refers to the process of designing and selecting nucleic acid base sequences that will hybridize to a target nucleic acid.

[0080] "Targeting" means having a nucleic acid base sequence that will enable hybridization to the target nucleic acid.

[0081] "Modulation" means a change in function, quantity, or activity. In certain embodiments, modulation means an increase in function, quantity, or activity. In certain embodiments, modulation means a decrease in function, quantity, or activity.

[0082] "Expression" refers to any function or step in which the encoded information of a gene is converted into a structure that exists within and operates within a cell.

[0083] "Nucleic acid base sequence" refers to the order of adjacent nucleic acid bases in an oligomeric compound or nucleic acid, and is typically enumerated in 5' to 3' orientation, regardless of any sugars, ligatures, and / or modifications of nucleic acid bases.

[0084] "Adjacent nucleic acid bases" refers to nucleic acid bases that are directly adjacent to each other within a nucleic acid.

[0085] "Nucleic acid base complementarity" refers to the ability of two nucleic acid bases to pair non-covalently via hydrogen bonds.

[0086] "Complementary" means that one nucleic acid can hybridize with another nucleic acid or oligonucleotide. In certain embodiments, complementary refers to an oligonucleotide that can hybridize with a target nucleic acid.

[0087] "Completely complementary" means that each nucleic acid base of an oligonucleotide can pair with a nucleic acid base at its corresponding position in the target nucleic acid. In certain embodiments, an oligonucleotide is completely complementary to the microRNA (also called 100% complementary). That is, each nucleic acid base of the oligonucleotide is complementary to the nucleic acid base at its corresponding position in the microRNA. A modified oligonucleotide may be completely complementary to the microRNA and have multiple ligated nucleosides shorter than the length of the microRNA. For example, an oligonucleotide having 16 ligated nucleosides, where each nucleic acid base of the oligonucleotide is complementary to the nucleic acid base at its corresponding position in the microRNA, is completely complementary to the microRNA. In certain embodiments, an oligonucleotide in which each nucleic acid base is complementary to a nucleic acid base within a region of the microRNA stem-loop sequence is completely complementary to the microRNA stem-loop sequence.

[0088] "Complementarity percentage" refers to the proportion of nucleic acid bases in an oligonucleotide that are complementary to the isolength portion of the target nucleic acid. The complementarity percentage is calculated by dividing the number of nucleic acid bases in the oligonucleotide that are complementary to the nucleic acid base at the corresponding position in the target nucleic acid by the total number of nucleic acid bases in the oligonucleotide.

[0089] "Identity percentage" refers to the number of nucleic acid bases in the first nucleic acid that are identical to the nucleic acid bases in the second nucleic acid at the corresponding positions, divided by the total number of nucleic acid bases in the first nucleic acid. In certain embodiments, the first nucleic acid is a microRNA, and the second nucleic acid is a microRNA. In certain embodiments, the first nucleic acid is an oligonucleotide, and the second nucleic acid is an oligonucleotide.

[0090] "Hybridizing" refers to the annealing of complementary nucleic acids that occurs due to the complementarity of nucleic acid bases.

[0091] A "mismatch" refers to a nucleic acid base of a first nucleic acid that cannot Watson-crick with the corresponding nucleic acid base of the second nucleic acid at its corresponding position.

[0092] In the context of nucleic acid base sequences, "identical" means having the same nucleic acid base sequence, regardless of sugars, ligations, and / or nucleic acid base modifications, and regardless of the methylation state of any pyrimidine present.

[0093] "MicroRNA" refers to endogenous non-coding RNA with a length of 18–25 nucleic acid bases, and is the product of cleavage of premicroRNA by an enzyme dicer. Examples of mature microRNA can be found in the microRNA database known as miRBase (microrna.sanger.ac.uk / ). In certain embodiments, microRNA is abbreviated as "miR".

[0094] "MicroRNA-regulated transcripts" refer to transcripts that are regulated by microRNAs.

[0095] A "seed-match sequence" refers to a nucleic acid sequence that is complementary to the seed sequence and has the same length as the seed sequence.

[0096] An "oligomeric compound" refers to a compound containing multiple linked monomer subunits. Oligomer compounds include oligonucleotides.

[0097] An "oligonucleotide" refers to a compound containing multiple linked nucleosides, each of which can be modified or unmodified independently of the others.

[0098] "Naturally occurring internucleoside linkages" refers to phosphodiester linkages between nucleosides from 3' to 5'.

[0099] "Natural sugars" refers to sugars found in DNA (2'-H) or RNA (2'-OH).

[0100] "Internucleoside linkage" refers to a shared linkage between adjacent nucleosides.

[0101] "Linked nucleosides" refer to nucleosides joined together by covalent linkage.

[0102] A "nucleic acid base" refers to a heterocyclic portion that can pair with another nucleic acid base in a non-covalent bond.

[0103] "Nucleoside" refers to a nucleic acid base linked to a sugar portion.

[0104] A "nucleotide" refers to a nucleoside that has a phosphate group covalently attached to the sugar portion of the nucleoside.

[0105] A compound containing a modified oligonucleotide consisting of a certain number of linked nucleosides means a compound containing a modified oligonucleotide having a specific number of linked nucleosides. Therefore, the compound may contain additional substituents or conjugates. Unless otherwise specified, the modified oligonucleotide is not hybridized to a complementary chain, and the compound does not contain any additional nucleosides beyond those of the modified oligonucleotide.

[0106] A "modified oligonucleotide" refers to a single-stranded oligonucleotide that has one or more modifications compared to naturally occurring terminal, sugar, nucleic acid base, and / or nucleoside linkages. Modified oligonucleotides may contain unmodified nucleosides.

[0107] "Modified nucleosides" refer to nucleosides that have any modifications from naturally occurring nucleosides. Modified nucleosides may have modified sugars and unmodified nucleic acid bases. Modified nucleosides may have modified sugars and modified nucleic acid bases. Modified nucleosides may have natural sugars and modified nucleic acid bases. In certain embodiments, modified nucleosides are bicyclic nucleosides. In certain embodiments, modified nucleosides are non-bicyclic nucleosides.

[0108] "Modified nucleoside linkages" refer to any changes from naturally occurring nucleoside linkages.

[0109] "Phosphothioate nucleoside linkage" refers to a linkage between nucleosides where one of the non-bridged atoms is a sulfur atom.

[0110] "Modified sugar portion" means substitution and / or any alteration from natural sugar.

[0111] "Unmodified nucleic acid bases" refer to naturally occurring heterocyclic bases of RNA or DNA, with purines based on adenine (A) and guanine (G), and pyrimidines based on thymine (T), cytosine (C) (including 5-methylcytosine), and uracil (U).

[0112] "5-methylcytosine" refers to cytosine containing a methyl group attached at the 5th position.

[0113] "Non-methylated cytosine" refers to cytosine that does not have a methyl group attached at position 5.

[0114] "Modified nucleic acid base" refers to any nucleic acid base that is not an unmodified nucleic acid base.

[0115] The term "sugar portion" refers to the naturally occurring furanosyl or modified sugar portion.

[0116] "Modified sugar portion" refers to the substituted sugar portion or sugar substitute.

[0117] "2'-O-methyl sugar" or "2'-OMe sugar" refers to a sugar that has an O-methyl modification at the 2' position.

[0118] "2'-O-methoxyethyl sugar" or "2'-MOE sugar" refers to a sugar that has an O-methoxyethyl modification at the 2' position.

[0119] "2'-Fluoro" or "2'-F" refers to a sugar that has a fluoro modification at the 2' position.

[0120] A “bicyclic sugar moiety” refers to a modified sugar moiety containing a 4- to 7-membered ring (including, but not limited to, a furanosyl) that includes a bridge that joins two atoms of a 4- to 7-membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4- to 7-membered ring is a sugar ring. In certain embodiments, the 4- to 7-membered ring is a furanosyl. In certain such embodiments, the bridge joins the 2'-carbon and 4'-carbon of the furanosyl. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

[0121] The term "locked nucleic acid (LNA) sugar moiety" refers to a substituted sugar moiety that contains a (CH2)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0122] The "ENA sugar portion" refers to the substituted sugar portion that contains a (CH2)2-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0123] The term "restricted ethyl (cEt) sugar moiety" refers to a substituted sugar moiety containing a CH(CH3)-O bridge between a 4'-furanose ring atom and a 2'-furanose ring atom. In certain embodiments, the CH(CH3)-O bridge is restricted in the S direction. In certain embodiments, the CH(CH3)-O bridge is restricted in the R direction.

[0124] The term "S-cEt sugar moiety" refers to a substituted sugar moiety that includes an S-restricted CH(CH3)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0125] The term "R-cEt sugar moiety" refers to a substituted sugar moiety that includes an R-constrained CH(CH3)-O bridge between the 4'-furanose ring atom and the 2'-furanose ring atom.

[0126] "2'-O-methyl nucleoside" refers to a 2'-modified nucleoside that has a 2'-O-methyl sugar modification.

[0127] "2'-O-methoxyethyl nucleoside" refers to a 2'-modified nucleoside having a 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleosides may contain modified or unmodified nucleic acid bases.

[0128] "2'-Fluoronucleoside" refers to a 2'-modified nucleoside that has a 2'-fluorosaccharide modification. 2'-Fluoronucleosides may contain modified or unmodified nucleic acid bases.

[0129] A "bicyclic nucleoside" refers to a 2'-modified nucleoside that has a bicyclic sugar moiety. Bicyclic nucleosides can have modified or unmodified nucleic acid bases.

[0130] "cEt nucleoside" refers to a nucleoside containing a cEt sugar moiety. cEt nucleosides may contain modified or unmodified nucleic acid bases.

[0131] "S-cEt nucleoside" refers to a nucleoside that contains an S-cEt sugar moiety.

[0132] "R-cEt nucleoside" refers to a nucleoside that contains the R-cEt sugar moiety.

[0133] "β-D-deoxyribonucleoside" refers to a naturally occurring DNA nucleoside.

[0134] "β-D-ribonucleoside" refers to a naturally occurring RNA nucleoside.

[0135] "LNA nucleoside" refers to a nucleoside that contains the LNA sugar portion.

[0136] "ENA nucleoside" refers to a nucleoside that contains the ENA sugar portion.

[0137] A "hydrogen bond acceptor" refers to a component of a hydrogen bond that does not supply the shared hydrogen atom.

[0138] A "hydrogen bond donor" refers to a bond or molecule that supplies the hydrogen atoms for a hydrogen bond.

[0139] References to values ​​or parameters “about” in this specification include (and are described) embodiments relating to the value or parameter itself. In certain embodiments, the term “about” includes the indicated amount ± 10%. In other embodiments, the term “about” includes the indicated amount ± 5%. In certain other embodiments, the term “about” includes the indicated amount ± 1%. Also, the term “about X” includes a description of “X.” Furthermore, the singular forms “a” and “the” include plural references unless otherwise explicitly indicated in the context. Thus, for example, a reference to “compound” includes multiple such compounds.

[0140] overview Polycystic kidney disease (PKD) is a genetic form of kidney disease characterized by the development of fluid-filled cysts in the kidneys, leading to renal failure and, in many cases, end-stage renal disease (ESRD). Certain forms of PKD are also characterized by nephroemmography. Excessive cystic proliferation is a characteristic pathological feature of PKD. In the management of PKD, the main goals of treatment are to manage symptoms such as hypertension and infections, maintain renal function, and prevent or delay the development of ESRD, thereby improving the life expectancy of individuals with PKD.

[0141] miR-17 has been identified as a therapeutic target for PKD. RGLS4326, an anti-miR-17 compound, was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutically sound properties. RGLS4326 preferentially distributes to cysts derived from the kidney and collection duct, displaces miR-17 from translationally active polysomes, and desuppresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in a human in vitro autosomal dominant polycystic kidney disease (ADPKD) model and several PKD mouse models after subcutaneous administration. A Phase 1 single-dose elevation (SAD) clinical trial in healthy volunteers (HVs) was initiated in December 2017, followed by a Phase 1 multiple-dose elevation (MAD) clinical trial in HVs in May 2018.

[0142] Following the initiation of the Phase 1b MAD clinical trial, non-clinical toxicity studies revealed CNS-related findings in mice with high doses of RGLS4326, including abnormal gait, reduced motor activity, and / or debilitation. RGLS4326 was found to be an antagonist of AMPA receptors (AMPA-R), glutamate receptors and ion channels on excitatory synapses in the central nervous system (CNS) that mediate rapid excitatory neurotransmission, and are therefore crucial components of all neuronal networks. Antagonism of AMPA receptors may explain the CNS-mediated findings observed with high doses of RGLS4326 in the non-clinical toxicity model. While such CNS-related findings were not observed in human subjects, avoiding AMPA receptor antagonism is still preferable. Therefore, a library of anti-miR-17 compounds was screened to identify compounds with comparable physicochemical and pharmacological properties to RGLS4326 that have a more favorable safety profile (e.g., those that can avoid AMPA receptor antagonism).

[0143] We identified one such compound, RG-NG-1015, and selected it as a candidate therapeutic agent for the treatment of ADPKD.

[0144] RG-NG-1015 and related compounds A compound containing a modified oligonucleotide, wherein the modified oligonucleotide has structure 5'-A S G S C M A F C F U F U M U S A S A compound having -3' and in which each cytosine is unmethylated cytosine is provided herein.

[0145] In certain embodiments, the compound consists of a modified oligonucleotide.

[0146] In certain embodiments, the pharmaceutically acceptable salt is a sodium salt.

[0147] The modified oligonucleotide named RG-NG-1015 has the following structure: [ka] The modified oligonucleotide described herein is provided.

[0148] pharmaceutically acceptable salts of the modified oligonucleotide RG-NG-1015 are also provided herein. Thus, in some embodiments, the modified oligonucleotide has the following structure: [ka] or a pharmaceutically acceptable salt thereof. Non-limiting and exemplary pharmaceutically acceptable salts of RG-NG-1015 include: [ka] It holds.

[0149] In some embodiments, the pharmaceutically acceptable salt of the modified oligonucleotide contains fewer cationic counterions (Na) per molecule than phosphorothioate and / or phosphodiester links. + (e.g., some phosphorothioates and / or phosphodiester links are protonated). In some embodiments, pharmaceutically acceptable salts of RG-NG-1015 contain fewer than eight cationic counterions (Na) per molecule of RG-NG-1015. + This includes, for example, RG-NG-1015. In other words, in some embodiments, a pharmaceutically acceptable salt of RG-NG-1015 may, on average, contain 1, 2, 3, 4, 5, 6, or 7 cationic counterions per molecule of RG-NG-1015, with the remaining phosphorothioate groups being protonated.

[0150] Where used herein, and unless there is a specific reference to a particular pharmaceutically acceptable salt of RG-NG-1015, any dosage should be understood to refer to the amount of RG-NG-1015 in its protonated form (i.e., not as a salt), whether expressed, for example, as mg / kg, mg, or by weight %.

[0151] 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 volume sufficient to extract 1 mL of RG-NG-1015 at a concentration of 150 mg / mL. In some embodiments, RG-NG-1015 is in a solution containing 0.3% physiological saline.

[0152] Treatment methods for polycystic kidney disease A method for inhibiting the activity of one or more members of the miR-17 family within a cell is provided herein, the method comprising contacting the cell with a compound provided herein that comprises a nucleic acid base sequence complementary to a miR-17 seed sequence.

