TW202539703A - Methods for treating polycystic kidney disease - Google Patents

Abstract

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

Description

Treatment methods for polycystic kidney disease

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

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

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

This disclosure relates to methods for treating polycystic kidney disease (PKD) and, as appropriate, somatic dominant polycystic kidney disease (ADPKD), including administering to individuals in need a therapeutically effective amount of a modified oligonucleotide or a pharmaceutically acceptable salt thereof.

Example 1. A method for treating polycystic kidney disease, comprising administering to an individual in need a modified oligonucleotide or a pharmaceutically acceptable salt _thereof at _a fixed dose between about 150 mg and about _{350 mg, wherein the modified oligonucleotide has the structure 5'-ASGSCMAFFCFUFUMUUSAS - 3 ' ,} wherein the nucleoside followed by the subscript "M" is a 2'-O _{- methyl} nucleoside; the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and wherein each cytosine is an unmethylated cytosine.

Example 2. The method of Example 1, wherein the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg.

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

Example 4. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to an individual in need at a fixed dose between about 150 mg and about 350 mg, wherein the modified oligonucleotide has the structure: a) b) or a medically acceptable salt thereof.

Example 5. The method of Example 4, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg.

Example 6. As in Example 4 or 5, wherein the pharmaceutically acceptable salt is a sodium salt.

Example 7. The method of any of Examples 1-6, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent.

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

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

Example 10. A method for treating polycystic kidney disease, comprising administering a modified oligonucleotide to an individual in need at a fixed dose between about 150 mg and about 350 mg, wherein the modified oligonucleotide has the following structure: .

Example 11. The method of Example 10, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg.

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

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

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

Example 15. The method of any of Examples 1-14, wherein the individual suffers from polycystic kidney disease.

Example 16. The method of any of Examples 1-15, wherein the individual has been diagnosed with polycystic kidney disease using clinical, histopathological and/or genetic criteria.

Example 17. The method of any of Examples 1-16, wherein the polycystic kidney disease is somatic dominant polycystic kidney disease (ADPKD).

Example 18. The method of Example 17, wherein the individual has ADPKD of Mayo Imaging Classification 1C, 1D or 1E.

Example 19. The method of any of Examples 1-18, wherein the individual has an estimated glomerular filtration rate (eGFR) between 30 and 90 mL/min/1.73 ^m² before administration of the modified oligonucleotide.

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

Example 21. The method of any of Examples 1-20, wherein the individual has a mutation selected from either the PKD1 gene or the PKD2 gene.

Example 22. The method of any of Examples 1-21, wherein the individual has an increased total renal volume.

Example 23. The method of any of Examples 1-22, wherein the individual suffers from hypertension.

Example 24. The method of any of Examples 1-23, wherein the individual has impaired renal function.

Example 25. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 150 mg.

Example 26. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 160 mg.

Example 27. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 170 mg.

Example 28. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 180 mg.

Example 29. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 190 mg.

Example 30. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 200 mg.

Example 31. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 210 mg.

Example 32. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 220 mg.

Example 33. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 230 mg.

Example 34. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 240 mg.

Example 35. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 250 mg.

Example 36. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 260 mg.

Example 37. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 270 mg.

Example 38. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 280 mg.

Example 39. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 290 mg.

Example 40. The method of any of Examples 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 300 mg.

Example 41. The method of any of claims 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 310 mg.

Example 42. The method of any of claims 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 320 mg.

Example 43. The method of any of claims 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 330 mg.

Example 44. The method of any of claims 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 340 mg.

Example 45. The method of any of claims 1-24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 350 mg.

Example 46. The method of any of Examples 1-45, wherein the method comprises administering the modified oligonucleotide once every two weeks.

Example 47. The method of any of Examples 1-46, wherein the method comprises administering the modified oligonucleotide at least 7 times.

Example 48. The method of any of Examples 1-47, wherein the modified oligonucleotide is administered subcutaneously.

Example 49. The method of any of Examples 1-48, wherein the treatment reduces the total renal volume in the individual.

Example 50. The method of any of Examples 1-49, wherein the treatment slows the rate of increase of total renal volume in the individual.

Example 51. The method of Example 49 or 50, wherein the total renal volume is the height-adjusted total renal volume (htTKV).

Example 52. The method of any of Examples 1-51, wherein the treatment slows the rate of decline in the glomerular filtration rate in the individual.

Example 53. The method of any of Examples 1-52, wherein the treatment increases the glomerular filtration rate in the individual.

Example 54. The method of Example 52 or 53, wherein the glomerular filtration rate is an estimated glomerular filtration rate.

Example 55. The method of any of Examples 1-54, wherein the treatment inhibits or slows the increase in cyst growth in the kidneys and/or liver of the individual.

Example 56. The method of Example 55, wherein the treatment inhibits or slows the increase in total cyst volume, number and/or size distribution.

Example 57. The method of any of Examples 1-56, wherein the treatment: a) improves or slows the rate of decrease in creatinine clearance in the individual; b) reduces or slows the rate of increase in the albumin:creatinine ratio in the individual; c) reduces or slows the rate of increase in blood urea nitrogen (BUN) levels in the individual; d) reduces or slows the rate of increase in serum creatinine (SCr) levels in the individual; e) increases polycystic protein-1 (PC1) in the individual's urine; f) increases polycystic protein-2 (PC2) in the individual's urine; g) reduces or slows the rate of increase in neutrophil gelatinase-associated lipocalin (NGAL) protein in the individual's urine; and/or h) Reduce or slow the rate of increase of kidney damage molecule-1 (KIM-1) protein in the urine of the individual.

Example 58. The method of any of Examples 1-57, wherein the administration: a) reduces or slows the rate of increase of mononuclear globulin-1 (MCP-1) in the urine of the individual; b) reduces or slows the rate of increase of β-2 microglobulin (B2M) in the urine of the individual; c) reduces or slows the rate of increase of complement cleavage products C3a and/or Bb in the plasma of the individual; d) reduces or slows the rate of increase of serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the individual; e) reduces or slows the rate of increase of serum CT-proAVP in the individual; f) To reduce or slow the rate of increase of serum N-acetylglucosamine in the individual; and/or g) to reduce or slow the rate of increase of acute-phase proteins in the individual.

Example 59. The method of any of Examples 1-58, wherein the treatment causes virtually no CNS damage in the individual.

Example 60. The method of Example 59, wherein the treatment causes almost no change in the individual’s dysregulation assessment and rating scale (SARA) test score.

Example 61. A method as described in any one of Examples 1-60, comprising: a) measuring the height-adjusted total renal volume (HtTKV) in the individual; b) measuring polycystic protein-1 (PC1) in the individual's urine; c) measuring polycystic protein-2 (PC2) in the individual's urine; d) measuring the blood urea nitrogen (BUN) level in the individual; e) measuring the serum creatinine (SCr) level in the individual; f) measuring the creatinine clearance rate in the individual; g) measuring the urinary albumin:creatinine ratio (UACR) in the individual; h) measuring the estimated glomerular filtration rate (eGFR) in the individual; i) j) Measure neutrophil gelatinase-associated lipotransferase (NGAL) protein in the individual's urine; k) Measure nephrotic molecule-1 (KIM-1) protein in the individual's urine; l) Measure β-2 microglobulin (B2M) in the individual's urine; m) Measure serum insulin-like growth factor-binding protein acid-instable subunit (IGFALS) in the individual; n) Measure serum CT-proAVP in the individual; o) Measure serum N-acetylglucosamine in the individual; p) Measure complement cleavage products C3a and/or Bb in the individual's plasma; and/or q) Measure the total cyst volume, number and/or size distribution in the individual; and/or r) measure the individual's SARA test score.

Example 62. The method of any of Examples 1-61, wherein the individual is a human individual.

Example 63. The method of any of Examples 1-62 has an acceptable safety and tolerability profile.

Example 64. A modified oligonucleotide or _a pharmaceutically acceptable salt thereof for the treatment of polycystic kidney disease, wherein the _{modified oligonucleotide has the structure 5'-ASGSCMAFFCFUFUMUUSAS - 3 '} , wherein the nucleoside followed by the _subscript "M _" is a 2'- _O - _methyl nucleoside; the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, wherein each cytosine is an unmethylated cytosine; and wherein the modified oligonucleotide is administered at a fixed dose between about 150 mg and about 350 mg.

Example 65. The modified oligonucleotide used in Example 64, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg.

Example 66. The modified oligonucleotide used in Examples 64 or 65, wherein the pharmaceutically acceptable salt is a sodium salt.

Example 67. A modified oligonucleotide used in any of Examples 64-66, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

Example 68. The modified oligonucleotide used in any of Examples 64-67, wherein the polycystic kidney disease is somatic dominant polycystic kidney disease (ADPKD).

Example 69. The modified oligonucleotide used in any of Examples 64-68, wherein the modified oligonucleotide is administered once every two weeks.

Example 70. The modified oligonucleotide used in any of Examples 64-69, wherein the modified oligonucleotide is administered at least seven times.

Example 71. Use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment of polycystic kidney disease, wherein the modified _{oligonucleotide} has the structure 5'- _{ASGSCMAFFCFUFUMUUSAS - 3} ', wherein the _nucleoside followed by _{the subscript} " _M " is a 2'-O- _methyl nucleoside; the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and wherein each cytosine is an unmethylated cytosine; wherein the modified oligonucleotide is formulated for administration at a fixed dose between about 150 mg and about 350 mg.

Example 72. As used in Example 71, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg.

Example 73. As in Examples 71 or 72, wherein the pharmaceutically acceptable salt is a sodium salt.

Example 74. As used in any of Examples 71-73, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution.

Example 75. As used in any of Examples 71-74, wherein the polycystic kidney disease is autosomal dominant polycystic kidney disease (ADPKD).

Example 76. As in any of Examples 71-75, wherein the modified oligonucleotide is administered at least once every two weeks.

Example 77. As used in any of Examples 71-76, wherein the modified oligonucleotide is administered at least seven times.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Unless specifically defined, the nomenclature, procedures, and techniques described herein in conjunction with analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry are those nomenclature, procedures, and techniques well-known and commonly used in the art. Where a term in this document has multiple definitions, the definition in this section shall prevail. Standard techniques are applicable to chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and individual treatment. Some of these techniques and procedures can be found in, for example, "Carbohydrate Modifications in Antisense Research," eds. Sanghvi and Cook, American Chemical Society, Washington D.C., 1994; and "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., 18th edition, 1990; and are incorporated herein by reference for any purpose. Where permitted, unless otherwise stated, all patents, patent applications, publications and disclosures, GENBANK sequences, websites and other public materials mentioned in this disclosure are incorporated herein by reference in their entirety. In the case of reference to URLs or other such identifiers or addresses, it should be understood that such identifiers are changeable, and specific information on the Internet is changeable, but equivalent information can be found by searching the Internet. It mentions that it proves the availability and public dissemination of such information.

Before disclosing and describing the compositions and methods of the present invention, it should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive. It must be noted that, unless the context expressly requires otherwise, the singular forms "a/an" and "the" as used in the specification and the appended claims include the plural indicators.

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

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

"Renal function markers" refer to medical parameters used to assess an individual's renal function. Non-limiting examples of renal function markers include glomerular filtration rate, blood urea nitrogen level, and serum creatinine level.

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

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

"Nephronophthisis" or "NPHP" refers to a type of somatic recessive cystic kidney disease characterized by cortico-medullary cysts, rupture of the renal tubular basement membrane, and tubulointerstitial kidney disease.

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

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

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

"Deterioration of hypertension" refers to changes in blood pressure that require the initiation or intensification of hypertension treatment.

"Fibrinization" refers to the formation or development of an excess of fibrous connective tissue in an organ or tissue. In some embodiments, fibrosis occurs as a repair or reaction process. In some embodiments, fibrosis occurs as a response to injury or damage. The term "fibrosis" should be understood as the formation or development of an excess of fibrous connective tissue in an organ or tissue as a repair or reaction process, which is the opposite of the formation of fibrous tissue as a normal component of an organ or tissue.

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

"Albuminuria" refers to the presence of excessive albumin in the urine, including but not limited to normoalbuminuria, hypernoralbuminuria, microalbuminuria, and macroalbuminuria. Normally, the glomerulus, composed of podocytes, the glomerular basement membrane, and endothelial cells, filters through the permeability barrier to prevent serum proteins from leaking into the urine. Albuminuria reflects damage to the glomerular filtration permeability barrier. Albuminuria can be calculated from 24-hour urine samples, overnight urine samples, or fresh urine samples.

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

"Microalbuminuria" means elevated albuminuria, characterized by: (i) 30 to 300 mg of albumin excreted in the urine every 24 hours and/or (ii) an albumin/creatinine ratio of 2.5 to <25 mg/mmol (or 20 to <200 mg/g) in men or 3.5 to <35 mg/mmol (or 30 to <300 mg/g) in women.

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

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

"Glomerular filtration rate" or "GFR" refers to the flow rate of filtered fluid through the kidney and is used as an indicator of an individual's renal function. In some embodiments, an individual's GFR is determined by calculating and estimating the glomerular filtration rate. In some embodiments, the inulin method is used to directly measure an individual's GFR.

"Estimated glomerular filtration rate" or "eGFR" refers to the degree to which the kidneys filter creatinine and is used to simulate glomerular filtration rate. Because direct measurement of GFR is complex, eGFR is frequently used in clinical practice. Normal results are in the ^range of 90-120 mL/min/1.73 m². A level below 60 mL/min/1.73 ^m² for three months or longer can be an indicator of chronic kidney disease. A level below 15 mL/min/1.73 ^m² can be an indicator of kidney failure.

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

"Blood urea nitrogen level" or "BUN level" refers to a measure of the amount of nitrogen in the blood in the form of urea. The liver produces urea in the urea cycle as a waste product of protein digestion, and urea is removed from the blood by the kidneys. Normal adult blood contains between 7 and 21 mg of urea nitrogen per 100 ml of blood (7-21 mg/dL). Blood urea nitrogen levels are used as an indicator of kidney health. If the kidneys cannot properly remove urea from the blood, an individual's BUN level will be elevated.

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

"End-stage renal disease (ESRD)" means that kidney function has completely or almost completely failed.

"Quality of life" refers to the degree to which an individual's physical, mental, and social functions are impaired due to illness and/or treatment. Individuals with polycystic kidney disease may experience a reduced quality of life.