[0153] A method for inhibiting the activity of one or more members of the miR-17 family in a subject, comprising administering a pharmaceutical composition provided herein to the subject, is provided herein. In certain embodiments, the subject has a disease associated with one or more members of the miR-17 family.

[0154] A method for treating polycystic kidney disease (PKD) is provided herein, comprising administering a compound provided herein, comprising a nucleic acid sequence complementary to a miR-17 seed sequence, to a subject in need thereof. In certain embodiments, the subject has polycystic kidney disease. In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD), autosomal recessive polycystic kidney disease (ARPKD), and nephronophthisis (NPHP). In certain embodiments, the polycystic kidney disease is selected from autosomal dominant polycystic kidney disease (ADPKD) and autosomal recessive polycystic kidney disease (ARPKD).

[0155] In certain embodiments, the subject has a disorder characterized by multiple non-renal indicators, and also by polycystic kidney disease. Such disorders include, for example, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Valday-Beedl syndrome (BBS). Therefore, a method for the treatment of polycystic kidney disease (PKD) is provided herein, comprising administering to a subject a compound provided herein containing a nucleic acid sequence complementary to the miR-17 seed sequence, wherein the subject is suspected to have Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Valday-Beedl syndrome (BBS). A method for the treatment of polycystic kidney disease (PKD) is provided herein, comprising administering to a compound provided herein containing a nucleic acid sequence complementary to the miR-17 seed sequence, wherein the subject is suspected to have Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Valday-Beedl syndrome (BBS).

[0156] In certain embodiments, polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is caused by mutations in the PKD1 or PKD2 gene. ADPKD is a progressive disease in which cystic formation and nephroematous enlargement lead to renal failure in 50% of patients by age 60, eventually leading to end-stage renal disease. Patients with ADPKD may require lifelong dialysis and / or kidney transplantation. ADPKD is the most frequent genetic cause of renal failure. Excessive cystic proliferation is a characteristic pathological feature of ADPKD. In the management of PKD, the main goal of treatment is to maintain renal function and prevent or delay the onset of end-stage renal disease (ESRD), thereby improving the life expectancy of subjects with PKD. Total kidney volume generally increases steadily in ADPKD patients, and this increase correlates with a decline in renal function. A method for treating ADPKD is provided herein, comprising administering a compound provided herein, comprising a nucleic acid base sequence complementary to the miR-17 seed sequence, to a subject having or suspected of having ADPKD.

[0157] In certain embodiments, polycystic kidney disease is autosomal recessive polycystic kidney disease (ARPKD). ARPKD is caused by mutations in the PKHD1 gene and is the cause of chronic kidney disease in children. The typical renal phenotype of ARPKD is kidney enlargement, but ARPKD has significant effects on other organs, particularly the liver. Patients with ARPKD progress to end-stage renal disease and require a kidney transplant at the young age of 15. A method for treating ARPKD is provided herein, comprising administering a compound provided herein, comprising a nucleic acid sequence complementary to the miR-17 seed sequence, to a subject having or suspected of having ARPKD.

[0158] In certain embodiments, polycystic kidney disease is nephronophthria (NPHP). Nephronophthria is an autosomal recessive cystic kidney disease that is a frequent cause of ESRD in children. NPHP is characterized by kidneys of normal or reduced size, cysts concentrated at the corticomedullary junction, and tubulointerstitial fibrosis. Mutations in one of several NPHP genes, e.g., NPHP1, have been identified in patients with NPHP. A method for the treatment of NPHP is provided herein, comprising administering to a subject having or suspected of having NPHP a compound provided herein, comprising a nucleic acid sequence complementary to a miR-17 seed sequence.

[0159] In certain embodiments, subjects with polycystic kidney disease also have Joubert syndrome and related disorders (JSRD). JSRD includes a wide range of prominent features, including abnormalities of the brain, retina, and skeleton. Certain subjects with JSRD have polycystic kidney disease in addition to the features of JSRD. Accordingly, a method for treating polycystic kidney disease in a subject with JSRD is provided herein, comprising administering to the subject with JSRD a compound provided herein comprising a nucleic acid sequence complementary to the miR-17 seed sequence. In certain embodiments, a subject is suspected of having JSRD.

[0160] In certain embodiments, a subject with polycystic kidney disease has Meckel's syndrome (MKS). MKS is a disorder 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, resulting in kidney enlargement. Accordingly, a method for treating MKS is provided herein, comprising administering to a subject having MKS a compound provided herein, comprising a nucleic acid base sequence complementary to the miR-17 seed sequence. In certain embodiments, a subject is suspected of having MKS.

[0161] In certain embodiments, a subject with polycystic kidney disease has Valday-Beedl syndrome (BBS). BBS is a disorder that affects many parts of the body, including the eyes, heart, kidneys, liver, and digestive system. A characteristic feature of BBS is the presence of renal cysts. Accordingly, a method for treating polycystic kidney disease in a subject with BBS is provided herein, comprising administering to the subject with BBS a compound provided herein that contains a nucleic acid base sequence complementary to the miR-17 seed sequence. In certain embodiments, the subject is suspected of having BBS.

[0162] In certain embodiments, the subject is diagnosed with PKD prior to administration of a compound containing modified oligonucleotides. The diagnosis of PKD may be achieved by assessment of parameters including, but not limited to, the subject's family history, clinical features (including, but not limited to, hypertension, albuminuria, hematuria, and impaired GFR), renal imaging studies (including, but not limited to, MRI, ultrasound, and CT scans), and / or histological analysis.

[0163] In some embodiments, subjects with ADPKD are classified as class 1C, 1D, or 1E according to the Mayo imaging classification of ADPKD. In some embodiments, subjects with ADPKD are 30-90 mL / min / 1.73 m 2 It is defined as an object having an estimated glomerular filtration rate (eGFR).

[0164] In some embodiments, a method for treating ADPKD is provided, comprising administering to a subject in need of such treatment a compound comprising a modified oligonucleotide considered herein, such as RG-NG-1015, or a pharmaceutically acceptable salt thereof, such as a sodium salt of RG-NG-1015, in amounts of 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. In some embodiments, compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof are administered in 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, compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof are administered in doses of about 1 mg / kg, about 2 mg / kg, or about 3 mg / kg. In some embodiments, compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof are administered in doses of 1 mg / kg, 2 mg / kg, or 3 mg / kg. In some embodiments, compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof are administered in doses of about 1 mg / kg or 1 mg / kg. In some embodiments, compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof are administered in doses of about 2 mg / kg or 2 mg / kg. In some embodiments, the compound comprising a modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a dose of approximately 3 mg / kg or 3 mg / kg. In some embodiments, the compound comprising a modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered subcutaneously.

[0165] In some embodiments, the compound comprising a modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered every two weeks (14 days). In some embodiments, the compound comprising 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, the subject is not administered tolvaptan within 28 days prior to administration of the compound comprising a modified oligonucleotide or a pharmaceutically acceptable salt thereof considered herein.

[0166] In certain embodiments, the diagnosis of PKD includes screening for mutations in one or more of the PKD1 or PKD2 genes. In certain embodiments, the diagnosis of ARPKD includes screening for mutations in the PKHP1 gene. In certain embodiments, the diagnosis of NPHP includes screening for one or more mutations in one or more of the NPHP1, NPHP2, NPHP3, NPHP4, NPHP5, NPHP6, NPHP7, NPHP8, or NPHP9 genes. In certain embodiments, the diagnosis of JSRD includes screening for mutations in the NPHP1, NPHP6, AHI1, MKS3, or RPGRIP1L genes. In certain embodiments, the diagnosis of MKS includes screening for mutations in the NPHP6, MKS3, RPGRIP1L, NPHP3, CC2D2A, BBS2, BBS4, BBS6, or MKS1 genes. In a particular embodiment, the diagnosis of BBS involves screening for mutations in the BBS2, BBS4, BBS6, MKS1, BBS1, BBS3, BBS5, BBS7, BBS7, BBS8, BBS9, BBS10, BBS11, or BBS12 genes.

[0167] In certain embodiments, the subject has increased total renal volume. In certain embodiments, total renal volume is height-adjusted total renal volume (HtTKV). In certain embodiments, the subject has hypertension. In certain embodiments, the subject has renal impairment. In certain embodiments, the subject requires improved renal function. In certain embodiments, the subject is identified as having renal impairment.

[0168] In certain embodiments, the level of one or more miR-17 family members is increased in the kidney of a subject having PKD. In certain embodiments, prior to administration, the subject is determined to have an increased level of one or more miR-17 family members in the kidney. The level of miR-17 family members may be measured from renal biopsy material. In certain embodiments, prior to administration, the subject is determined to have an increased level of one or more miR-17 family members in the subject's urine or blood. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) or polycystin-2 (PC2) in the subject's urine. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) or polycystin-2 (PC2) in the subject's urine. In certain embodiments, prior to administration, the subject is determined to have a decreased level of polycystin-1 (PC1) and / or polycystin-2 (PC2) in the subject's urine.

[0169] In some embodiments, prior to administration, the subject is determined to have elevated levels of neutrophil gelatinase-binding lipocalin (NGAL) and / or kidney injury molecule 1 (KIM-1) in the subject's urine. In some embodiments, prior to administration, the subject is determined to have elevated levels of neutrophil gelatinase-binding lipocalin (NGAL) and kidney injury molecule 1 (KIM-1) in the subject's urine. In some embodiments, prior to administration, the subject is determined to have elevated levels of neutrophil gelatinase-binding lipocalin (NGAL) or kidney injury molecule 1 (KIM-1) in the subject's urine.

[0170] In any of the embodiments provided herein, the subject may undergo certain tests before, during, and / or after administration to diagnose polycystic kidney disease in the subject, for example, to determine the cause of polycystic kidney disease, to assess the extent of polycystic kidney disease in the subject, and / or to determine the subject's response to treatment. Such tests may evaluate markers of polycystic kidney disease. Certain tests, such as glomerular filtration rate (GFR) and blood urea nitrogen (BUN) levels, are also indicators of renal function. Markers for polycystic ovary syndrome (PCOS) include, but are not limited to, measurement of total kidney volume and height-adjusted total kidney volume (htTKV) in subjects, measurement of hypertension in subjects, assessment of renal pain in subjects, measurement of fibrosis in subjects, measurement of polycystin-1 (PC1) in subjects' urine, measurement of polycystin-2 (PC2) in subjects' urine, measurement of blood urea nitrogen (BUN) levels in subjects, measurement of serum creatinine (SCr) levels in subjects, measurement of creatinine clearance in subjects, measurement of albuminuria in subjects, measurement of albumin:creatinine ratio in subjects, measurement of glomerular filtration rate (GFR) and estimated GFR (eGFR) in subjects, measurement of hematuria in subjects, measurement of NGAL protein in subjects' urine, and / or measurement of KIM-1 protein in subjects' urine. Unless otherwise indicated 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 of the blood (whole blood or serum, etc.) in question.

[0171] In some embodiments, the subject may also undergo certain tests, such as the measurement of monocyte chemotactic protein 1 (MCP-1) and / or beta-2 microglobulin (B2M) in the subject's urine before, during, and / or after administration; the measurement of insulin-like growth factor-binding protein acid unstable subunits (IGFALS), copeptin (CT-proAVP), and / or N-acetyl-1-methylhistidine in the subject's serum; complement degradation products C3a and / or Bb in the subject's plasma; and / or the measurement of total cyst volume, number, and / or size distribution of cysts in the subject.

[0172] Markers for polycystic kidney disease (PCD) are determined by laboratory testing. Reference ranges for individual markers may vary from laboratory to laboratory. This variation can be due, for example, to differences in the specific assay used. Therefore, the upper and lower limits of the normal distribution of markers within a population (also known as the upper normal limit (ULN) and lower normal limit (LLN), respectively) may vary from laboratory to laboratory. For any given marker, a healthcare professional may determine which levels outside the normal distribution are clinically relevant and / or indicate disease. For example, a healthcare professional may determine the glomerular filtration rate, which may indicate a decline in the rate of renal function in subjects with PCD.

[0173] In certain embodiments, administration of the compounds provided herein results in one or more clinically beneficial outcomes. In certain embodiments, the administration improves the renal function of the subject. In certain embodiments, the administration slows the rate of decline in renal function in the subject. In certain embodiments, the administration reduces the total renal volume of the subject. In certain embodiments, the administration slows the rate of increase in total renal volume in the subject. In certain embodiments, the administration reduces height-adjusted total renal volume (HtTKV). In certain embodiments, the administration slows the rate of increase in HtTKV.

[0174] In certain embodiments, the administration increases polycystin-1 (PC1) in the subject's urine. In certain embodiments, the administration increases polycystin-2 (PC2) in the subject's urine. In certain embodiments, the administration increases both polycystin-1 (PC1) and polycystin-2 (PC2) in the subject's urine.

[0175] In certain embodiments, the administration inhibits the growth of the cysts in the subject (total cyst volume, number, and / or size distribution). In certain embodiments, the administration slows down the rate of increase in the growth of the cysts in the subject (total cyst volume, number, and / or size distribution). In some embodiments, the cysts are located in the kidneys of the subject. In some embodiments, the cysts are located in organs other than the kidneys, such as the liver.

[0176] In certain embodiments, the administration alleviates the subject's kidney pain. In certain embodiments, the administration slows the increase in kidney pain in the subject. In certain embodiments, the administration delays the onset of kidney pain in the subject.

[0177] In certain embodiments, the administration reduces the hypertension of the subject. In certain embodiments, the administration slows the worsening of hypertension in the subject. In certain embodiments, the administration delays the onset of hypertension in the subject.

[0178] In certain embodiments, the administration reduces fibrosis in the target kidney. In certain embodiments, the administration slows the progression of fibrosis in the target kidney.

[0179] In certain embodiments, the administration delays the onset of end-stage renal disease in the subject. In certain embodiments, the administration delays the time to dialysis in the subject. In certain embodiments, the administration delays the time to kidney transplantation in the subject. In certain embodiments, the administration improves the life expectancy of the subject.

[0180] In certain embodiments, the administration reduces albuminuria in the subject. In certain embodiments, the administration slows the worsening of albuminuria in the subject. In certain embodiments, the administration delays the onset of albuminuria in the subject. In certain embodiments, the administration reduces hematuria in the subject. In certain embodiments, the administration slows the worsening of hematuria in the subject. In certain embodiments, the administration delays the onset of hematuria in the subject. In certain embodiments, the administration reduces or slows the rate of increase in blood urea nitrogen (BUN) levels in the subject. In certain embodiments, the administration reduces or slows the rate of increase in serum creatinine (SCr) levels in the subject. In certain embodiments, the administration improves or slows the rate of decrease in creatinine clearance in the subject. In certain embodiments, the administration reduces or slows the rate of increase in the urinary albumin:creatinine ratio in the subject.

[0181] In certain embodiments, the administration improves the glomerular filtration rate in the subject. In certain embodiments, the administration slows the rate of decline of the glomerular filtration rate in the subject. In certain embodiments, the glomerular filtration rate is the estimated glomerular filtration rate (eGFR). In certain embodiments, the glomerular filtration rate is the measured glomerular filtration rate (mGFR).