"Impaired kidney function" means reduced kidney function compared to normal kidney function.

"Slow the worsening of" and "slow worsening" mean reducing the rate at which a medical condition progresses to an advanced stage.

"Delaying dialysis time" means maintaining sufficient kidney function to delay the need for dialysis treatment.

"Delaying kidney transplantation" means maintaining sufficient kidney function to postpone the need for a kidney transplant.

"Increasing life expectancy" means extending an individual's life by treating one or more symptoms of an illness.

"Individual" refers to a human or non-human animal selected for treatment or therapy.

"Individuals in need" refers to individuals who have been identified as needing therapy or treatment.

An individual "suspected of having" is an individual who exhibits one or more clinical indicators of the disease.

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

"Giving" means providing an individual with a medicine or combination thereof, including but not limited to giving by a medical professional and giving by oneself.

"Non-intestinal administration" means administration via injection or infusion.

Non-intestinal administration includes, but is not limited to, subcutaneous administration, intravenous administration, and intramuscular administration.

"Subcutaneous throwing" means throwing directly under the skin.

"To be given within the vein" means to give it to the vein.

"Concomitant administration" refers to the co-administration of two or more drugs in any manner in which the pharmacological effects of two of them are simultaneously observed in the patient. Concomitant administration does not require the two drugs to be administered as a single drug combination, in the same dosage form, or via the same route of administration. The effects of the two drugs do not need to be simultaneous. These effects only need to overlap for a period of time and do not need to be of the same extent.

"Duration" refers to the period during which an activity or event continues. In some implementations, the duration of treatment is the period during which a certain dose of a drug or pharmaceutical combination is administered.

"Therapy" means a method of treating a disease. In some practices, therapy includes, but is not limited to, administering one or more medications to an individual suffering from a disease.

"Treatment" means the application of one or more specific procedures to improve at least one indicator of disease. In some embodiments, the specific procedure is the administration of one or more medications. In some embodiments, treatment of PKD includes, but is not limited to, reducing total renal volume, improving renal function, lowering blood pressure, and/or reducing renal pain.

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

"At risk of developing..." means that an individual is predisposed to developing an illness or disease. In some embodiments, an individual at risk of developing an illness or disease exhibits one or more symptoms of that illness or disease, but not in a number sufficient to diagnose the illness or disease. In some embodiments, an individual at risk of developing an illness or disease exhibits one or more symptoms of that illness or disease, but to a degree less than necessary to diagnose the illness or disease.

"Prevention of the onset of..." means preventing the onset of the disease or ailment in an individual who is at risk of developing it. In some embodiments, an individual at risk of developing the disease or ailment receives treatment similar to that received by an individual who already has the disease or ailment.

"Delaying the onset of..." means preventing the onset of the disease or ailment in an individual who is at risk of developing it. In some embodiments, an individual at risk of developing the disease or ailment receives treatment similar to that received by an individual who already has the disease or ailment.

"Dosage" refers to the specified amount of medication delivered in a single administration. In some embodiments, the dosage may be administered in two or more bolus, tablet, or injection doses. For example, in some embodiments where subcutaneous administration is required, the volume needed is not easily provided by a single injection. In such embodiments, two or more injections may be used to achieve the desired dosage. In some embodiments, the dosage may be administered in two or more injections to minimize individual injection site reactions. In some embodiments, the dosage is administered via slow infusion.

"Dosage unit" refers to the form in which the drug is provided. In some embodiments, the dosage unit is a vial containing freeze-dried oligonucleotides. In some embodiments, the dosage unit is a vial containing reconstituted oligonucleotides.

"Therapeutic effective dose" refers to the amount of medication that provides therapeutic benefits to an animal.

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

"Medicine" means a substance that provides a therapeutic effect when administered to an individual.

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

"Pharmaceutically acceptable salt" means a physiologically and pharmaceutically acceptable salt of the compounds provided herein, that is, a salt that retains the desired biological activity of the compound when administered to an individual and does not have undesirable toxicological effects. Non-limiting exemplary pharmaceutically acceptable salts of the compounds provided herein include sodium and potassium salts. Unless otherwise expressly indicated, the terms "compound," "oligonucleotide," and "modified oligonucleotide" as used herein include their pharmaceutically acceptable salts.

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

"Improved organ function" means that organ function is moving towards the normal range. In some implementations, organ function is assessed by measuring molecules found in an individual's blood or urine. For example, in some implementations, improvements in renal function are measured by a decrease in blood urea nitrogen levels, a reduction in proteinuria, or a decrease in albuminuria.

"Acceptable safety profile" refers to the pattern of side effects that are acceptable within clinical limits.

"Side effects" refer to physiological responses attributable to the treatment, rather than the intended effect. In some practices, side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. These side effects can be detected directly or indirectly. For example, elevated serum aminotransferase levels can indicate hepatotoxicity or abnormal liver function. For example, elevated bilirubin can indicate hepatotoxicity or abnormal liver function.

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

"Anti-miR" refers to an oligonucleotide with a nucleobase sequence that complements microRNA. In some embodiments, anti-miR is a modified oligonucleotide.

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

"miR-17" means a mature miRNA with the nucleobase sequence 5'-CAAAGUGCUUACAGUGCAGGUAG-3' (SEQ ID NO: 1).

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

"miR-20b" means a mature miRNA with the nucleobase sequence 5'-CAAAAGUGCUCAUAGUGCAGGUAG-3' (SEQ ID NO: 3).

"miR-93" means a mature miRNA with the nucleobase sequence 5'- CAAAGUGCUGUUCGUGCAGGUAG-3' (SEQ ID NO: 4).

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

"miR-106b" means a mature miRNA with the nucleobase sequence 5'-UAAAAGUGCUGACAGUGCAGAU-3' (SEQ ID NO: 6).

"miR-17 seed sequence" refers to the nucleobase sequence 5'-AAAGUG-3' present in each member of the miR-17 family.

"MiR-17 family member" means a mature miRNA that contains a seed sequence of miR-17 and is selected from miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b.

The "miR-17 family" refers to the following group of miRNAs: miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b, each of which has a nucleotide sequence containing the miR-17 seed sequence.

"Targeted nucleic acid" refers to the design of nucleic acids that are hybridized with oligomeric compounds.

"Targeting" refers to the process of designing and selecting nucleobase sequences that are hybridized with target nucleic acids.

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

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

"Expression" refers to any function and process that translates the coding information of genes into structures that exist in and function within cells.

"Nucleobase sequence" refers to the order of linked nucleosides in oligomers or nucleic acids, usually listed in a 5' to 3' direction, and is unrelated to any sugars, bonds, and/or nucleobase modifications.

"Linked nucleobases" refers to the adjacent nucleobases in nucleic acids.

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

"Complementary" means that one nucleic acid can hybridize with another nucleic acid or oligonucleotide. In some embodiments, complementarity refers to an oligonucleotide that can hybridize with a target nucleic acid.

"Complete complementarity" means that each nucleobase of the oligonucleotide can pair with the corresponding nucleobases in the target nucleic acid. In some embodiments, the oligonucleotide is completely complementary to microRNA (also known as 100% complementarity), meaning that each nucleobase of the oligonucleotide is complementary to the corresponding nucleobases in the microRNA. Modified oligonucleotides can be completely complementary to microRNA and have a number of linking nucleotides smaller than the length of the microRNA. For example, an oligonucleotide with 16 linking nucleotides is completely complementary to microRNA, where each nucleobase of the oligonucleotide is complementary to the corresponding nucleobases in the microRNA. In some embodiments, oligonucleotides in which each nucleobase is complementary to the nucleobase in the region of the microRNA stem-loop sequence are completely complementary to the microRNA stem-loop sequence.

"Complementarity percentage" refers to the percentage of nucleosides in an oligonucleotide that complement the corresponding nucleosides in the target nucleic acid. The complementarity percentage is calculated by dividing the number of nucleosides in the oligonucleotide that complement the corresponding nucleosides in the target nucleic acid by the total number of nucleosides in the oligonucleotide.

"Percentage of identity" means the number of nucleotides in the first nucleic acid that are identical to those in the second nucleic acid at corresponding positions, divided by the total number of nucleotides in the first nucleic acid. In some embodiments, both the first and second nucleic acids are microRNAs. In some embodiments, both the first and second nucleic acids are oligonucleotides.

"Hybridization" refers to the annealing of complementary nucleic acids resulting from nucleobase complementarity.

"Mismatch" means that the nucleobase of the first nucleic acid cannot be paired with the corresponding nucleobase of the second nucleic acid using Watson-Crick pairing.

In the context of nucleobase sequences, "consistent" means having the same nucleobase sequence, regardless of sugars, bonds, and/or nucleobase modifications, and regardless of the methylation state of any pyrimidines present.

"MicroRNA" refers to endogenous non-coding RNA with a length between 18 and 25 nucleotides, which is a product of microRNA before it is cleaved by the Dicer enzyme. Examples of mature microRNAs have been found in a microRNA database called miRBase (microrna.sanger.ac.uk/). In some implementations, microRNA is abbreviated as "miR".

"MicroRNA-regulated transcripts" means transcripts regulated by microRNAs.

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

"Oligomers" are compounds that contain a plurality of linked monomer subunits. Oligomeric compounds include oligonucleotides.

"Oligonucleotide" means a compound containing a plurality of linked nucleosides, each of which may be modified or unmodified independently.

"Naturally occurring nucleoside bonds" refers to 3' to 5' phosphodiester bonds between nucleosides.

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

"Nucleoside linkage" refers to the covalent link between adjacent nucleosides.

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

"Nucleobase" refers to a heterocyclic part that can non-covalently pair with another nucleobase.

"Nucleoside" refers to a nucleobase that is attached to the sugar portion.

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

"A compound comprising a modified oligonucleotide consisting of a specified number of linked nucleotides" means a compound comprising a modified oligonucleotide having a specified number of linked nucleotides. Therefore, the compound may include additional substituents or complexes. Unless otherwise indicated, the modified oligonucleotide is not hybridized with complementary strands, and the compound does not contain any additional nucleotides other than those of the modified oligonucleotide.

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

"Modified nucleosides" means nucleosides that have undergone any changes relative to naturally occurring nucleosides. Modified nucleosides may have modified sugars and unmodified nucleobases. Modified nucleosides may have modified sugars and modified nucleobases. Modified nucleosides may have natural sugars and modified nucleobases. In some embodiments, modified nucleosides are bicyclic nucleosides. In some embodiments, modified nucleosides are non-bicyclic nucleosides.

"Modified nucleoside linkage" means any variation of the naturally occurring nucleoside linkage.

"Thiophosphate nucleoside linkage" refers to the linkage between nucleosides in which one of the atoms is a sulfur atom.

"Modified sugar portion" means any alteration that replaces and/or is relative to natural sugar.

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

"5-Methylcytosine" means cytosine containing a methyl group connected to the 5th position.

"Unmethylated cytosine" means cytosine that does not have a methyl group connected to the 5th position.

"Modified nucleobase" means any nucleobase that is not an unmodified nucleobase.

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

"Modified sugar portion" means the sugar portion that has been replaced or a sugar substitute.

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

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

"2'-F" or "2'-F" means a sugar with fluorine modification at the 2' position.

"Bicyclic sugar moiety" means a modified sugar moiety comprising 4 to 7 member rings (including but not limited to furanyl glycosyl groups), comprising a bridge connecting two atoms of the 4 to 7 member rings to form a second ring, thereby creating a bicyclic structure. In some embodiments, the 4 to 7 member rings are sugar rings. In some embodiments, the 4 to 7 member rings are furanyl glycosyl groups. In some such embodiments, the bridge connects the 2'-carbon and 4'-carbon of the furanyl glycosyl group. Non-limiting exemplary bicyclic sugar moieties include LNA, ENA, cEt, S-cEt, and R-cEt.

"Locked nucleoside (LNA) sugar moiety" refers to a substituted sugar moiety containing a ( _CH2 )-O bridge between the 4' and 2' furanose ring atoms.

"ENA sugar moiety" refers to a substituted sugar moiety containing a ( _CH2 ) ₂ -O bridge between the 4' and 2' furanose ring atoms.

"Constrained ethyl (cEt) sugar moiety" means a substituted sugar moiety containing a CH( _CH3 )-O bridge between the 4' and 2' furanose ring atoms. In some embodiments, the CH( _CH3 )-O bridge is constrained to the S orientation. In some embodiments, the CH( _CH3 )-O is constrained to the R orientation.

"S-cEt sugar moiety" refers to a substituted sugar moiety containing an S-bound CH( _CH3 )-O bridge between the 4' and 2' furanose ring atoms.

"R-cEt sugar moiety" refers to a substituted sugar moiety containing an R-bound CH( _CH3 )-O bridge between the 4' and 2' furanose ring atoms.

"2'-O-methyl nucleoside" means a 2'-modified nucleoside with 2'-O-methyl sugar modification.

"2'-O-methoxyethyl nucleoside" means a 2'-modified nucleoside with 2'-O-methoxyethyl sugar modification. 2'-O-methoxyethyl nucleoside may contain modified or unmodified nucleobases.

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

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

"cEt nucleoside" means a nucleoside that contains the cEt sugar moiety. cEt nucleosides may contain modified or unmodified nucleobases.

"S-cEt nucleoside" means a nucleoside that contains the S-cEt sugar moiety.

"R-cEt nucleoside" means a nucleoside that contains the R-cEt sugar moiety.

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

"β-D-ribonucleoside" refers to a naturally occurring DNA nucleoside.

"LNA nucleoside" means a nucleoside that contains the LNA sugar portion.

"ENA nucleoside" means a nucleoside that contains the ENA sugar portion.

"Hydrogen bond acceptor" means a hydrogen bond component that does not provide a shared hydrogen atom.

"Hydrogen bond donor" refers to a bond or molecule that provides hydrogen atoms for hydrogen bonds.

References to the value or parameter “about” in this document include (and describe) embodiments relating to that value or parameter itself. In some embodiments, the term “about” includes an indication of ±10%. In other embodiments, the term “about” includes an indication of ±5%. In some still embodiments, the term “about” includes an indication of ±1%. Furthermore, the term “about X” includes a description of “X”. Additionally, unless the context clearly requires otherwise, the singular forms “a” and “the” include plural references. Thus, for example, a reference to “the compound” includes plural compounds of that kind.