[0182] In certain embodiments, the administration reduces or slows the rate of increase of neutrophil gelatinase-binding lipocalin (NGAL) protein in the subject's urine. In certain embodiments, the administration reduces or slows the rate of increase of kidney injury molecule 1 (KIM-1) protein in the subject's urine.

[0183] In certain embodiments, the administration reduces or slows the rate of increase of monocyte chemotactic protein 1 (MCP-1) in the subject's urine. In certain embodiments, the administration reduces or slows the rate of increase of beta-2 microglobulin (B2M) in the subject's urine.

[0184] In certain embodiments, the administration reduces or slows down the rate of increase of complement degradation products C3a and / or Bb in the plasma of the subject.

[0185] In certain embodiments, the administration reduces or slows down the rate of increase of acute-phase proteins (i.e., Alb, fibrinogen, and / or highly sensitive C-reactive proteins).

[0186] In certain embodiments, the administration reduces or slows the rate of increase of serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the serum of the subject. In certain embodiments, the administration reduces or slows the rate of increase of copeptin (CT-proAVP) in the serum of the subject. In certain embodiments, the administration reduces or slows the rate of increase of N-acetyl-1-methylhistidine in the serum of the subject.

[0187] In any of the embodiments provided herein, the subject may be subjected to certain tests to assess the degree of disease in the subject. Such tests include: measurement of total kidney volume in the subject; measurement of htTKV in the subject; measurement of hypertension in the subject; measurement of renal pain in the subject; measurement of fibrosis in the kidney of the subject; measurement of blood urea nitrogen (BUN) levels in the subject; measurement of serum creatinine (SCr) levels in the subject; measurement of blood creatinine clearance in the subject; measurement of albuminuria in the subject; measurement of albumin:creatinine ratio in the subject; measurement of glomerular filtration rate (GFR) in the subject (GFR is an estimate or measurement); measurement of urinary porcine in the subject. This includes, but is not limited to, the measurement of lystistin-1 (PC1) and / or polycystistin-2 (PC2), the measurement of neutrophil gelatinase-binding lipocalin (NGAL) protein in the subject's urine and / or the measurement of kidney injury molecule 1 (KIM-1) protein in the subject's urine, the measurement of MCP-1 and / or B2M in the subject's urine, the measurement of IGFALS in the subject's serum, the measurement of CT-proAVP and / or the measurement of N-acetyl-1-methylhistidine, and the measurement of total cyst volume, number, and / or size distribution of cysts.

[0188] In some embodiments, administration of compounds containing modified oligonucleotides or pharmaceutically acceptable salts thereof, as considered herein, results in little to no CNS failure in the subject. In some embodiments, CNS failure is measured by the Scale for the Assessment and Rating of Ataxia (SARA) test, a tool for assessing ataxia. In some embodiments, the SARA test is performed on the subject before administration. In some embodiments, administration results in little to no change in the Scale for the Assessment and Rating of Ataxia (SARA) test score for the subject compared to before treatment.

[0189] In some embodiments, the subject may be subjected to certain tests before, during, and / or after administration to evaluate the pharmacokinetics of compounds including modified oligonucleotides or pharmaceutically acceptable salts thereof discussed herein. Pharmacokinetic analysis is performed to measure and compare one or more of the following parameters: maximum observed concentration (C max ), time to reach maximum observed concentration (T max ), Area under the concentration-time curve (AUC) up to 24 hours after drug administration 0-24 ), area under the concentration-time curve (AUC) up to the last quantifiable concentration 0-t ), area under the concentration-time curve (AUC) over the dosing interval. tau ), area under the concentration-time curve extrapolated to infinity (AUC inf ), half-life (t 1 / 2 ), apparent clearance (CL / F), apparent distribution volume (V z / F), the proportion of the unchanged compound excreted in the urine (fe), and / or the total amount of the unchanged compound excreted in the urine (Ae).

[0190] In some embodiments, during and / or after administration of a compound containing a modified oligonucleotide or a pharmaceutically acceptable salt thereof as discussed herein, the subject may be subjected to a specific test to evaluate the development of anti-drug antibodies (ADAs) in the subject's plasma.

[0191] In certain embodiments, subjects with polycystic kidney disease experience a reduction in quality of life. For example, subjects with polycystic kidney disease may experience kidney pain, which can reduce their quality of life. In certain embodiments, administration improves the subject's quality of life.

[0192] In any of the embodiments provided herein, the subjects are human subjects. In certain embodiments, the human subjects are adults. In certain embodiments, adults are at least 21 years of age. In certain embodiments, the human subjects are child subjects, i.e., subjects are under 21 years of age. The child population may be defined by regulatory authorities. In certain embodiments, the human subjects are adolescents. In certain embodiments, adolescents are at least 12 years of age and under 21 years of age. In certain embodiments, the human subjects are children. In certain embodiments, children are at least 2 years of age and under 12 years of age. In certain embodiments, the human subjects are infants. In certain embodiments, infants are at least 1 month of age and under 2 years of age. In certain embodiments, the subjects are neonates. In certain embodiments, neonates are less than 1 month of age. In certain embodiments, the subjects are between 18 and 70 years of age.

[0193] Any of the compounds described herein may be for use in therapy. Any of the compounds provided herein may be for use in the treatment of polycystic kidney disease. In certain embodiments, the polycystic kidney disease is autosomal dominant polycystic kidney disease. In certain embodiments, the polycystic kidney disease is autosomal recessive polycystic kidney disease. In certain embodiments, the polycystic kidney disease is nephronophthisis. In certain embodiments, the subject has Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS).

[0194] Any of the modified oligonucleotides described herein may be for use in therapy. Any of the modified oligonucleotides provided herein may be for use in the treatment of polycystic kidney disease.

[0195] Any of the compounds provided herein may be for use in the preparation of a medicament. Any of the compounds provided herein may be for use in the preparation of an agent for the treatment of polycystic kidney disease.

[0196] Any of the modified oligonucleotides provided herein may be for use in the preparation of a medicament. Any of the modified oligonucleotides provided herein may be for use in the preparation of an agent for the treatment of polycystic kidney disease.

[0197] Any of the pharmaceutical compositions provided herein may be for use in the treatment of polycystic kidney disease.

[0198] In some embodiments, the treatment method has an acceptable safety and tolerance profile. In some embodiments, the treatment method is generally safe and well-tolerated.

[0199] Certain additional therapies Treatment for polycystic kidney disease or any of the conditions listed herein may comprise two or more therapies. Accordingly, in certain embodiments, a method for treating a subject having or suspected to have polycystic kidney disease is provided herein, comprising administering a compound provided herein comprising a nucleic acid sequence complementary to the miR-17 seed sequence, in addition to administering at least one therapy.

[0200] In certain embodiments, at least one additional therapy comprises a pharmaceutical agent. In certain embodiments, the pharmaceutical agent is an antihypertensive agent. The antihypertensive agent is used to control the blood pressure of the subject.

[0201] In certain embodiments, the pharmaceutical agent is a vasopressin receptor 2 antagonist. In certain embodiments, the vasopressin receptor 2 antagonist is tolvaptan.

[0202] In certain embodiments, the pharmaceutical agent comprises an angiotensin II receptor blocker (ARB). In certain embodiments, the angiotensin II receptor blocker is candesartan, irbesartan, olmesartan, losartan, valsartan, telmisartan, or eprosartan.

[0203] In certain embodiments, the pharmaceutical agent comprises an angiotensin II-converting enzyme (ACE) inhibitor. In certain embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benazepril, quinapril, fosinopril, or ramipril.

[0204] In certain embodiments, the pharmaceutical agent is a diuretic. In certain embodiments, the pharmaceutical agent is a calcium channel blocker.

[0205] In certain embodiments, the pharmaceutical agent is a glucosylceramide synthase inhibitor. In certain embodiments, the glucosylceramide synthase inhibitor is benglustat.

[0206] In certain embodiments, the pharmaceutical agent is an antihyperglycemic agent. In certain embodiments, the antihyperglycemic agent is a biguanide. In certain embodiments, the biguanide is metformin.

[0207] In certain embodiments, the pharmaceutical agent is a kinase inhibitor. In certain embodiments, the kinase inhibitor is bosutinib or KD019.

[0208] In certain embodiments, the pharmaceutical agent is an adrenaline receptor antagonist.

[0209] In certain embodiments, the pharmaceutical agent is an aldosterone receptor antagonist. In certain embodiments, the aldosterone receptor antagonist is spironolactone. In certain embodiments, spironolactone is administered in a dose ranging from 10 to 35 mg daily. In certain embodiments, spironolactone is administered in a dose of 25 mg daily.

[0210] In certain embodiments, the pharmaceutical agent is a mammalian target (mTOR) inhibitor of rapamycin. In certain embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

[0211] In certain embodiments, the pharmaceutical agent is a hormone analog. In certain embodiments, the hormone analog is somatostatin or adrenocorticotropic hormone.

[0212] In certain embodiments, the pharmaceutical agent is an antifibrotic agent. In certain embodiments, the antifibrotic agent is a modified oligonucleotide complementary to miR-21.

[0213] In certain embodiments, the additional therapy is dialysis. In certain embodiments, the additional therapy is kidney transplantation.

[0214] In certain embodiments, the pharmaceutical agent comprises an anti-inflammatory agent. In certain embodiments, the anti-inflammatory agent is a steroidal anti-inflammatory agent. In certain embodiments, the steroidal anti-inflammatory agent is a corticosteroid. In certain embodiments, the corticosteroid is prednisone. In certain embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug. In certain embodiments, the non-steroidal anti-inflammatory agent is ibuprofen, a COX-I inhibitor, or a COX-2 inhibitor.

[0215] In certain embodiments, the pharmaceutical agent is an agent that blocks one or more responses to fibrotic signals.

[0216] In certain embodiments, the additional therapy can be a pharmaceutical agent that enhances the body's immune system, including low-dose cyclophosphamide, thymostimulin, vitamins, and dietary supplements (e.g., antioxidants including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q-10, and echinacea), and vaccines, such as an immunostimulating complex (ISCOM) comprising a vaccine formulation combining multimeric presentation of an antigen and an adjuvant.

[0217] In certain embodiments, the additional therapy is selected to treat or alleviate the side effects of one or more pharmaceutical compositions provided herein. Such side effects include, but are not limited to, injection site reactions, liver function abnormalities, kidney function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, an increase in serum aminotransferase levels can indicate hepatotoxicity or liver function abnormalities. For example, an increase in bilirubin can indicate hepatotoxicity or liver function abnormalities.

[0218] A certain microRNA nucleic acid base sequence The miR-17 family includes miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b. Each member of the miR-17 family has a nucleic acid sequence containing the miR-17 seed sequence, which is the nucleic acid sequence 5'-AAAGUG-3' or the nucleic acid sequence at positions 2-7 of SEQ ID NO: 1. In addition, each member of the miR-17 family shares some nucleic acid sequence identity outside the seed region. Therefore, modified oligonucleotides containing nucleic acid sequences complementary to the miR-17 seed sequence can target not only miR-17 but also other microRNAs of the miR-17 family.

[0219] In a particular embodiment, the modified oligonucleotide contains the nucleic acid base sequence 5'-AGCACUUUA-3'.

[0220] In certain embodiments, each cytosine is independently selected from unmethylated cytosine and 5-methylcytosine. In certain embodiments, at least one cytosine is unmethylated cytosine. In certain embodiments, each cytosine is unmethylated cytosine. In certain embodiments, at least one cytosine is 5-methylcytosine. In certain embodiments, each cytosine is 5-methylcytosine.

[0221] In certain embodiments, the number of linked nucleosides in the modified oligonucleotide is shorter than the length of the target microRNA. The modified oligonucleotide is considered to have a number of linked nucleosides shorter than the length of the target microRNA, and each nucleic acid base of the modified oligonucleotide is complementary to the nucleic acid base at the corresponding position of the target microRNA, and the modified oligonucleotide is considered to have a nucleic acid base sequence that is perfectly complementary (also called 100% complementary) to the region of the target microRNA sequence. For example, the modified oligonucleotide consists of nine linked nucleosides, and each nucleic acid base is complementary to the corresponding position of miR-17 and is perfectly complementary to miR-17.

[0222] In certain embodiments, the modified oligonucleotide has a nucleic acid sequence with one mismatch with respect to the nucleic acid sequence of the target microRNA. In certain embodiments, the modified oligonucleotide has a nucleic acid sequence with two mismatches with respect to the nucleic acid sequence of the target microRNA. In certain such embodiments, the modified oligonucleotide has a nucleic acid sequence with two or fewer mismatches with respect to the nucleic acid sequence of the target microRNA. In certain such embodiments, the mismatched nucleic acid bases are consecutive. In certain such embodiments, the mismatched nucleic acid bases are not consecutive.

[0223] The sequence listings attached to this application identify each nucleic acid sequence as either "RNA" or "DNA" as necessary, although in practice, these sequences may be modified by combinations of chemical modifications specified herein. Those skilled in the art will readily understand that the designations such as "RNA" or "DNA" used to describe modified oligonucleotides in the sequence listings are somewhat arbitrary. For example, a modified oligonucleotide containing a nucleoside with a 2'-O-methoxyethyl sugar moiety and a thymine base may be listed as a DNA residue in the sequence listings even if the nucleoside is modified and not a natural DNA nucleoside.

[0224] Therefore, the nucleic acid sequences provided in the sequence listing are intended to include nucleic acids containing any combination of natural or modified RNA and / or DNA, including but not limited to nucleic acids having modified nucleic acid bases. Further examples, but not limited to, a modified oligonucleotide having the nucleic acid base sequence "ATCGATCG" in the sequence listing, whether modified or unmodified, including but not limited to RNA bases such as those having the sequence "AUCGAUCG", and those having some DNA bases and some RNA bases such as "AUCGATCG", and "AT me CGAUCG( meThis includes any oligonucleotide having a nucleic acid base sequence, such as compounds containing oligonucleotides with other modified bases, such as C (which represents 5-methylcytosine).

[0225] A certain modifier In certain embodiments, the oligonucleotides provided herein may comprise one or more modifications to the nucleic acid bases, sugars, and / or nucleoside linkages, and thus may be modified oligonucleotides. For example, modified nucleic acid bases, sugars, and / or nucleoside linkages may be preferred over unmodified forms for desirable properties such as enhanced cellular uptake, increased affinity to other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases.

[0226] In certain embodiments, the modified oligonucleotide comprises one or more modified nucleosides.

[0227] In certain embodiments, the modified nucleoside is a sugar-modified nucleoside. In certain such embodiments, the sugar-modified nucleoside may further comprise a natural or modified heterocyclic base moiety and / or be bonded to another nucleoside via natural or modified nucleoside linkages and / or include further modifications independent of sugar modification. In certain embodiments, the sugar-modified nucleoside is a 2'-modified nucleoside, where the sugar ring is modified at the 2' carbon derived from natural ribose or 2'-deoxy-ribose.

[0228] In certain embodiments, the 2'-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is an alpha-configured D sugar. In certain such embodiments, the bicyclic sugar moiety is a beta-configured D sugar. In certain such embodiments, the bicyclic sugar moiety is an alpha-configured L sugar. In certain such embodiments, the bicyclic sugar moiety is a beta-configured L sugar.