Polycystic nephropathy (PKD) is a hereditary form of kidney disease characterized by fluid-filled cysts in the kidneys, leading to renal insufficiency and often resulting in end-stage renal disease (ESRD). Some PKD cases also feature kidney enlargement. Excessive cyst proliferation is a hallmark pathological feature of PKD. In the management of PKD, the primary goals of treatment are to manage symptoms such as hypertension and infection, maintain renal function, and prevent or delay the onset of ESRD, thereby extending the life expectancy of individuals with PKD.

miR-17 has been identified as a target for PKD treatment. The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed anti-miR-17 oligonucleotide library for optimal therapeutic properties. RGLS4326 preferentially distributes to renal and collecting duct-derived cysts, induces miR-17 autotransformation-active polyribosome shift, and de-inhibits multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, subcutaneous administration of RGLS4326 attenuated cyst growth in a human in vivo exochromosomal dominant polycystic kidney disease (ADPKD) model and several PKD mouse models. The Phase 1 single-dose (SAD) clinical trial in healthy volunteers (HV) began in December 2017, followed by the Phase 1 multiple-dose (MAD) study in HV in May 2018.

Following the initiation of the Phase 1b MAD clinical trial, non-clinical toxicology studies revealed CNS-related findings in mice under high doses of RGLS4326, including abnormal gait, decreased motor activity, and/or collapse. RGLS4326 was found to be an antagonist of AMPA receptors (AMPA-R), ion channels at excitatory synapses in the central nervous system (CNS) that mediate rapid excitatory neurotransmission and are therefore key components of all neuronal networks. The antagonistic effect of AMPA receptors explains the CNS-mediated findings observed in non-clinical toxicology models under high doses of RGLS4326. Although no CNS-related findings have been observed in human individuals, AMPA receptor antagonism is still better avoided. Therefore, a library of anti-miR-17 compounds was screened to identify compounds with physicochemical and pharmacological properties comparable to RGLS4326, which also have a more favorable safety profile (e.g., the ability to avoid AMPA receptor antagonism).

A compound of this class, RG-NG-1015, was identified and selected as a candidate treatment for ADPKD. The structural names “RG-NG-1015” and “RGLS8429” are used interchangeably in this document.

RG-NG-1015 and related compounds This article provides a compound comprising a modified oligonucleotide having the following structure 5'- _{AS GSCMAFCFUMUUMUSAS - 3 '} , _wherein each cytosine _is a _{nonmethylated cytosine} .

In some embodiments, the compound consists of modified oligonucleotides.

In some implementations, pharmaceutically acceptable salts are sodium salts.

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

This document also provides a pharmaceutically acceptable salt of the modified oligonucleotide RG-NG-1015. Therefore, in some embodiments, the modified oligonucleotide has the following structure: Or a pharmaceutically acceptable salt thereof. A non-limiting exemplary pharmaceutically acceptable salt of RG-NG-1015 has the following structure: .

In some embodiments, each molecule of the modified oligonucleotide pharmaceutically acceptable salt contains fewer than cation-paired ions (such as Na ⁺ ) linked by thiophosphate and/or phosphodiester bonds (i.e., some thiophosphate and/or phosphodiester bonds are protonated). In some embodiments, each molecule of the pharmaceutically acceptable salt of RG-NG-1015 contains fewer than 8 cation-paired ions (such as Na ⁺ ). That is, in some embodiments, each molecule of the pharmaceutically acceptable salt of RG-NG-1015 may contain an average of 1, 2, 3, 4, 5, 6, or 7 cation-paired ions, wherein the remaining thiophosphate groups are protonated.

As used herein, and unless a specific pharmaceutically acceptable salt of RG-NG-1015 is specifically mentioned, any dosage (whether expressed as, for example, mg/kg, mg, or % by weight) shall be regarded as referring to the amount of RG-NG-1015 in its protonated form (i.e., not in its salt form).

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

Methods for treating polycystic kidney disease This article provides methods for inhibiting the activity of one or more members of the miR-17 family in cells, which include contacting cells with a compound provided herein containing a nucleobase sequence complementary to the miR-17 seed sequence.

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

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

In some embodiments, an individual suffers from a condition characterized by multiple non-renal indicators and also by polycystic kidney disease. Such conditions include, for example, Joubert syndrome and related disorder (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). Therefore, this document provides a method for treating polycystic kidney disease (PKD) comprising administering to an individual a compound provided herein, the compound comprising a nucleobase sequence complementary to a miR-17 seed sequence, wherein the individual suffers from Joubert syndrome and related disorder (JSRD), Meckel syndrome (MKS), or Bardet-Biedl syndrome (BBS). This article provides a method for treating polycystic kidney disease (PKD) comprising administering a compound provided herein containing a nucleobase sequence complementary to a miR-17 seed sequence in an individual suspected of having Jubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), or Bald-Beder syndrome (BBS).

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

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

In some embodiments, polycystic kidney disease is called nephropathy (NPHP). Nephropathy is a somatic recessive cystic kidney disease and a common cause of ESRD in children. NPHP is characterized by normal or reduced kidney size, cysts concentrated at the corticomedullary junction, and tubulointerstitial fibrosis. Mutations in several NPHP genes (e.g., NPHP1 ) have been identified in patients with NPHP. This article provides a method for treating NPHP, which involves administering the compound provided herein to individuals with or suspected of having NPHP, the compound containing a nucleobase sequence complementary to the miR-17 seed sequence.

In some embodiments, individuals with polycystic kidney disease (PCD) also have Jupiter syndrome and related disorders (JSRD). JSRD encompasses a wide range of hallmark features, including abnormalities of the brain, retina, and bones. In addition to the hallmark features of JSRD, some individuals with JSRD also have PCD. Therefore, this document provides a method for treating PCD in individuals with JSRD, comprising administering to the individual with JSRD a compound provided herein, the compound comprising a nucleobase sequence complementary to the miR-17 seed sequence. In some embodiments, the individual is suspected of having JSRD.

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

In some embodiments, individuals with polycystic kidney disease (PCD) may also have Bald-Beder syndrome (BBS). BBS is a condition that affects many parts of the body, including the eyes, heart, kidneys, liver, and digestive system. A hallmark feature of BBS is the presence of kidney cysts. Therefore, this article provides a method for treating PCD in individuals with BBS, comprising administering to the individual with BBS a compound provided herein containing a nucleobase sequence complementary to the miR-17 seed sequence. In some embodiments, the individual may be suspected of having BBS.

In some embodiments, an individual has been diagnosed with PKD prior to administration of a compound containing a modified oligonucleotide. The diagnosis of PKD can be made by evaluating parameters including, but not limited to, an individual'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.

In some embodiments, individuals with ADPKD are classified into categories 1C, 1D, or 1E based on the Mayo Cognitive Classification of ADPKD. In some embodiments, individuals with ADPKD are defined as those with an estimated glomerular filtration rate (eGFR) between 30 and 90 mL/min/1.73 ^m² .

In some embodiments, a method of treating ADPKD is provided, which includes administering to an individual in need a solid dose of a compound containing a modified oligonucleotide, such as RG-NG-1015, or a pharmaceutically acceptable salt thereof, such as sodium salt of RG-NG-1015, in a dose between about 150 mg and about 350 mg, between about 150 mg and about 300 mg, between about 150 mg and about 250 mg, between about 150 mg and about 200 mg, between about 200 mg and about 350 mg, between about 200 mg and about 300 mg, between about 200 mg and about 250 mg, between about 250 mg and about 350 mg, or between about 250 mg and about 300 mg, as discussed herein. In some embodiments, compounds containing modified oligonucleotides or their pharmaceutically acceptable salts are administered at fixed doses between 150 mg and 350 mg, between 150 mg and 300 mg, between 150 mg and 250 mg, between 150 mg and 200 mg, between 200 mg and 350 mg, between 200 mg and 300 mg, between 200 mg and 250 mg, between 250 mg and 350 mg, or between 250 mg and 300 mg. In some embodiments, compounds containing modified oligonucleotides or their pharmaceutically acceptable salts are administered at fixed doses of about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, or about 350 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, or 350 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 150 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 160 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 170 mg or 170 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 180 mg or 180 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 190 mg or 190 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 200 mg or 200 mg. In some embodiments, the compound containing the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of about 210 mg or 210 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 220 mg or 220 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 230 mg or 230 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 240 mg or 240 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 250 mg or 250 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 260 mg or 260 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 270 mg or 270 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 280 mg or 280 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 290 mg or 290 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 300 mg or 300 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 310 mg or 310 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 320 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 330 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 340 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered at a fixed dose of about 350 mg. In some embodiments, the compound containing the modified oligonucleotide or a pharmaceutically acceptable salt thereof is administered subcutaneously.

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

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

In some embodiments, the individual has an increased total renal volume. In some embodiments, the total renal volume is the height-adjusted total renal volume (HtTKV). In some embodiments, the individual has hypertension. In some embodiments, the individual has impaired renal function. In some embodiments, the individual requires improvement in renal function. In some embodiments, the individual is diagnosed with impaired renal function.

In some embodiments, elevated levels of one or more miR-17 family members are observed in the kidneys of individuals with PKD. In some embodiments, elevated levels of one or more miR-17 family members in the kidneys are identified prior to administration. The levels of miR-17 family members can be measured from renal biopsy materials. In some embodiments, elevated levels of one or more miR-17 family members in the urine or blood are identified prior to administration. In some embodiments, decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine are identified prior to administration. In some embodiments, decreased levels of polycystic protein-1 (PC1) or polycystic protein-2 (PC2) in the urine are identified prior to administration. In some embodiments, prior to administration, it is determined that the individual has reduced levels of polycystic protein-1 (PC1) and/or polycystic protein-2 (PC2) in the individual's urine.

In some embodiments, prior to administration, individuals are identified as having increased levels of neutrophil gelatinase-associated lipotransferase (NGAL) and/or kidney damage molecule-1 (KIM-1) in their urine.

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

In some embodiments, individuals may also undergo additional testing before, during, and/or after administration, such as measuring mononuclear globulin-1 (MCP-1) and/or β-2 microglobulin (B2M) in the individual's urine; measuring insulin-like growth factor-binding protein acid unstable subunits (IGFALS), CT-proAVP, and/or N-acetylglucosamine in the individual's serum; measuring complement mitotic products C3a and/or Bb in the individual's plasma; and/or measuring the total cyst volume, number, and/or size distribution of cysts in the individual.

Biomarkers for polycystic kidney disease are identified through laboratory testing. Reference ranges for individual biomarkers can vary from laboratory to laboratory. This variation can be attributed to, for example, differences in the specific analysis used. Therefore, the upper and lower limits of the normal distribution of biomarkers within a population, also known as the upper limit of normal (ULN) and lower limit of normal (LLN), can vary from laboratory to laboratory. For any given biomarker, healthcare professionals can determine which levels outside the normal distribution are clinically relevant and/or indicative of disease. For example, healthcare professionals can determine glomerular filtration rate, which indicates a decline in renal function in individuals with polycystic kidney disease.

In some embodiments, administration of the compounds provided herein produces one or more clinically beneficial results. In some embodiments, administration improves renal function in an individual. In some embodiments, administration slows the rate of decline in renal function in an individual. In some embodiments, administration reduces total renal volume in an individual. In some embodiments, administration slows the rate of increase in total renal volume in an individual. In some embodiments, administration reduces height-adjusted total renal volume (HtTKV). In some embodiments, administration slows the rate of increase in HtTKV.

In some embodiments, polycystic protein-1 (PC1) is administered to increase the amount of polycystic protein-1 (PC1) in an individual's urine. In some embodiments, polycystic protein-2 (PC2) is administered to increase the amount of polycystic protein-1 (PC1) and polycystic protein-2 (PC2) in an individual's urine.

In some embodiments, treatment involves inhibiting cyst growth in an individual (total cyst volume, number, and/or size distribution). In some embodiments, treatment involves slowing the rate of increase in cyst growth in an individual (total cyst volume, number, and/or size distribution). In some embodiments, the cysts are present in the individual's kidneys. In some embodiments, the cysts are present in organs other than the kidneys, such as the liver.

In some embodiments, the treatment aims to alleviate kidney pain in the individual. In some embodiments, the treatment aims to slow the increase of kidney pain in the individual. In some embodiments, the treatment aims to delay the onset of kidney pain in the individual.

In some implementations, medication is administered to lower an individual's high blood pressure. In some implementations, medication is administered to slow the worsening of an individual's high blood pressure. In some implementations, medication is administered to delay the onset of an individual's high blood pressure.

In some embodiments, the treatment aims to reduce fibrosis in the individual's kidneys. In some embodiments, the treatment aims to slow the worsening of fibrosis in the individual's kidneys.

In some embodiments, interventions delay the onset of end-stage renal disease in individuals. In some embodiments, interventions delay the duration of dialysis in individuals. In some embodiments, interventions delay the time to kidney transplantation in individuals. In some embodiments, interventions extend the life expectancy of individuals.

In some embodiments, the treatment aims to reduce albuminuria in individuals. In some embodiments, it aims to slow the worsening of albuminuria in individuals. In some embodiments, it aims to delay the onset of albuminuria in individuals. In some embodiments, it aims to reduce hematuria in individuals. In some embodiments, it aims to slow the worsening of hematuria in individuals. In some embodiments, it aims to delay the onset of hematuria in individuals. In some embodiments, it aims to reduce or slow the rate of increase in blood urea nitrogen (BUN) levels in individuals. In some embodiments, it aims to reduce or slow the rate of increase in serum creatinine (SCr) levels in individuals. In some embodiments, it aims to improve or slow the rate of decrease in creatinine clearance rate in individuals. In some implementations, the administration reduces or slows the rate of increase in the urinary albumin:creatinine ratio in an individual.

In some embodiments, treatment aims to increase the glomerular filtration rate (GFR) in an individual. In some embodiments, treatment aims to slow the rate of decline in the GFR in an individual. In some embodiments, the GFR is the estimated GFR (eGFR). In some embodiments, the GFR is the measured GFR (mGFR).

In some embodiments, administration reduces or slows the rate of increase of neutrophil gelatinase-associated lipotransferase (NGAL) protein in individual urine. In some embodiments, administration reduces or slows the rate of increase of renal injury molecule-1 (KIM-1) protein in individual urine.