[0229] Nucleosides containing such a bicyclic sugar moiety are referred to as bicyclic nucleosides or BNAs. In certain embodiments, bicyclic nucleosides include (A) α-L-methyleneoxy(4'-CH2-O-2')BNA, (B) β-D-methyleneoxy(4'-CH2-O-2')BNA, (C) ethyleneoxy(4'-(CH2)2-O-2')BNA, (D) aminooxy(4'-CH2-ON(R)-2')BNA, (E) oxyamino(4'-CH2-N(R)-O-2')BNA, and (F) methyl(methyleneoxy)(4'-CH This includes, but is not limited to, (CH3)-O-2')BNA (also known as restrained ethyl or cEt), (G) methylene-thio(4'-CH2-S-2')BNA, (H) methylene-amino(4'-CH2-N(R)-2')BNA, (I) methyl carboncyclic (4'-CH2-CH(CH3)-2')BNA, (J) c-MOE(4'-CH(CH2-OMe)-O-2')BNA, and (K) propylene carboncyclic (4'-(CH2)3-2')BNA. [ka] In the formula, Bx is the nucleic acid base portion, and R is independently H, a protecting group, or C1-C 12 It is alkyl.

[0230] In certain embodiments, the 2'-modified nucleoside includes a 2'-substituent selected from F, OCF3, O-CH3 (also referred to as "2'-OMe"), OCH2CH2OCH3 (also referred to as "2'-O-methoxyethyl" or "2'-MOE"), 2'-O(CH2)2SCH3, O-(CH2)2-ON(CH3)2, -O(CH2)2O(CH2)2N(CH3)2, and O-CH2-C(=O)-N(H)CH3.

[0231] In certain embodiments, the 2'-modified nucleoside includes a 2'-substituted group selected from F, O-CH3, and OCH2CH2OCH3.

[0232] In certain embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In certain embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. The 4'-thio-modified nucleoside has a β-D-ribonucleoside, where 4'-O is substituted with 4'-S. The 4'-thio-2'-modified nucleoside is a 4'-thio-modified nucleoside in which 2'-OH is substituted with a 2'-substituent. Preferred 2'-substituents include 2'-OCH3, 2'-OCH2CH2OCH3, and 2'-F.

[0233] In certain embodiments, the modified oligonucleotide comprises one or more internucleoside modifications. In certain such embodiments, each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. In certain embodiments, the modified internucleoside linkage comprises a phosphorus atom.

[0234] In certain embodiments, the modified oligonucleotide includes at least one phosphorothioate nucleoside linkage. In certain embodiments, each nucleoside linkage of the modified oligonucleotide is a phosphorothioate nucleoside linkage.

[0235] In certain embodiments, the modified oligonucleotide comprises one or more modified nucleic acid bases. In certain embodiments, the modified nucleic acid bases are selected from 5-hydroxymethylcytosine, 7-deazaguanine, and 7-deazaadenine. In certain embodiments, the modified nucleic acid bases are selected from 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. In certain embodiments, the modified nucleic acid bases are selected from 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.

[0236] In certain embodiments, the modified nucleic acid base includes a polycyclic heterocycle. In certain embodiments, the modified nucleic acid base includes a tricyclic heterocycle. In certain embodiments, the modified nucleic acid base includes a phenoxazine derivative. In certain embodiments, the phenoxazine may be further modified to form a nucleic acid base known in the art as a G-clamp.

[0237] In certain embodiments, the modified oligonucleotide is conjugated to one or more moieties that enhance the activity, cell distribution, or cell uptake of the resulting antisense oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety. In certain embodiments, the moiety is a lipid moiety. Additional moieties for conjugation include carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and pigments. In certain embodiments, the carbohydrate moiety is N-acetyl-D-galactosamine (GalNac). In certain embodiments, the conjugation group is directly attached to the oligonucleotide. In certain embodiments, the conjugate group is attached to the modified oligonucleotide by a linkage selected from amino, azide, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, the substituent is selected from hydroxyl, amino, alkoxy, azide, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl, and alkynyl.

[0238] In certain such embodiments, the compound comprises a modified oligonucleotide in which one or more stabilizing groups are attached to one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. The stabilizing groups include cap structures. These terminal modifications may protect the modified oligonucleotide from exonuclease degradation and 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. The cap structures include, for example, reverse deoxydebasic caps.

[0239] A certain pharmaceutical composition Pharmaceutical compositions comprising a compound or modified oligonucleotide provided herein and a pharmaceutically acceptable diluent are provided herein. In certain embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In certain embodiments, the aqueous solution is physiological saline. As used herein, the pharmaceutically acceptable diluent is understood to be a sterile diluent. Preferred routes of administration include, but are not limited to, intravenous and subcutaneous administration. In certain embodiments, administration is intravenous. In certain embodiments, administration is subcutaneous. In certain embodiments, administration is oral.

[0240] In certain embodiments, the pharmaceutical composition is administered in units of medication. For example, in certain embodiments, the units of medication may be in the form of tablets, capsules, or bolus injections.

[0241] In certain embodiments, the pharmaceutical preparation is a modified oligonucleotide, which is prepared in a suitable diluent, adjusted to pH 7.0–9.0 with an acid or base during preparation, and then lyophilized under sterile conditions. The lyophilized modified oligonucleotide is then reconstituted in a suitable diluent, such as an aqueous solution like water, or a physiologically compatible buffer such as saline, Hanks' solution, or Ringer's solution. The reconstituted product is administered by subcutaneous injection or intravenous infusion. The lyophilized preparation may be packaged in a 2 mL clear glass vial (ammonium sulfate treated), stoppered with a bromobutyl rubber closure, and sealed with an aluminum overseal.

[0242] In certain embodiments, the pharmaceutical compositions provided herein may further contain other auxiliary components conventionally found in pharmaceutical compositions at their established levels of use in the art. Thus, for example, the compositions may include additional suitable pharmaceutically active materials, such as antipruritics, astringents, topical anesthetics, or anti-inflammatory agents.

[0243] In some embodiments, the pharmaceutical compositions provided herein may contain additional materials useful for physically formulating various dosage forms of the compositions provided herein, such as dyes, flavorings, 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, silicic acid, viscous paraffin, hydroxymethylcellulose, and polyvinylpyrrolidone. In various embodiments, such materials, when added, should not unduly interfere with the biological activity of the components of the compositions provided herein. The formulations may be sterilized and, if necessary, mixed with adjuvants that do not adversely interact with the oligonucleotide(s) of the formulation, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, fragrances, and / or aromatic substances. Certain pharmaceutical compositions for injection are suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent to enable the preparation of highly concentrated solutions.

[0244] Lipid moieties are used in nucleic acid therapy in various ways. In one method, nucleic acids are introduced into pre-formed liposomes or lipoplexes made from a mixture of cationic and neutral lipids. In another method, DNA complexes with monocationic or polycationic lipids are formed without the presence of neutral lipids. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to specific cells or tissues. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to adipose tissue. In certain embodiments, lipid moieties are selected to increase the distribution of a pharmaceutical agent to muscle tissue.

[0245] In certain embodiments, the pharmaceutical compositions provided herein include a polyamine compound or lipid moiety complexed with a nucleic acid. In certain embodiments, such preparations include one or more compounds each having individually a structure defined by formula (Z) or a pharmaceutically acceptable salt thereof. [ka] In the formula, each a and X b C is independent of each occurrence. 1~6 It is an alkylene, where n is 0, 1, 2, 3, 4, or 5, each R independently is H, and at least about 80% of the R portions of the compound of formula (Z) in the preparation, at least n+2 are not H, m is 1, 2, 3, or 4, and Y is O, NR 2 , or S, R 1 is alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents, R 2is H, alkyl, alkenyl, or alkynyl, each of which is optionally substituted with one or more substituents, except that if n is 0, at least n+3 R moieties are not H. Such preparations are described in PCT Publication WO / 2008 / 042973, which is incorporated herein by reference in its entirety for the disclosure of lipid preparations. Certain additional preparations are described in Akinc et al., Nature Biotechnology 26, 561-569 (May 1, 2008), which is incorporated herein by reference in its entirety for the disclosure of lipid preparations.

[0246] In certain embodiments, the pharmaceutical compositions provided herein are prepared using known techniques, including but not limited to mixing, dissolving, granulation, sugar coating, polishing, emulsification, encapsulation, capture, or tableting processes.

[0247] In certain embodiments, the pharmaceutical compositions provided herein are solid (e.g., powders, tablets, and / or capsules). In certain embodiments of such a configuration, the solid pharmaceutical composition comprising one or more oligonucleotides is prepared using components known in the art, including but not limited to starches, sugars, diluents, granulators, lubricants, binders, and disintegrants.

[0248] In certain embodiments, the pharmaceutical compositions provided herein are formulated as depot preparations. Certain such depot preparations typically act for a longer period than non-depot preparations. In certain embodiments, such preparations are administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. In certain embodiments, the depot preparations are prepared using suitable polymers or hydrophobic materials (e.g., emulsions in acceptable oils) or ion exchange resins, or as sparingly soluble derivatives, for example, as sparingly soluble salts.

[0249] In certain embodiments, the pharmaceutical compositions provided herein include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions, including pharmaceutical compositions containing hydrophobic compounds. In certain embodiments, certain organic solvents, such as dimethyl sulfoxide, are used.

[0250] In certain embodiments, the pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver one or more pharmaceutical agents provided herein to a specific tissue or cell type. For example, in certain embodiments, the pharmaceutical composition comprises liposomes coated with tissue-specific antibodies.

[0251] In certain embodiments, the pharmaceutical compositions provided herein include a sustained-release system. A non-limiting example of such a sustained-release system is a semipermeable matrix of a solid hydrophobic polymer. In certain embodiments, the sustained-release system may release the pharmaceutical agent over several hours, days, weeks, or months, depending on its chemical properties.

[0252] Certain pharmaceutical compositions for injection are provided in unit dosage forms, for example, in ampoules or presented in multi-dose containers.

[0253] In certain embodiments, the pharmaceutical composition provided herein comprises a therapeutically effective amount of a modified oligonucleotide. In certain embodiments, the therapeutically effective amount is sufficient to prevent, reduce, or alleviate the symptoms of a disease, or to extend the survival time of the subject being treated.

[0254] In certain embodiments, one or more modified oligonucleotides provided herein are formulated as prodrugs. In certain embodiments, at in vivo administration, the prodrug is chemically converted to a more biologically, pharmaceutically, or therapeutically active form of the oligonucleotide. In certain embodiments, the prodrug is useful because it is easier to administer than the corresponding active form. For example, in certain cases, the prodrug may be more biologically available than the corresponding active form (e.g., by oral administration). In certain cases, the prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, the prodrug is less water-soluble than the corresponding active form. In certain cases, such a prodrug has excellent permeability across cell membranes where water solubility impairs mobility. In certain embodiments, the prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to a carboxylic acid at administration. In certain cases, the carboxylic acid-containing compound is the corresponding active form. In certain embodiments, the prodrug contains a short peptide (polyamino acid) bonded to an acid group. In certain such embodiments, the peptide is cleaved at administration to form the corresponding active form.

[0255] In certain embodiments, prodrugs are produced by modifying a pharmaceutically active compound so that the active compound is regenerated upon in vivo administration. Prodrugs can be designed to alter the metabolic stability or transport properties of a drug, to mask side effects or toxicity, to improve the flavor of a drug, or to alter other characteristics or properties of a drug. With knowledge of pharmacodynamic processes and drug metabolism in vivo, those skilled in the art can design prodrugs of a compound if the pharmaceutically active compound is known (see, for example, Nogrady (1985) Medicinal Chemistry: A Biochemical Approach, Oxford University Press, New York, pp. 388-392).

[0256] Additional routes of administration include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, inhalation, intrathecal, intracardiac, ventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intrathecal administration. In certain embodiments, the pharmaceutical is administered intrathecally to achieve local exposure rather than systemic exposure. For example, the pharmaceutical composition may be injected directly into the area where the effect is desired (e.g., the liver).

[0257] A certain kit Kits are also provided. In some embodiments, the kit comprises one or more compounds, including modified oligonucleotides disclosed herein. In some embodiments, the kit may be used to administer the compounds to a target.

[0258] In certain embodiments, the kit includes a pharmaceutical composition ready for administration. In some embodiments, the compounds provided herein are contained in vials. Multiple vials, such as 10, may be contained in a dispensing pack, for example. In some embodiments, the vials are manufactured for use with syringes. The kit may also include instructions for using the compounds.

[0259] In some embodiments, the kit contains a pharmaceutical composition that is present not in a vial, but in a pre-filled syringe (e.g., a single-dose syringe having a 27-gauge, 1 / 2-inch needle with a needle guard). Multiple pre-filled syringes, such as 10, may be present in a dispensing pack, for example. The kit may also include instructions for administering compounds containing modified oligonucleotides disclosed herein.

[0260] In some embodiments, the kit comprises a modified oligonucleotide provided herein as a lyophilized formulation and a pharmaceutically acceptable diluent. During preparation for administration to a subject, the lyophilized formulation is reconstituted in the pharmaceutically acceptable diluent.

[0261] In some embodiments, in addition to the compounds containing the modified oligonucleotides disclosed herein, the kit may further include one or more of the following: a syringe, an alcohol swab, a cotton ball, and / or a gauze pad.

[0262] A specific experimental model In certain embodiments, methods are provided for using and / or testing modified oligonucleotides provided herein in an experimental model. Those skilled in the art can select and modify protocols for such experimental models to evaluate pharmaceutical agents provided herein.

[0263] Generally, modified oligonucleotides are first tested in cultured cells. Suitable cell types include those related to the cell type for which in vivo delivery of the modified oligonucleotide is desired. For example, suitable cell types for the study of the method described herein include primary or cultured cells.

[0264] In certain embodiments, the extent to which a modified oligonucleotide interferes with the activity of one or more miR-17 family members is assessed in cultured cells. In certain embodiments, inhibition of microRNA activity may be assessed by measuring the levels of one or more predicted or validated microRNA regulatory transcripts. Inhibition of microRNA activity may result in an increase in miR-17 family member regulatory transcripts and / or proteins encoded by miR-17 family member regulatory transcripts (i.e., the miR-17 family member regulatory transcripts are desuppressed). Furthermore, in certain embodiments, certain phenotypic outcomes may be measured.

[0265] Several animal models are available to those skilled in the art for studying one or more miR-17 family members in models of human diseases. Models of polycystic kidney disease include, but are not limited to, models with mutations and / or deletions in Pkd1 and / or Pkd2, as well as models with mutations in other genes. Non-limiting exemplary models of PKD with mutations and / or deletions in Pkd1 and / or Pkd2 include low-phenotype models, e.g., models with missense mutations in Pkd1, and models with reduced or unstable expression of Pkd2, inducible conditional knockout models, and conditional knockout models. Non-limiting exemplary PKD models with mutations in genes other than Pkd1 and Pkd2 include models with mutations in Pkhd1, Nek8, Kif3a, and / or Nphp3. The PKD model is outlined, for example, in Shibazaki et al., Human Mol. Genet., 2008;17(11):1505-1516, Happe and Peters, Nat Rev Nephrol., 2014;10(10):587-601, and Patel et al., PNAS, 2013;110(26):10765-10770.

[0266] A specific quantitative assay In certain embodiments, microRNA levels are quantified in cells or tissues, either in vitro or in vivo. In certain embodiments, changes in microRNA levels are measured by microarray analysis. In certain embodiments, changes in microRNA levels are measured by one of several commercially available PCR assays, such as the TaqMan® MicroRNA Assay (Applied Biosystems).