In some embodiments, administration is given to reduce or slow the rate of increase of mononuclear globulin-1 (MCP-1) in an individual's urine. In some embodiments, administration is given to reduce or slow the rate of increase of β-2 microglobulin (B2M) in an individual's urine.

In some embodiments, administration reduces or slows the rate of increase in complement cleavage products C3a and/or Bb in an individual's plasma.

In some embodiments, administration is given to reduce or slow down the rate of increase of acute-phase proteins (i.e., Alb, cellulose, and/or highly sensitive C-reactive proteins).

In some embodiments, administration reduces or slows the rate of increase in insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in individual serum. In some embodiments, administration reduces or slows the rate of increase in CT-proAVP in individual serum. In some embodiments, administration reduces or slows the rate of increase in N-acetylglucosamine in individual serum.

In any of the embodiments provided herein, an individual may undergo certain tests to assess the severity of their disease. Such tests include, but are not limited to, measuring total renal volume; measuring total kidney volume (HTKV); measuring hypertension; measuring renal pain; measuring renal fibrosis; measuring blood urea nitrogen (BUN) levels; measuring serum creatinine (SCr) levels; measuring creatinine clearance in the blood; measuring albuminuria; measuring the albumin:creatinine ratio; measuring glomerular filtration rate (GFR), where the GFR is estimated or measured; measuring polycystic protein-1 (PC1) and/or polycystic protein-2 in the urine. (PC2); Measure neutrophil gelatinase-associated lipotransferase (NGAL) protein in individual urine; and/or measure renal injury molecule-1 (KIM-1) protein in individual urine; measure MCP-1 and/or measure B2M in individual urine; measure IGFALS, measure CT-proAVP in individual serum and/or measure N-acetylglucosamine; measure total cyst volume, number and/or size distribution of cysts.

In some embodiments, such as those discussed herein, administration of compounds containing modified oligonucleotides or their pharmaceutically acceptable salts causes virtually no CNS damage in individuals. In some embodiments, CNS damage is measured using the Disorder Assessment and Rating Scale (SARA), a tool used to assess disorder. In some embodiments, individuals are tested for SARA prior to administration. In some embodiments, administration causes virtually no change in an individual's Disorder Assessment and Rating Scale (SARA) score compared to pre-treatment levels.

In some embodiments, individuals may perform certain tests before, during, and/or after administration to assess the pharmacokinetics of compounds containing modified oligonucleotides or their pharmaceutically acceptable salts, as discussed herein. Perform pharmacokinetic analysis to measure and compare one or more of the following parameters: maximum observed concentration ( _Cmax ), time to reach maximum observed concentration ( _Tmax ), concentration-time under-curve up to 24 hours after administration (AUC _0-24 ), concentration-time under-curve up to the final quantifiable concentration (AUC _0-t ), concentration-time under-curve over the dosing interval (AUC _τ ), concentration-time under-curve extrapolated to infinity (AUC _inf ), half-life (t _½ ), apparent clearance (CL/F), apparent volume of distribution ( _Vz /F), fraction of unmodified compound excreted in urine (fe), and/or total amount of unmodified compound excreted in urine (Ae).

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

In some implementations, individuals with polycystic kidney disease experience a reduced quality of life. For example, individuals with polycystic kidney disease may experience kidney pain, which can reduce their quality of life. In some implementations, measures are taken to improve the individual's quality of life.

In any of the embodiments provided herein, the individual is a human individual. In some embodiments, the human individual is an adult. In some embodiments, an adult is at least 21 years old. In some embodiments, the human individual is a pediatric individual, i.e., an individual under 21 years old. The pediatric population may be defined by a governing body. In some embodiments, the human individual is an adolescent. In some embodiments, an adolescent is at least 12 years old and under 21 years old. In some embodiments, the human individual is a child. In some embodiments, a child is at least two years old and under 12 years old. In some embodiments, the human individual is an infant. In some embodiments, an infant is at least one month old and under two years old. In some embodiments, the individual is a newborn. In some embodiments, a newborn is under one month old. In some implementations, the individuals are between 18 and 70 years old.

Any of the compounds described herein may be used in therapy. Any of the compounds provided herein may be used to treat polycystic kidney disease. In some embodiments, polycystic kidney disease is somatic dominant polycystic kidney disease. In some embodiments, polycystic kidney disease is somatic recessive polycystic kidney disease. In some embodiments, polycystic kidney disease is nephropathy. In some embodiments, the individual has Jupiter syndrome and related disorders (JSRD), Meckel's syndrome (MKS), or Bald-Bieder syndrome (BBS).

Any modified oligonucleotide described herein may be used in therapy. Any modified oligonucleotide provided herein may be used to treat polycystic kidney disease.

Any of the compounds provided herein may be used in the preparation of pharmaceutical preparations. Any of the compounds provided herein may be used in the preparation of pharmaceutical preparations for the treatment of polycystic kidney disease.

Any of the modified oligonucleotides provided herein may be used in the preparation of pharmaceutical preparations. Any of the modified oligonucleotides provided herein may be used in the preparation of pharmaceutical preparations for the treatment of polycystic kidney disease.

Any of the pharmaceutical combinations described herein may be used to treat polycystic kidney disease.

In some implementations, the treatment has an acceptable safety and tolerability profile. In other implementations, the treatment is generally safe and well-tolerated.

Certain additional therapies for polycystic kidney disease or any of the disorders listed herein may include more than one therapy. Therefore, in some embodiments, this document provides methods for treating individuals who have or are suspected of having polycystic kidney disease, methods that include, in addition to administering the compounds provided herein, administering at least one therapy comprising a nucleobase sequence complementary to the miR-17 seed sequence.

In some embodiments, at least one additional treatment includes a medication. In some embodiments, the medication is an antihypertensive agent. The antihypertensive agent is used to control an individual's blood pressure.

In some embodiments, the agent is an angiotensin II receptor 2 antagonist. In some embodiments, the angiotensin II receptor 2 antagonist is tolvaptan.

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

In some embodiments, the agent includes angiotensin II converting enzyme (ACE) inhibitors. In some embodiments, the ACE inhibitor is captopril, enalapril, lisinopril, benzazepril, quinapril, fosinopril, or ramipril.

In some embodiments, the agent is a diuretic. In some embodiments, the agent is a calcium channel blocker.

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

In some embodiments, the medication is an antihyperglycemic agent. In some embodiments, the antihyperglycemic agent is biguanide. In some embodiments, the biguanide is metformin.

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

In some implementations, the drug is an adrenaline receptor antagonist.

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

In some embodiments, the agent is a mammalian rapamycin-targeted (mTOR) inhibitor. In some embodiments, the mTOR inhibitor is everolimus, rapamycin, or sirolimus.

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

In some embodiments, the agent is an antifibrillating agent. In some embodiments, the antifibrillating agent is a modified oligonucleotide complementary to miR-21.

In some implementations, the additional treatment is dialysis. In some implementations, the additional treatment is kidney transplantation.

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

In some embodiments, the agent is an agent that blocks one or more responses to fiberization signals.

In some embodiments, adjunctive therapies may be agents that enhance the body’s immune system, including low-dose cyclophosphamide, thymosin, vitamins and nutritional supplements (such as antioxidants, including vitamins A, C, E, beta-carotene, zinc, selenium, glutathione, coenzyme Q10 and pennywort) and vaccines, such as immunostimulatory complexes (ISCOMs), which are vaccine formulations containing multimer presenters of combined antigens and adjuvants.