[0267] The regulation of microRNA activity by anti-miRs or microRNA mimes can be evaluated by microarray profiling of mRNA. The mRNA sequences regulated (either increased or decreased) by anti-miRs or microRNA mimes are then screened for microRNA seed sequences to compare the regulation of target mRNAs with that of non-target mRNAs. In this way, the interaction between anti-miRs and their target microRNAs, or between microRNA mimes and their targets, can be evaluated. In the case of anti-miRs, mRNAs whose expression levels increase are screened for mRNA sequences containing seed matches with microRNAs to which the anti-miR is complementary.

[0268] The modulation of microRNA activity by anti-miR compounds can be assessed by measuring the level of the microRNA's messenger RNA target, either by measuring the level of the messenger RNA itself or the protein transcribed from it. Antisense inhibition of microRNA generally results in an increase in the level of the microRNA's messenger RNA and / or the protein of its messenger RNA target; that is, anti-miR therapy results in the desuppression of one or more target messenger RNAs. [Examples]

[0269] The following embodiments are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the present invention.

[0270] Those skilled in the art will readily adapt the principles underlying this discovery to design various compounds without departing from the spirit of the present invention.

[0271] Example 1: The role of miR-17 in PKD The miR-17 family members of the miR-17~92 cluster of microRNAs are upregulated in mouse models of PKD. Genetic deletion of the miR-17~92 cluster in mouse models of PKD reduces renal cyst growth, improves renal function, and prolongs survival (Patel et al., PNAS, 2013;110(26):10765-10770). The miR-17~92 cluster contains six distinct microRNAs, each with a different sequence: miR-17, miR-18a, miR-19a, miR-19-b-1, and miR-92a-1.

[0272] The miR-17~92 cluster contains two microRNAs, miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Each member of this family shares seed sequence identity and varying degrees of sequence identity outside the seed region. Other members of the miR-17 family are miR-20b, miR-93, miR-106a, and miR-106b. miR-20b and miR-106a are located within the miR-106a~363 cluster on human X chromosome, while miR-93 and miR-106b are located within the miR-106b~25 cluster on human chromosome 7. The sequences of the miR-17 family members are shown in Table 1. [Table 1]

[0273] The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed library of anti-miR-17 oligonucleotides for optimal pharmaceutically appropriate properties. RGLS4326 preferentially distributes to renal and vascular cysts, displaces miR-17 from translationally active polysomes, and desuppresses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in a human in vitro ADPKD model and multiple PKD mouse models after subcutaneous administration. A Phase 1 single-dose elevation (SAD) clinical trial of RGLS4326 in healthy volunteers was initiated in December 2017, followed by a Phase 1 multiple-dose elevation (MAD) clinical trial in healthy volunteers in May 2018. We initiated a Phase 1b clinical trial of RGLS4326 in October 2020 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD).

[0274] Following the initiation of the Phase 1 MAD clinical trial, non-clinical toxicity studies revealed central nervous system (CNS) related findings, including abnormal gait, reduced motor activity, and / or debilitation, with high doses of RGLS4326. To identify potential off-target pharmacological candidates, a panel of 174 targets, including G protein-binding receptors, transporters, ion channels, nuclear receptors, and cytokine receptors, was evaluated in vitro for possible interactions with RGLS4326. RGLS4326 was found to be an antagonist of AMPA glutamate receptors, with a 50% inhibitory concentration (IC50) of 4.6 μM (14.2 μg / mL) based on ligand binding and a functional IC50 of 300–600 nM (0.9–1.8 μg / mL) based on patch-clamp activity. AMPA receptors are ion channels on excitatory synapses in the CNS, which mediate rapid excitatory neurotransmission and are therefore critical components of all neuronal networks. Such interactions with AMPA receptors can explain the CNS-mediated findings observed with high doses of RGLS4326 in nonclinical toxicity models.

[0275] Example 2: Screening of anti-miR-17 compounds with reduced AMPA receptor binding. 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 S (In the formula, the nucleoside following the subscript "M" is a 2'-O-methylnucleoside, the nucleoside following the subscript "F" is a 2'-fluoronucleoside, the nucleoside following the subscript "S" is an S-cEt nucleoside, each cytosine is unmethylated cytosine, and all links are phosphorothioate links). Chemical modifications and length variants of RGLS4326 were designed and screened to identify compounds that retain the potency and pharmacokinetic profile of RGLS4326 and show reduced binding to the AMPA receptor (AMPA-R).

[0276] For RGLS4326, we designed a library of compounds with various chemical modifications, nucleic acid sequences, and lengths. [Table 2-1] [Table 2-2]

[0277] The activity of anti-miR-17 compounds was increased in the presence of higher concentrations of anti-miR-17 compounds to AMPA-R present on rat brain synaptic membranes. 3 The binding of the [H]AMPA ligand was evaluated by a radioligand binding assay. Anti-miR-17 compounds with affinity for AMPA-R were evaluated by [ 3 It binds to the [H]AMPA ligand and competes for its binding.

[0278] The assay was performed according to previously published methods (Honore et al., J Neurochem., 1982, 38(1):173-178, Olsen et al., Brain Res., 1987, 402(2):243-254). 5.0 nM ligand [ 3 [H]AMPA, 1.0 mM nonspecific ligand L-glutamic acid, and uM concentrations of anti-miR compounds were incubated for 90 minutes in synaptic membranes prepared from Wistar rat cerebral cortex. The compounds shown in Table 2 were tested in three experiments. Anti-miR compounds targeting microRNAs other than miR-17 were used as control compounds (RG5124 targeting miR-33a; RG5365 targeting let-7a; RG8093 targeting miR-214). RGLS4326 and RG-NG-1001 were also tested in each experiment, as they were demonstrated to bind to AMPA-R and inhibit its activity. 3 The amount of [H]AMPA ligand was quantified by radioactive ligand binding and is shown in Tables 3, 4, and 5. As the data shows, the compounds differ in their ability to inhibit the binding of radiolabeled ligands to AMPA-R. [Table 3] [Table 4] [Table 5]

[0279] To evaluate the functional antagonism of anti-miR-17 oligonucleotides against AMPA-R, specific oligonucleotides were tested using manual whole-cell patch-clamp techniques that recorded membrane current as a measure of AMPA-R activity.

[0280] Manual whole-cell patch-clamp studies were performed by Metrion Biosciences (Cambridge, UK). Whole-cell voltage clamp experiments were performed at room temperature (18–21°C) using an EPC10 patch-clamp amplifier with Patchmaster software (HEKA Elektronik). Glass patch pipettes were fabricated from borosilicate glass capillaries (Harvard Apparatus) with resistances ranging from 1.4–2.5 MΩ. Membrane currents were recorded using whole-cell patch-clamp techniques. ChanTest® GluA1 / GluA4 EZCells were clamped at a holding potential of -80 mV, and membrane currents delivered by 10 μM(S)-AMPA were extracted using a VC38 perfusion system (ALA Scientific Instruments). Minimum current amplitude values ​​were measured for each application of 10 μM(S)-AMPA. The fractional change in current amplitude generated by each compound concentration was calculated and compared to the control current (prior compound), and expressed as the percentage change (inhibition%) for each cell. The compounds tested are shown in Table 6. RGLS4326 was tested using a separate test from all the other compounds in Table 6.

[0281] As shown in Table 6, compared to RGLS4326, compounds RG-NG-1015, RG-NG-1016, and RG-NG-1017 exhibited reduced functional antagonism to AMPA-R based on manual whole-cell patch-clamp studies in human ChanTest® GluA1 / GluA4 EZ-Cells. [Table 6]

[0282] Example 3: Relationship between nucleic acid base properties and AMPA-R binding As demonstrated by AMPA-R binding and whole-cell patch-clamp studies, the presence of guanosine at the 3' end of anti-miR-17 oligonucleotides in a position complementary to the first nucleotide of miR-17 affects the functional antagonism of AMPA-R. Although adenosine is a purine, similar to guanosine, adenosine did not inhibit AMPA-R. Because guanosine and adenosine are similar in several properties except for hydrogen bonding, we evaluated the differences in hydrogen bonding at positions 1, 2, and 6 of purine bases. The purine nucleic acid bases tested are shown in Figure 1 and Table 7. In the "Purine Position" column of Table 7, "A" indicates the position of purine as a hydrogen acceptor, and "D" indicates the position of purine as a hydrogen donor. In the "Purine Position" column of Table 7, "N" indicates the natural position that is neither a hydrogen acceptor nor a donor. We also tested various 2'-sugar moieties on purine nucleic acid bases to evaluate the effect of 2'-sugar subchemistry on the ability of purine nucleic acid bases to inhibit the sugar AMPA-R. [Table 7]

[0283] The compound was tested using the radioligand binding assay described herein to determine the anti-miR-17 compound, [ 3 The ability to bind to the [H]AMPA ligand and the ability to compete with it were determined. As shown in Table 8, a correlation was observed between inhibition of ligand binding to [H]AMPA-R and the presence of a hydrogen bond acceptor at the 6th position of the purine in the 3' terminal nucleic acid base of the oligonucleotide. For example, compounds with guanosine or inosine at the 3' terminal resulted in inhibition of ligand binding to AMPA-R. Compounds with a 3' terminal nucleic acid base having a hydrogen bond acceptor at the 6th position of the purine, such as RG-NG-1037 and RG-NG-1039, were less likely to inhibit ligand binding to AMPA-R. [Table 8]

[0284] Example 4: Anti-miR-17 compounds with reduced binding and inhibition of AMPA-R did not exhibit CNS toxicity in high-dose studies. RG-NG-1015, RG-NG-1016, and RG-NG-1017 were tested in high-dose mouse toxicity studies. Each compound was tested with a single dose of 2000 mg / kg and with escalating doses (100, 450, and 2000 mg / kg). As shown in Table 9, escalating doses of RG-NG-1001 and RGLS4326 resulted in ataxia and coma, and in the case of RGLS4326, unconsciousness occurred at the highest dose. No CNS toxicity was observed for RG-NG-1015, RG-NG-1016, or RG-NG-1017. [Table 9]

[0285] Example 5: Maximum Allowable Dose (MTD) Test and Comparative Dose Evaluation of Different Compounds Data from the following studies further support the idea that AMPA-R antagonism is responsible for the CNS toxicity and mortality observed in previous toxicity studies of RGLS4326.

[0286] Study 1: Maximum tolerated dose (MTD) testing and comparative dose evaluation of RG-NG-1017, RGLS4326, and RG-NG-1001. The compounds (RG-NG-1017, RGLS4326, and RG-NG-1001) were evaluated in a pilot maximum tolerated dose (MTD) test (described below). RG-NG-1017, RGLS4326, and RG-NG-1001 were initially evaluated at four dose levels each. RG-NG-1017 was included for evaluation as a non-AMPA-R binding compound compared to RGLS4326 and RG-NG-1001, which bind to AMPA-R. Six-to-seven-week-old C57Bl / 6J male mice (Jackson Laboratories) were used in this study. Mice were randomly assigned to the treatment group, and the study was blinded. Animals were acclimatized for at least five days and housed in a 12-hour light / dark cycle (lights on at 7am). Each cage in a ventilated cage rack system housed no more than four mice. The diet consisted of standard rodent feed and free water.

[0287] MTD Pilot Study The following parameters were used in this study: 1. Route of administration (multiple routes possible): Intraventricular (ICV) administration of RG-NG-1017, RG-NG-1001, and RGLS4326. 2. Dosage volume (multiple options allowed): 4 μL 3. Preparations (multiple preparations are possible): Vehicle, Ca 2+ and Mg 2+ dPBS free 4. Medication frequency: Once a day 5. Exam period: 8 days 6. Number of groups: 3 7. Number of animals per group: (2-4 per group) 8. Total number of animals: 54

[0288] For ICV administration, mice were anesthetized and positioned for injection. The skin on the skull was incised, and a small hole was drilled into the skull above the target using a micro-drill. The stereotactic coordinates were anterior-posterior (AP) -0.4 mm, medial-lateral (ML) + / - 1.0-1.5 mm, and dorsal-ventral (DV) -3.0 mm, for injection into both the left and right lateral ventricles from the anterior aspect of the skull (Hironaka et al, 2015). 4 μl was unilaterally injected into the right lateral ventricle of the animal. 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 closed with sutures, wound clips, or VetBond.

[0289] After ICV treatment (day 0), animals were monitored for 7 days, with daily health checks, weight, and mortality recorded. On day 7, the brain and kidneys were collected, fixed (in 10% formalin), and retained histological types were preserved.

[0290] The results of the MTD study are shown in Table 10 and Figure 3. All animal deaths were reported to occur within the first 5-8 hours after ICV infusion. Mice injected with 2.5 μg of RG4326 were reported to show some immediate signs of respiratory distress, and a heating pad was provided. RG-NG-1017 (non-AMPA-R conjugated compound) was well-tolerated at high doses, and no established MTD was found for this compound (0 deaths at 600 μg, 100 μg, or 50 μg; 1 death at 300 μg). For RG4326 and RG-NG-1001 (e.g., 600, 300, 100 μg), 100% mortality was observed at high doses, and in addition, 100% mortality was observed at 50 μg and 25 μg for both AMPA-R conjugated compounds. The RG-NG-1001 MTD was not achieved in this study and was predicted to be less than 2.5 μg. The MTD for RG4326 was predicted by ICV to be approximately 2.5 < 5.0 μg. All animals were reported to have fully recovered by the second day of observation. [Table 10]

[0291] RGLS4326 Maximum Tolerable Dose (MTD) Study A second MTD (Method-to-Dose) trial of RGL4326 was conducted using ICV to evaluate dose selection for evaluating the compound in disease models (Table 11). Different mouse strains were evaluated in this study (Swiss: Rjorl male mice, 5 weeks old, sourced from Janvier). Mice were placed under isoflurane anesthesia (5% for induction, 2% for maintenance, <100% O2) and administered 5 mg / kg of sc-carprofen (Rimadyl®). 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 (outer diameter 0.51 mm) was stereotactically placed in the left ventricle at the following coordinates: +0.5 posterior to the parietal region, L ± 0.7 mm, V = -2.7 mm. After a 2-minute delay to allow brain tissue to slide along the cannula, 4 μL of a solution containing 0.625 mg / mL of RG4326 was slowly infused over 2 minutes. Following infusion, the cannula was left in place for an additional 5 minutes to prevent backflow of the solution along the cannula track. Mice were administered 5 mg / kg of sc-carprofen (Rimadyl®) at 24 and 48 hours post-surgery. Mice were monitored for 3–7 days post-surgery (starting 24 hours after ICV administration), and their health status was assessed by daily weight measurements. For mice monitored for 7 days, weight was measured on day 1 and day 7 post-surgery to assess their health status. [Table 11]

[0292] In Study 1, six mice were injected with 4 μL of a 0.625 mg / mL solution (2.5 μg total per ICV; Table 10). At the end of anesthesia, the mice remained lying on one side. They were still, and scratching occurred during the first few hours postoperatively. No toxic effects were observed in the six administered mice at 24, 48, or 72 hours. In Study 2, four mice were injected with four different doses of RGLS4326 (0.75, 1.0, 1.25, and 1.875 mg / mL, 4 μL volume). One mouse that received the highest dose (1.875 mg / mL, i.e., 7.5 μg / mouse) died approximately 24 hours after ICV injection. All other mice remained in good health until the end of the pilot study (7 days post-administration).

[0293] The combined results of studies 1 and 2 showed that RG4326 was generally well-tolerated in the study subjects, but only at significantly lower doses than RG-NG-1017 (see Figure 3).

[0294] Table 12 summarizes the MTD data for RGLS4326 in the mouse models from Studies 1 and 2. Based on these results from Study 2, an MTD of approximately 4 μg for RGLS4326 in the Swiss:Rjorl mouse strain was predicted. [Table 12]

[0295] In summary, compounds RG-NG-1017, RGLS4326, and RG-NG-1001 were evaluated across two MTD studies, showing significant differences in tolerability between the non-AMPA-R conjugated compound (RG-NG-1017) and the AMPA-R conjugated compounds (RGLS4326, RG-NG-1001) (see Figure 3). Despite one death at the 300 μg ICV dose, no deaths occurred at the higher test dose of 600 μg, so an MTD for RG-NG-1017 could not be established. In addition, no effect on mortality was observed for RG-NG-1017 at doses of 100 and 50 μg. In comparison, a clear effect on mortality was evident for the AMPA-R conjugated compounds RGLS4326 and RG-NG-1001, with no animals surviving across the test dose range of 25 μg to 600 μg. The trend towards improved survival rates was observed with lower doses of RGLS4326 (10 μg), with a 50% survival rate in animals treated with RGLS4326 at 5 μg and a 100% survival rate at 2.5 μg. Similarly, in the case of RG-NG-1001 (which exhibits stronger AMPA-R binding compared to RGLS4326), a 100% mortality rate was evident at the lower dose of 5 μg, with a trend toward improved survival at 2.5 μg. The results for RGLS4326 from Study 1 were further confirmed in a second MTD study (Study 2) using different mouse strains. This study found that slight differences in the tolerability of RGLS4326 between strains may exist, and survival was observed to be affected only in mice at the highest dose of 7.5 μg versus mice at 5 μg in Study 1 using C57 / Bl / 6J. However, these results still support the idea that the MTD for 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 for non-AMPA-R-bound RG-NG-1017 (at least >40 times or more) (Figure 3).

[0296] Example 6: In vitro and in vivo efficacy of anti-miR-17 compounds The in vitro potency of a specific compound was evaluated in tandem against two fully complementary miR-17 binding sites within the 3'-UTR of the luciferase gene using a miR-17 luciferase sensor assay with a luciferase reporter vector for miR-17. HeLa cells were co-transfected with a luciferase reporter vector and an exogenous miR-17 expression vector acting to suppress luciferase signaling. HeLa cells were then individually treated with anti-miR-17 oligonucleotides at concentrations of 0.045, 0.137, 0.412, 1.23, 3.70, 11.1, 33.3, 100, and 300 nM. Luciferase activity was measured at the end of the 18–24 hour transfection period. RG5124 was included as a control compound. As shown in Table 13, these compounds exhibited similar EC values ​​compared to in vitro RGLS4326. 50 The value inhibited miR-17 function and released the suppression of miR-17 luciferase reporter activity. [Table 13]

[0297] As shown in Figure 4, RG-NG-1015 exhibits similar EC compared to RGLS4326 in vitro. 50 In luciferase assays in HeLa cells, it inhibited miR-17, as well as miR-20a, miR-106a, and miR-93.

[0298] RG-NG-1015 also has similar EC values ​​compared to RGLS4326 in vitro. 50 The values ​​also released the repression of luciferase sensors containing the full-length 3' untranslated region (UTR) of the miR-17 direct target genes PKD1 and PKD2.

[0299] The activity of a specific compound was evaluated using a mouse miR-17 pharmacodynamic signature (miR-17 PD-Sig), consisting of the expression of 18 unique miR-17 target genes normalized by six reference housekeeping genes, providing an unbiased and comprehensive assessment of miR-17 activity. The mouse miR-17 PD-Sig score was the calculated mean of the individual log2-fold changes (normalized by six housekeeping genes) of the 18 genes compared to mock transfection (Lee et al., Nat. Commun., 2019, 10, 4148).

[0300] As shown in Table 13, the tested oligonucleotides had similar EC values ​​compared to RGLS4326 in vitro. 50 The values ​​indicated that the compound inhibited miR-17 function and desuppressed the expression of several direct miR-17 target genes in normal and PKD kidney cell lines (both mouse and human) (measured by miR-17 PD signature). The PD-Sig of RGLS4326 (77.2, indicated by "*") in mIMCD3 cells was not generated in this experiment. The values ​​in Table 14 are reported by Lee et al., Nat. Commun., 2019, 10, 4148. Blank cells in the table indicate that the compound was not tested in a particular cell line. [Table 14]

[0301] The in vivo efficacy was evaluated using a microRNA polysome shift assay (miPSA). This assay was used to determine the extent to which the compound directly binds to the miR-17 target 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) polysomes, while inhibited miRNAs reside in low molecular weight (LMW) polysomes. Treatment with anti-miRs results in a shift of microRNAs from HMW polysomes to LMW polysomes. Therefore, miPSA provides a direct measurement of microRNA target involvement by complementary anti-miRs (Androsavich et al., Nucleic Acids Research, 2015, 44:e13).

[0302] Wild-type mice were administered a single dose of 0.3 mg / kg, 3 mg / kg, or 30 mg / kg. Kidney tissue was collected after 7 days and subjected to miPSA. The mean displacement scores for each treatment are shown in Table 15 (PBS, n=17; RGLS4326 30 mg / kg, n=10; all other treatments, n=4-5). The tested oligonucleotides displaced miR-17 from translationally active polysomes (measured by miPSA) in the kidneys of normal mice. [Table 15]

[0303] Furthermore, as shown in Table 16 and Figures 5A-5D, RGLS4326 and RG-NG-1015 exhibit similar pharmacokinetic and target engagement (measured by miPSA) profiles after a single subcutaneous administration in C57BL6 mice. [Table 16]

[0304] Example 7: Efficacy of RG-NG-1015 in an experimental model of ADPKD. The efficacy of RG-NG-1015 was evaluated in the KspCre / Pkd1F / RC (Pkd1-F / RC) mouse model. Pkd1-F / RC is an orthologous ADPKD model that includes a germline hypomorphic Pkd1 mutation (a mouse equivalent of the human PKD1-R3277C (RC mutation) on one allele and the loxP region adjacent to Pkd1 exons 2 and 4 on the other allele). Using KspCre-mediated recombination, a phloxed Pkd1 exon was deleted, creating compound mutant mice with a renal tubule-specific somatic null mutation on one allele and a germline hypomorphic mutation on the other. This is an aggressive but long-lived model of ADPKD (Hajarnis et al., Nat.Commun., 2017, 8, 14395).

[0305] At 8, 10, 12, and 15 days of age, sex-matched Pkd1-F / RC mice were administered subcutaneously at a dose of 20 mg / kg (n=8, 4 males and 4 females per treatment group) of RGLS4326, 20 mg / kg (n=8) of RG5124, 20 mg / kg (n=8) of RG-NG-1015, or PBS (n=8). Mice were sacrificed at 18 days of age, and kidney weight, body weight, cyst index, serum creatinine levels, and blood urea nitrogen (BUN) levels were measured. BUN levels are a marker of renal function. Higher BUN levels correlate with decreased renal function; therefore, a decrease in BUN levels is an indicator of reduced kidney injury and damage, as well as improved function. Statistical significance was calculated by one-way ANOVA with Dunnett's multiple correction.

[0306] 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. In Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, the mean ratio of kidney weight to body weight (KW / BW ratio) was significantly lower than that of Pkd1-F / RC mice treated with PBS (Figure 2A). Mean BUN levels were significantly reduced in Pkd1-F / RC mice treated with RGLS4326 and RG-NG-1015, respectively, compared to mice treated with PBS (Figure 2B). Compared to mice treated with PBS, mean serum creatinine levels in Pkd1-F / RC mice were reduced in mice treated with RGLS4326 and RG-NG-1015, respectively, but the reduction was not statistically significant (Figure 2C). Treatment with the control oligonucleotide RG5124 did not reduce kidney weight-to-body weight ratio, serum creatinine, or serum BUN, indicating that the results observed with RGLS4329 and RG-NG-1015 were specific to the inhibition of miR-17. [Table 17]

[0307] The efficacy of RG-NG-1015 was also evaluated in Pcy / DBA mouse models, both with PKD alone and in combination with tolvaptan. Pcy / DBA mice exhibit slowly progressing PKD caused by missense mutations in the Nphp3 gene, which is involved in adolescent nephronophthisis in humans (Takahashi et al., J Am Soc Nephrol 1991, 1:980-989; Olbrich et al., Nat Genet 2003, 34:455-459). In Pcy mice, cysts originate from the distal duct, and all nephron segments are often sporadically occupied by cysts associated with disease progression by 30 weeks of age, often coinciding with the development of ESRD (Nagao et al., Exp Anim 2012, 61:477-488). In particular, male Pcy / DBA mice have been used to characterize the pharmacological profiles of many research products for the treatment of ADPKD, including the first-generation anti-miR-17s tolvaptan and RGLS4326 (Aihara et al., J Pharmacol Exp Ther 2014 May;349(2):258-67 and Lee et al., Nat.Commun., 2019, 10, 4148). Studies in these mice typically involve initiating treatment at around 5 weeks of age and continuing until 15–30 weeks of age.

[0308] As outlined in Figures 6A and 6B, five groups of male Pcy / DBA mice (n=13 per treatment group) were subcutaneously treated with 25, 5, 1, or 0.2 mg / kg of PBS or RG-NG-1015 once every two weeks (Q2W). Two groups of male Pcy / DBA mice (n=13 per group) were also treated with RG-NG-1015 at 50 mg / kg once every four weeks (Q4W) or at 12.5 mg / kg once weekly (QW). Four other groups of male Pcy / DBA mice (n=13 per group) were subcutaneously treated with PBS or 25, 5, or 1 mg / kg of RG-NG-1015 (Q2W), optionally combined with 0.3% (w / w feed) tolvaptan. A group of male WT-BDA / 2J mice that received subcutaneous injections of PBS Q2W were included in the study as a baseline for the normal range. Mice were randomized to the treatment group at 5 weeks of age, and treatment was administered for 17 weeks, starting at 6 weeks of age and sacrificed 7 days after the final treatment. Kidney weight, body weight, renal cyst index, and urinary Ngal to creatinine ratio (Ngal / Cr) were measured. Urinary Ngal / Cr is a marker of kidney injury.

[0309] As shown in Figures 6C–6E and Tables 18–20, RG-NG-1015 is effective in Pcy / DBA mouse models of PKD at various dosages and regimens, and provides additive or synergistic effects when used in combination with tolvaptan. In particular, RG-NG-1015 treatment significantly reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index in a dose-dependent manner in Pcy / DBA mice (Table 18 and Figures 6C–6E). In addition, RG-NG-1015 treatment with different drug regimens (including QW, Q2W, and Q4W) at similar total doses (212.5–250 mg total per mouse during the study period) reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index to similar levels in Pcy / DBA mice (Table 19; Figures 6C–6E). Treatment with tolvaptan alone reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index in Pcy / DBA mice, and the combination of RG-NG-1015 + tolvaptan further reduced mean KW / BW, urinary Ngal / Cr, and renal cyst index (Table 20; Figures 6C-6E). Bliss additiveity analysis showed that the observed effects of drug combinations on KW / BW, urinary Ngal / Cr, and renal cyst index were synergistic, mainly additive, and less additive, respectively (Table 20). [Table 18] [Table 19] [Table 20]

[0310] Example 8: Metabolites of RG-NG-1015 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 weight (MW) of RG-NG-1015 and potential metabolites was calculated from MS signals and compared to theoretical values.

[0311] The in vitro metabolic stability of RG-NG-1015 was evaluated in mouse, monkey, and human tissues (i.e., kidney and liver lysates), as well as serum. RG-NG-1015 was incubated at a concentration of 5 μM in these matrices with kidney and liver homogenates (corresponding to 307 μg / g tissue) or serum samples (corresponding to 15.3 μg / mL) at 37°C for 24 hours. RG-NG-1015 and metabolites were then extracted and analyzed by HPLC-TOF.

[0312] In vivo metabolism of RG-NG-1015 was evaluated in the liver and kidneys of CD-1 mice after a single dose, and in the plasma, tissue, and urine of monkeys after single and / or repeated administration. CD-1 mice received a single SC dose of 2000 mg / kg of RG-NG-1015, while monkeys received SC doses of 15, 75, or 150 mg / kg for up to 5 weeks. RG-NG-1015 and its metabolites were then extracted and analyzed by HPLC-TOF.

[0313] RG-NG-1015 undergoes sequential hydrolysis from both its 3' and 5' ends to produce chain-shortening metabolites (see Table 21). Nine possible metabolites were identified, as shown in Table 21 below: 5'N-1, 5'N-2, 5'N-3, 5'N-4, 3'N-1, 3'N-2, 3'N-3, 3'N-4, and 3'N-5. Unlike RG-NG-1015, all metabolites are terminated at hydroxyl groups at the 3' and 5' ends by sequential removal of terminal nucleotides. No 5'-terminus shortmers (N-5 to N-8) or 3'-terminus shortmers (N-6 to N-8) were observed. [Table 21]

[0314] Example 9: Clinical study to evaluate the safety, tolerability, pharmacodynamics, and pharmacokinetics of RG-NG-1015 in patients with autosomal dominant polycystic kidney disease. A. Overview of the research design This is a Phase Ib, double-blind, placebo-controlled, multiple escalation-dose (MAD) study in which RG-NG-1015 or placebo will be administered via subcutaneous (SC) injection to approximately 36 subjects with a diagnosis of autosomal dominant polycystic kidney disease (ADPKD) with Mayo imaging class 1C, 1D, or 1E (based on either an MRI obtained during screening or a previous MRI obtained within 5 years of screening with recorded Mayo classification). Subjects will be required to sign an informed consent form (ICF) and will be evaluated against inclusion / exclusion criteria during the screening period. Subjects meeting all inclusion / exclusion criteria will be centrally randomized in a 3:1 ratio to receive either RG-NG-1015 or placebo via subcutaneous (SC) injection in seven doses every two weeks (Q2W). Cohort 1 (12 subjects): 1 mg / kg of RG-NG-1015 or placebo Cohort 2 (12 subjects): 2 mg / kg of RG-NG-1015 or placebo Cohort 3 (12 subjects): 3 mg / kg of RG-NG-1015 or placebo.

[0315] The investigational drug will be administered by SC injection by the principal investigator or other qualified and trained site staff who have undergone at least 4 hours of safety monitoring by site staff.

[0316] Participants will take part in the study for a maximum of 141 days. The study consists of a screening period of up to 28 days (-28 to -1), followed by an 85-day treatment period (1 to 86), and then a 28-day follow-up period (92 to 113). During the treatment period, clinic visits will be made on day 1 and / or day 2 (medication 1 on day 1), day 15 (medication 2), day 29 (medication 3), day 43 (medication 4), day 57 (medication 5), day 71 (medication 6), and day 85 and / or day 86 (medication 7 on day 85). During the follow-up period, clinic visits will be made on day 92, day 99, and day 113 (end of study visits).

[0317] B. Target group This study consists of three consecutive cohorts of 12 participants each, who will be centrally randomized in a 3:1 ratio to receive either RG-NG-1015 1 mg / kg, 2 mg / kg, or 3 mg / kg via SC injection every other week (Q2W) for 7 doses, or placebo (total of 36 participants).

[0318] Inclusion Criteria To participate in this study, participants must meet all of the following inclusion criteria. 1) The person must be between 18 and 70 years old at the time of signing the informed consent form; 2) Diagnosed with ADPKD (1C, 1D, or 1E according to the Mayo imaging classification based on magnetic resonance imaging (MRI) obtained during screening, or a previous MRI obtained within 5 years of screening with a recorded Mayo classification); 3) Estimated glomerular filtration rate (eGFR) of 30-90 mL / min / 1.73M 2 Being; 3) Body Mass Index (BMI) of 18-35 kg / m² 2 Being; 4) If the subject has hypertension, the antihypertensive regimen must have been stable for at least 28 days prior to randomization, and blood pressure must be adequately controlled prior to randomization; 5) The following hematological and clinical chemistry screenings: a) Platelets are within the normal range. b) Unless the elevated bilirubin is associated with a known benign condition (e.g., Gilbert's syndrome), total bilirubin and direct bilirubin are less than 1.5 times the upper limit of normal (ULN). c) Alanine aminotransferase (ALT) is less than 1.5 times ULN. d) Aspartate aminotransferase (AST) is less than 1.5 times the ULN. e) Alkaline phosphatase (ALP) is less than 1.5 times ULN. f) Gamma-glutamyltransferase (GGT) levels are less than 1.5 times the ULN level. 6) The participant understands and agrees to the research procedures described in the Informed Consent Form (ICF), and is willing and able to follow the protocol. 7) Women of childbearing age must not be breastfeeding and must not plan to become pregnant during the study period up to 28 days after the last dose of the investigational drug. Heterosexual women of childbearing age must agree to use one of the following contraceptive methods considered highly effective (i.e., resulting in a failure rate of less than 1% when used consistently and correctly) from the screening up to 28 days after the last dose of the investigational drug: a) Having had an intrauterine device (IUD) or intrauterine system (IUS) for at least 3 months prior to randomization. b. The partner has undergone a vasectomy. A vasectomy by the partner is considered highly effective only if the partner is the sole sexual partner of a woman of childbearing age and has undergone a vasectomy at least 6 months prior to randomization. c. Stable hormonal contraception associated with ovulation inhibition (by an approved oral, transdermal, or depo regimen) for at least three months prior to randomization. 8) Women who are not capable of becoming pregnant must have undergone one of the following infertility procedures at least six months prior to the first dose of the investigational drug: a. Hysterectomy b. Bilateral oophorectomy c. Bilateral fallopian tube occlusion d. Bilateral salpingectomy Alternatively, the patient must be postmenopausal and have not had a menstrual period for at least one year prior to the first dose of the investigational drug. 9) Heterosexual men who have not undergone vasectomy must consent to the use of spermicide-treated condoms. (There are no restrictions for heterosexual men who have undergone vasectomy, provided they had the procedure at least six months prior to the start of the study. Heterosexual men who underwent vasectomy less than six months prior to the start of the study are subject to the same restrictions as heterosexual men who have not undergone vasectomy.) 10) Male and female participants agree not to provide sperm or eggs (OVAs) from day 1 until 28 days after the final dose of the investigational drug. 11) You agree not to donate blood within 28 days prior to randomization, or to provide plasma within 7 days prior to randomization, until the end of your study visit (EOS).

[0319] Exclusion criteria Subjects meeting any of the following criteria will be excluded from the study: 1) The patient has received tolvaptan for 28 days prior to randomization; 2) The subject is mentally incapacitated or has serious emotional problems; 3) In the opinion of the principal investigator, there is any medical or social condition that makes it unlikely the subject will complete the study or comply with the study procedures and requirements, or that may endanger the subject's safety; 4) A history of or current history of alcohol dependence or substance abuse within the past two years prior to screening; 5) There is an active infection of the urinary tract (e.g., kidneys, bladder, etc.); 6) Known to have hepatitis B, hepatitis C, or human immunodeficiency virus (HIV) infection; 7) Having only one kidney or having undergone a kidney transplant; 8) A history of malignant tumors other than squamous cell carcinoma or basal cell carcinoma or skin cancer that has been successfully treated; 9) The principal investigator has a history of clinically significant reactions to oligonucleotide compounds; 10) There are tattoos or scars at or near the SC injection site, or other conditions that, in the opinion of the principal investigator, could interfere with the examination of the injection site; 11) Prior to administration of the investigational drug, within 28 days of administration of the investigational drug or within 5 half-lives, whichever is longer, the participant has participated in another clinical trial and / or been exposed to any investigational drug or therapy approved for investigational use. The 28-day or 5 half-life period is calculated from the last dose of the previous study to day 1 of the current study.

[0320] C. Drug Products RG-NG-1015 is provided in a 2 mL clear glass vial containing a volume sufficient to extract 1 mL of the indicated volume of 150 mg / mL RG-NG-1015 in 0.3% physiological saline. The placebo injection is provided in a 2 mL clear glass vial containing a volume sufficient to extract 1 mL of the indicated volume of 1.5 μg / mL riboflavin in 0.9% sodium chloride. Both the RG-NG-1015 solution and the placebo solution are clear and colorless to pale yellow.

[0321] D. Administration RG-NG-1015 and placebo are administered via subcutaneous (SC) injection as anterior abdominal bolus, following the facility's standard care procedures. Each injection(s) on each medication day are rotated across different quadrants of the abdomen. The investigational drug is administered by qualified and trained facility staff. Because the amount of investigational drug varies significantly depending on the dose level, the following guidelines must be followed: The maximum amount per injection should not exceed 2 mL (for example, a 6 mL dose requires three 2 mL injections in the same quadrant of the abdomen).

[0322] E. Evaluation Items The main objectives and evaluation criteria of this study are as follows: TIFF2026513775000031.tif76170

[0323] The secondary objectives and evaluation items of this study are as follows: TIFF2026513775000032.tif180170

[0324] The exploratory objectives and evaluation criteria for this study are as follows: TIFF2026513775000033.tif113170

[0325] F. Rating Clinical and safety assessments at screening and pre-specified time points during the study include: ● Demographics, medical history, and concomitant medications, ●Measurement of height and weight, ●Vital signs (body temperature, systolic and diastolic blood pressure, heart rate, and retinal rate), ● Physical examination (complete and limited), ●SARA evaluation to detect the possibility of CNS damage caused by investigational drugs, ●Safety clinical tests (hematological complete blood count, chemical metabolism panel, urinalysis, coagulation, and lipids), ●12-lead ECG, ● ADPKD gene test, ● Plasma sample testing for C3a and Bb complement, ● Plasma sample testing of anti-drug antibodies, ● Urine biomarker tests (PC1, PC2, NGAL, KIM-1), ● Renal function tests (eGFR calculation, as well as UACR, SCr, and BUN tests), ○eGFR is calculated during screening using the 2021 CKD-EPI Creatinine-Cystatin C Age, Sex Equation (Inker, 2021). ● Pharmacokinetic studies of plasma and urine ○Using plasma and urine concentration-time data, derive the following PK parameters: C max , T max AUC 0-24 AUCinf (If computable), AUC tau , t 1 / 2 CL / F, V z (F, fe, and Ae, as well as additional PK parameters as appropriate), ○Pre-administration plasma PK samples on day 1 and day 85 should be obtained within 60 minutes prior to administration. Post-administration plasma samples on days 1 and 2, and days 85 and 86 should be obtained within the following time margins: 2, 4, 6, and 8 hours ± 15 minutes, 12 hours ± 30 minutes, and 24 hours ± 60 minutes. ○ Pre-medication PK samples are collected on days 15, 43, and 71. Pre-medication samples and samples taken 4 hours ± 15 minutes after administration are collected on days 29 and 57. PK samples are collected at EOS visits on days 99 and 113. ○24-hour urine collection for PK analysis will begin immediately after administration of the investigational drug on day 1 (0-24 hours) and day 71 (0-24 hours). Participants should urinate immediately before administration (before the start of 24-hour urine collection). The last urination for 24-hour collection will be 24 hours after administration. ●MRI to determine the change in htTKV from baseline. ● Exploratory renal biomarker testing in residual urine (MCP-1 and B2M) and residual serum (e.g., IGFALS, CT-proAVP, N-acetyl-1-methylhistidine, and others); as well as exploratory image-based biomarkers (e.g., total cyst volume, number, and / or size distribution, etc.). ● Examination of acute phase responses (Alb, fibrinogen, and highly sensitive C-reactive protein) in plasma samples.

[0326] G. Data Analysis / Statistics Summary and analysis results are generated by dose levels (pooled across all cohorts) for RG-NG-1015 versus placebo. Descriptive statistics by dose level and treatment group are compiled for each visit.

[0327] The following analysis will be conducted: Safety Analysis: Safety data (frequency of TEAEs (Triaded Adverse Events) and SAEs (Serious Adverse Events), safety laboratory tests, vital signs, ECG, and SARA test scores) are descriptively summarized by dose level and treatment group as appropriate. Analysis of safety laboratory tests, vital signs, and ECG includes time-series summary statistics, with descriptive summaries of changes from baseline. Changes from baseline in SARA test scores are descriptively summarized over time for each category and the total score. The SARA test has eight categories, with a cumulative score ranging from 0 (no ataxia) to 40 (most severe ataxia). When completing the outcome assessment, each category is evaluated and scored accordingly. The scores for the eight items are as follows: 1. Walking (0-8 points) 2. Posture (0-6 points), 3. Seating (0-4 points) 4. Speech impairment (0-6 points) 5. Finger tracking (0-4 points) 6. Nasal finger test (0-4 points) 7. Move your hands quickly and alternately (0-4 points) 8. Heel-knee slide (0-4 points). ○ After evaluating each of the eight categories, calculate the total to determine the severity of ataxia;

[0328] Pharmacodynamic (biomarker) analysis: Characterize the response of urinary biomarkers (PC1, PC2, NGAL, KIM-1). Compare the changes in urinary biomarkers over time (i.e., PC1, PC2, NGAL, KIM-1) with the baseline (samples at screening and day 1), and use analysis of covariance to adjust for the baseline biomarker values and treatment groups, and compare between each RG-NG-1015 dose level and placebo (pooled placebo across cohorts 1 - 3). The analysis is performed on data collected on days 29, 57, 85, 86, 92, 99, and 113, but the main comparisons will be made at the time points of days 86 and 113. Use plots to evaluate the relationship between the change from baseline in biomarkers (PC1, PC2, NGAL, KIM-1) and htTKV and plasma exposure (e.g., AUC tau C max C min ) and dose level.

[0329] MRI analysis: Summarize the absolute change and percent change from baseline in htTKV using descriptive statistics. Analyze the change from baseline in htTKV values using analysis of covariance, adjusting for the baseline htTKV value, baseline Mayo classification (1C, 1D, or 1E), and treatment group, and compare between treatment groups at each dose level and between all subjects receiving RG-NG-1015 and all subjects receiving placebo (pooled placebo across cohorts 1 - 3).

[0330] Kidney function analysis: Analyze and descriptively summarize the changes from baseline in eGFR (calculated using the CKD-EPI equation without race, using creatinine and cystatin-C), UACR, SCr, and BUN.

[0331] Pharmacokinetic analysis: Since the clinical laboratory analyzing the PK samples is unblinded, PK analysis is performed only on subjects receiving RG-NG-1015. Use plasma and urine concentration-time data to derive the following PK parameters: C max T max AUC0-24 、AUC inf (if calculable), AUC tau 、t 1 / 2 、CL / F, V z / F, fe, and Ae. Descriptive statistics of plasma concentrations are summarized by time point and cohort. Using the non-compartmental method, the following PK parameters are derived using plasma and urine concentration vs. time data: Cmax, Tmax, AUC0-24, AUCinf (if calculable), AUCtau, t1 / 2, CL / F, Vz / F, fe, and Ae. Dose proportionality is explored using power models of C max 、AUC 0-24 、AUC inf 、and AUC tau as data permits.

[0332] ADA analysis: Aggregate the incidence and titer of ADA in subjects who developed ADA at any time point during the study by cohort. Evaluate the impact of ADA on PK parameters by subgroup analysis (e.g., calculate PK parameters in subjects with and without ADA).

[0333] Exploratory analysis: Aggregate by visit for exploratory kidney biomarkers in urine (MCP-1 and B2M) and serum (e.g., IGFALS, CT-proAVP, N-acetyl-1-methylhistidine, and others); and for 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, deletions, or missense mutations, etc.) and biomarker responses (e.g., PC1 levels and PC2 levels).

[0334] Interim analysis: After the subjects in each cohort have completed dosing with the investigational drug and the EOS visit, the sponsor analyzes the unblinded safety, biomarker, kidney function, and PK data.

[0335] A CRO medical monitor will continuously review safety data. The sponsor and the CRO medical monitor will conduct monthly blinded safety reviews of adverse events (AEs) and safety laboratory test results to monitor safety during the study. A cohort dose escalation meeting will be held at least four weeks after the last subject of the enrolled cohort received the first dose (day 1). The sponsor will review the available safety data to make a dose escalation decision for cohort 2, and repeat this process to make a dose escalation decision for cohort 3.

[0336] H. Results The RG-NG-1015 procedure achieves one or more of the primary, secondary, and / or exploratory endpoints while maintaining acceptable safety and tolerability. [Table 22] The registered population exhibits significant disease burden through kidney size and reduced eGFR.

[0337] This study demonstrated that RG-NG-1015 (RGLS8429) administered at doses of 1 mg / kg and 2 mg / kg every two weeks for 12 weeks was well-tolerated with no significant safety findings. [Table 23]

[0338] With repeated bi-weekly dosing, no accumulation of RG-NG-1015 was observed in either plasma or urine. AUC plasma exposure in patients administered 1 mg / kg of RG-NG-1015 was nearly twice that of healthy volunteers, consistent with an approximately 35% reduction in renal excretion. AUC plasma exposure increased at 2 mg / kg compared to 1 mg / kg.

[0339] Urinary polycystin (PC) measurement: Measurement of PC1 and PC2 in urinary exosomes clearly distinguishes between healthy subjects and patients with ADPKD, and is inversely correlated with disease severity. See Figures 7A and 7B.

[0340] Multiple analytical methods were used to assess urinary PC levels: ● Absolute changes in urinary PC1 and PC2 levels from baseline, including best-fit regression models over the treatment period (see Figures 8A-8C, 9A-9C, and 10A-10B); ●Percentage change in urinary PC1 and PC2 levels from baseline (see Figures 11A and 11B); ● Mean change in urinary PC1 and PC2 levels from baseline 3 months after administration of RG-NG-1015 (see Figures 12A-B and 13A-13B).

[0341] The study demonstrated the absolute change and percentage increase in urinary polycystin (PC1 and PC2) levels following treatment with 1 mg / kg and 2 mg / kg of RG-NG-1015 (RGLS8429) from baseline. See Figures 8A-C, 9A-9C, 10A-10B, and 11A-11B. As shown in Figure 11A, for 1 mg / kg of RG-NG-1015, a statistically significant increase from baseline (mean of three separate pre-treatment samples) in urinary PC1 was observed at 12 weeks of treatment (36% and 41% at days 85 and 86, respectively). As seen in Figure 11B, for 1 mg / kg of RG-NG-1015, an increase from baseline was also observed in urinary PC2 at 12 weeks, but the change did not reach statistical significance (see Figures 10A and 10B for statistical analysis). The polycystin pattern is consistent with tissue PK profiles in preclinical studies. This study also shows that treatment with 2 mg / kg RG-NG-1015 increased the absolute change in PC1 and PC2 levels compared to 1 mg / kg RG-NG-1015. See Figures 8A-C, 9A-9C, and 10A-10B. In Figure 10A, for example, statistically significant increases from baseline in urinary PC1 were observed at 57, 86, 99, and 113 days of treatment with 2 mg / kg RG-NG-1015. In Figure 10B, statistically significant increases from baseline in urinary PC2 were observed at 57 days with 2 mg / kg RG-NG-1015.

[0342] This study also showed that mean polycystin levels increased 3 months after treatment with RG-NG-1015 (RGLS8429) at doses of 1 mg / kg and 2 mg / kg, respectively, for absolute changes in PC1 and PC2 (Figures 12A-B) and percentage changes in PC1 and PC2 (Figures 13A-B). RG-NG-1015 at 2 mg / kg showed a greater increase in mean polycystin levels compared to RG-NG-1015 at 1 mg / kg.

[0343] Urinary measurements of PC1 and PC2 showed that the biological activity of RG-NG-1015 (RGLS8429) at 2 mg / kg was superior to that of placebo, and this was most pronounced after 3 months of treatment. Based on urinary polycystin analysis, a mechanical dose-response was observed at the 2 mg / kg dose level.

[0344] This study also demonstrated that polycystin is an effective pharmacodynamic marker (i.e., for dose ranges) because urinary polycystin exhibited a suitable PK / PD correlation to function as a pharmacodynamic biomarker for ADPKD. See Figures 14A and 14B. As shown in Figures 14A-14B, novel dose responses were observed with RGLS4326 (NCT04536688; data in file) at 0.3 mg / kg and 1 mg / kg, as well as with RG-NG-1015 (RGLS8429) at 1 mg / kg. Combining the RGLS4326 and RG-NG-1015 datasets yielded PC1 and both PK parameters (C) measured. max and AUC last A positive correlation was observed between RGLS 4326 and RG-NG-1015. Furthermore, similar pharmacodynamic responses were observed between RGLS 4326 and RG-NG-1015 at a dose level of 1 mg / kg.

[0345] Renal function parameters and renal MRI measurements: Based on published longitudinal studies, patients with ADPKD experience approximately 6% kidney growth per year, so an increase of about 1-2% in kidney volume is expected over 12 weeks.

[0346] The impact of novel imaging biomarkers characterizing cystic structures was evaluated using MRI data collected at the end of the study. Exploratory results of MRI image analysis are shown in Figures 15A-C and 16A-C.

[0347] Figures 15A-15B show the changes in height-adjusted total kidney volume (htTKV) and total renal cystic volume (TKCV) in subjects receiving 1 mg / kg of RG-NG-1015, 2 mg / kg of RG-NG-1015, and placebo. Figure 15C shows the correlation between the change in TKCV and the change in htTKV. [Table 24] [Table 25]

[0348] Figures 16A and 16B show the changes in total liver volume (TLV) and total liver cyst volume (TLCV) in subjects receiving 1 mg / kg of RG-NG-1015, 2 mg / kg of RG-NG-1015, and placebo. Figure 16C shows the correlation between changes in TLCV and changes in TLV. [Table 26] [Table 27]

[0349] Figure 17A shows exploratory correlations between changes in PC1 compared to changes in HtTKV. Table 28 shows simple linear regressions of RG-NG-1015 for placebo, 1 mg / kg, and 2 mg / kg. [Table 28]

[0350] Figure 17B shows exploratory correlations between changes in PC1 compared to changes in eGFR. Table 29 shows simple linear regressions of RG-NG-1015 at placebo, 1 mg / kg, and 2 mg / kg. [Table 29]

[0351] Figure 17C shows exploratory correlations between changes in PC2 compared to changes in HtTKV. Table 30 shows simple linear regressions of RG-NG-1015 at placebo, 1 mg / kg, and 2 mg / kg. [Table 30]

[0352] Figure 17D shows exploratory correlations between changes in PC2 compared to changes in eGFR. Table 31 shows simple linear regressions of RG-NG-1015 at placebo, 1 mg / kg, and 2 mg / kg. [Table 31]

[0353] For Cohort 1, renal function parameters did not show significant changes over the short 12-week course of medication. Baseline measurements were consistent with the ADPKD Mayo classification stage. No significant changes were observed in renal function measurements over the 12 weeks (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM1).

[0354] For Cohort 2, the exploratory results of MRI image analysis are as follows: ●The mean change in htTKV was -0.84% ​​in the 2 mg / kg group, compared to 0.52% in the placebo group. ● Of the 11 subjects receiving 2 mg / kg, 4 showed a reduction of more than 2% in htTKV, a reduction in TKCV, and an increase in both urinary PC1 and PC2. ● Changes in TKCV correlated with changes in htTKV; ● Reductions in liver volume and liver cyst volume were observed in some patients treated with RG-NG-1015; ● No significant changes were observed in kidney function measurements over the 12-week period (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM1), which was expected given the short duration of treatment and the small number of subjects.

[0355] Here are some key points from different cases: ●Target 1: The highest increase between PC1 and PC2* ○A 47-year-old man diagnosed in 2006 ○Baseline eGFR was 66 mL / min, and htTKV was 941 mL / m². ○D113 MRI: htTKV decreased by 4.96%, TKCV decreased by 4.34%; ●Target 2: The second highest increase in PC1* ○A 44-year-old woman diagnosed in 2019 ○Baseline eGFR was 65 mL / min, and htTKV was 1253 mL / m². ○D113 MRI: htTKV decreased by 6.28%, TKCV decreased by 6.93%; ●Target 3: The second highest increase in PC2* ○ A 29-year-old male diagnosed in 2020 ○Baseline eGFR was 88 mL / min, and htTKV was 1162 mL / m². ○D113 MRI: htTKV decreased by 4.22%, TKCV decreased by 2.73%; ●Of the four active subjects in Cohort 2 whose htTKV decreased by more than 2%, all four had an increase in both PC1 and PC2. *Average percentage change between day 85 and day 113 of polycystin (PC) intake.

[0356] Results from Cohort 2 suggest numerical improvements in total renal and hepatic volume and cystic volume in patients with ADPKD compared to placebo.

[0357] Modeling of target engagement research data for RG-NG-1015 The efficacy of RG-NG-1015 (RGLS8429) was evaluated in the KspCre;Pkd1F / RC (Pkd1-F / RC) mouse model. Pkd1-F / RC mice were administered subcutaneously at various doses of RG-NG-1015, 20 mg / kg of a control oligonucleotide, or PBS (N=8-13 per group) on postnatal days 8, 10, 12, and 15. Separate groups of Pkd1-F / RC mice were administered subcutaneously at 20 mg / kg of RG-NG-1015 on postnatal days 8 and 12. Mice were sacrificed at 18 days of age. The left kidney was perfused with cold PBS and 4% PFA before collection. All other mouse kidneys were collected using a standard protocol, fixed in 10% formalin, dehydrated, and embedded in paraffin. The samples were sectioned to a thickness of 5 μm and subjected to hematoxylin and eosin staining.

[0358] As shown in Figures 18A-18B, RGLS8429 demonstrated efficacy in reducing kidney weight-to-body weight ratio (KW / BW) in a dose-response manner (Figure 18B left, individual KW / BW grouped by dose level and regime; right, individual calculated inhibition percentage (CPI) of KW / BW plotted against individual kidney concentrations of RG-NG-1015. Estimated kidney concentration corresponding to 50% inhibition of KW / BW, i.e., IC50 value). After administration of RGLS8429, both kidney size and cyst number were reduced in a dose-response manner. See Figure 18A.

[0359] Wild-type C57BL6 mice received single SC doses of RGLS8429 or RGLS4326 at 0.003, 0.03, 0.1, 0.3, 1, 3, 10, 30, or 300 mg / kg. Mice were sacrificed 7 days after administration. Kidney samples were collected, and target engagement (displacement of miR-17 from high molecular weight polysomes) was measured by miPSA assay (Androsavich, Nucleic Acids Res. 44, e13 (2016)). Individual calculated inhibition percentages (CPI) of target engagement in mouse kidneys were plotted against individual kidney concentrations of RGLS8429. Estimated kidney concentrations corresponding to 80% inhibition of miR-17, i.e., IC80 values, are shown. See Figure 18C. We predicted an AUC of approximately 12,494 h*ug / g and used it as a benchmark for renal exposure that drives maximum target engagement (approximately 80% inhibition of miR-17).

[0360] Based on recent results from cohorts 1 and 2 (RGLS8429-02), the 1 mg / kg dose (cohort 1) and 2 mg / kg dose (cohort 2) of RG-NG-1015 in ADPKD patients are predicted to result in approximately 6,052 h*ug / g and 8,900 h*ug / g of renal exposure, respectively (less than the predicted benchmark renal exposure of approximately 12,494 h*ug / g). Based on these results, the 3 mg / kg dose (cohort 3) is expected to result in a predicted human renal exposure close to or slightly above approximately 12,494 h*ug / g.

[0361] Plasma-to-tissue modeling suggests that 1 mg / kg represents less than half of the dose-response curve. The data suggest that higher doses may indicate a greater urinary polycystin response.

[0362] Based on extensive nonclinical analyses and PK / PD modeling, renal exposure associated with peak miR-17 target engagement is expected to be achieved in humans at doses exceeding 2.4 mg / kg.

Claims

1. A method for treating polycystic kidney disease, comprising administering to a subject in need of such treatment a modified oligonucleotide or a pharmaceutically acceptable salt thereof in 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. where the 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', and The method wherein the nucleoside following the subscript "M" is a 2'-O-methyl nucleoside, the nucleoside following the subscript "F" is a 2'-fluoro nucleoside, the nucleoside following the subscript "S" is an S-cEt nucleoside, and each cytosine is an unmethylated cytosine.

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

3. The method according to 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 of such treatment 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, wherein the modified oligonucleotide has the following structure: 【Chemistry 1】 The method comprising a pharmaceutically acceptable salt thereof.

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

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

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

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

9. The method according to claim 8, wherein the sterile aqueous solution is physiological saline.

10. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to a subject in need of such treatment 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, wherein the modified oligonucleotide has the following structure: 【Chemistry 2】 The method comprising the above.

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

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

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

14. The method according to claim 13, wherein the sterile aqueous solution is physiological saline.

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

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

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

18. The method according to claim 17, wherein the subject has ADPKD Mayo image classification 1C, 1D, or 1E.

19. The subjects described above had an estimated glomerular filtration rate (eGFR) of 30–90 mL / min / 1.73 m² prior to the administration of the modified oligonucleotide. ２ The method according to any one of claims 1 to 18.

20. The method according to any one of claims 1 to 19, wherein it is determined that the subject has decreased levels of polycystin-1 (PC1) and / or polycystin-2 (PC2) in the subject's kidney, urine, or blood prior to administration of the modified oligonucleotide.

21. The method according to 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 according to any one of claims 1 to 21, wherein the subject is an increase in total kidney volume.

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

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

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

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

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

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

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

33. The method according to claim 31 or 32, wherein the total renal volume is height-adjusted total renal volume (htTKV).

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

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

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

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

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

39. With the aforementioned treatment, a) The rate of decrease in creatinine clearance in the subject is improved or slowed down. b) The rate of increase in the albumin:creatinine ratio in the subject is reduced or slowed down. c) The rate of increase in blood urea nitrogen (BUN) levels in the subject is reduced or slowed down. d) The rate of increase in serum creatinine (SCr) levels in the subject is reduced or slowed down. e) The amount of polycystin-1 (PC1) in the urine of the subject increases. f) The amount of polycystin-2 (PC2) in the urine of the subject increases. g) The rate of increase of neutrophil gelatinase-binding lipocalin (NGAL) protein in the urine of the subject is reduced or slowed, and / or h) The method according to any one of claims 1 to 38, wherein the rate of increase of kidney injury molecule 1 (KIM-1) protein in the urine of the subject is reduced or slowed down.

40. By administering the above-mentioned dose, a) The rate of increase of monocyte chemotactic protein 1 (MCP-1) in the urine of the subject is reduced or slowed down. b) The rate of increase of beta-2 microglobulin (B2M) in the urine of the subject is reduced or slowed down. c) The rate of increase of complement degradation products C3a and / or Bb in the plasma of the subject is reduced or slowed down. d) The rate of increase of serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the subject is reduced or slowed down. e) The rate of increase of serum copeptin (CT-proAVP) in the subject is reduced or slowed down. f) The rate of increase of serum N-acetyl-1-methylhistidine in the subject is reduced or slowed, and / or g) The method according to any one of claims 1 to 39, wherein the rate of increase of acute-phase protein in the subject is reduced or slowed down.

41. The method according to any one of claims 1 to 40, wherein CNS deficiency in the subject is almost or completely eliminated.

42. The method according to claim 41, wherein the treatment results in little to no change in the Scale for the Assessment and Rating of Ataxia (SARA) test score for the subject.

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

44. The method according to 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 an acceptable safety and tolerability profile.

46. A modified oligonucleotide or a pharmaceutically acceptable salt thereof for use in the treatment of polycystic kidney disease, wherein the modified oligonucleotide has structure 5'-A Ｓ G Ｓ C Ｍ A Ｆ C Ｆ U Ｆ U Ｍ U Ｓ A Ｓ It has -3', Nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, nucleosides followed by the subscript "S" are S-cEt nucleosides, and each cytosine is an unmethylated cytosine. The modified oligonucleotide for use, administered in 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, or a pharmaceutically acceptable salt thereof.

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

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

49. The modified oligonucleotide for use according to any one of claims 46 to 48, wherein the modified oligonucleotide is present in a pharmaceutical composition containing sterile physiological saline.

50. The modified oligonucleotide for use according to 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 according to any one of claims 46 to 50, wherein the modified oligonucleotide is administered once every two weeks.

52. The modified oligonucleotide for use according to any one of claims 46 to 51, wherein the modified oligonucleotide is administered at least seven times.

53. Use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof for the preparation of a pharmaceutical for the treatment of polycystic kidney disease, wherein the modified oligonucleotide has structure 5'-A Ｓ G Ｓ C Ｍ A Ｆ C Ｆ U Ｆ U Ｍ U Ｓ A Ｓ It has -3', Nucleosides followed by the subscript "M" are 2'-O-methyl nucleosides, nucleosides followed by the subscript "F" are 2'-fluoro nucleosides, nucleosides followed by the subscript "S" are S-cEt nucleosides, and each cytosine is an unmethylated cytosine. The use wherein the modified oligonucleotide is 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 according to claim 53, wherein the modified oligonucleotide is administered in a dose of 1 mg / kg, 2 mg / kg, or 3 mg / kg.

55. The use according to 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 containing sterile physiological saline.

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

58. The use according to any one of claims 53 to 57, wherein the modified oligonucleotide is administered at least once every two weeks.

59. The use according to any one of claims 53 to 58, wherein the modified oligonucleotide is administered at least seven times.

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