In some embodiments, additional therapies are selected to treat or improve the side effects of one or more of the pharmaceutical combinations described herein. Such side effects include, but are not limited to, injection site reactions, abnormal liver function tests, abnormal kidney function, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, and myopathy. For example, elevated serum levels of aminotransferases can indicate hepatotoxicity or abnormal liver function. For example, elevated bilirubin can indicate hepatotoxicity or abnormal liver function.

The miR-17 family of microRNA nucleobase sequences includes miR-17, miR-20a, miR-20b, miR-93, miR-106a, and miR-106b. Each member of the miR-17 family possesses a nucleobase sequence containing the 5'-AAAGUG-3' nucleobase sequence or the miR-17 seed sequence, which is the nucleobase sequence at positions 2 to 7 of SEQ ID NO: 1. Furthermore, members of the miR-17 family share some nucleobase sequence identity outside the seed region. Therefore, modified oligonucleotides containing nucleobase sequences complementary to the miR-17 seed sequence can target other microRNAs in the miR-17 family besides miR-17.

In some embodiments, the modified oligonucleotide contains the nucleobase sequence 5'-AGCACUUUA-3'.

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

In some embodiments, the number of linking nucleotides in the modified oligonucleotide is less than the length of its target microRNA. Modified oligonucleotides having a number of linking nucleotides less than the length of the target microRNA are considered to be modified oligonucleotides having a nucleotide sequence that is completely complementary (also known as 100% complementary) to a region of the target microRNA sequence, wherein each nucleotide of the modified oligonucleotide is complementary to the nucleotide at a corresponding position in the target microRNA. For example, a modified oligonucleotide consisting of 9 linking nucleotides is completely complementary to miR-17, wherein each nucleotide is complementary to the nucleotide at a corresponding position in miR-17.

In some embodiments, the modified oligonucleotide has one mismatched nucleotide sequence relative to the target microRNA's nucleotide sequence. In some embodiments, the modified oligonucleotide has two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some of these embodiments, the modified oligonucleotide has at most two mismatched nucleotide sequences relative to the target microRNA's nucleotide sequence. In some of these embodiments, the mismatched nucleotides are adjacent. In some of these embodiments, the mismatched nucleotides are not adjacent.

Although the sequence listing accompanying this application identifies each nucleobase sequence as "RNA" or "DNA" as needed, in practice, these sequences can be modified with combinations of chemical modifications specified herein. Those skilled in the art will readily appreciate that the use of names such as "RNA" or "DNA" to describe modified oligonucleotides in the sequence listing is somewhat arbitrary. For example, a modified oligonucleotide containing a nucleotide with a 2'-O-methoxyethyl sugar moiety and a thymine base can be described in the sequence listing as a DNA residue, even if the nucleotide is modified and not a native DNA nucleotide.

Therefore, the nucleic acid sequences provided in the sequence listing are intended to cover nucleic acids containing any combination of natural or modified RNA and/or DNA, including but not limited to nucleic acids having modified nucleotide bases. For example, and not limited to, the modified oligonucleotides in the sequence listing containing the nucleotide sequence "ATCGATCG" cover any oligonucleotides having this nucleotide sequence, whether modified or unmodified, including but not limited to compounds containing RNA bases, such as those having the sequence "AUCGAUCG" and those having some DNA bases and some RNA bases (such as "AUCGATCG") and oligonucleotides having other modified bases, such as "AT ^me CGAUCG", where ^me C indicates 5-methylcytosine.

Certain modifications : In some embodiments, the oligonucleotides provided herein may contain one or more modifications to the nucleotide bases, sugars, and/or internucleotide links, and are therefore modified oligonucleotides. The modified nucleotide bases, sugars, and/or internucleotide links may be selected for their preferred properties, such as enhanced cellular uptake, enhanced affinity for other oligonucleotides or nucleic acid targets, and increased stability in the presence of nucleases, compared to the unmodified form.

In some embodiments, the modified oligonucleotide contains one or more modified nucleosides.

In some embodiments, the modified nucleotide is a sugar-modified nucleotide. In some such embodiments, the sugar-modified nucleotide may further comprise a natural or modified heterocyclic base moiety, and/or may be linked to another nucleotide via an inter-nucleotide bond, and/or may include other modifications independent of the sugar modification. In some embodiments, the sugar-modified nucleotide is a 2'-modified nucleotide, wherein the sugar ring is modified at the 2' carbon of either natural ribose or 2'-deoxyribose.

In some embodiments, the 2'-modified nucleotide has a dicyclic sugar moiety. In some such embodiments, the dicyclic sugar moiety is a D sugar in an α configuration. In some such embodiments, the dicyclic sugar moiety is a D sugar in a β configuration. In some such embodiments, the dicyclic sugar moiety is an L sugar in an α configuration. In some such embodiments, the dicyclic sugar moiety is an L sugar in a β configuration.

Nucleosides containing this type of dicyclic sugar moiety are called dicyclic nucleosides or BNAs. In some embodiments, the bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy(4'- _CH₂ -O-2') BNA; (B) β-D-methyleneoxy(4'- _CH₂ -O-2') BNA; (C) ethoxy(4'-( _CH₂ ) _₂ -O-2') BNA; (D) aminooxy(4'- _CH₂ -ON(R)-2') BNA; (E) oxyamino(4'- _CH₂ -N(R)-O-2') BNA; (F) methyl(methyleneoxy)(4'-CH( _CH₃ )-O-2') BNA (also known as bound ethyl or cEt); (G) methylene-thio(4'- _CH₂ -S-2') BNA; and (H) methylene-amino(4'-CH₂-N(R)-2'). BNA; (I) methyl carbocyclic (4'- _CH2 -CH( _CH3 )-2') BNA; (J) c-MOE (4'-CH( _CH2 -OMe)-O-2') BNA and (K) propyl carbocyclic (4'-( _CH2 ) _3-2 ') BNA, as described below. Where Bx is the nucleobase moiety and R is independently H, a protecting group, or a _C1 - _C12 alkyl group.

In some embodiments, the 2'-modified nucleoside comprises a 2'-substituent selected from the following: F, _OCF3 , O- _CH3 (also known as "2'-OMe"), _OCH2CH2OCH3 (also known as "2'-O- _methoxyethyl " _or "2'-MOE"), 2'-O( _CH2 ) _2SCH3 , _{O-(CH2)2 - ON} ( _CH3 ) ₂ , -O( _CH2 ) _2O ( _CH2 ) _{2N(CH3)2 and O} - _CH2 -C(=O)-N(H) _CH3 .

In some embodiments, the 2'-modified nucleoside contains a 2' _{- substituent} selected from F, O- _CH3 and _OCH2CH2OCH3 .

In some embodiments, the sugar-modified nucleoside is a 4'-thio-modified nucleoside. In some embodiments, the sugar-modified nucleoside is a 4'-thio-2'-modified nucleoside. The 4'-thio-modified nucleoside has a β-D-ribonucleoside, wherein the 4'-O is replaced by a 4'-S. The _4' -thio-2'-modified nucleoside is a 4'-thio-modified nucleoside with the 2'-OH replaced by a 2'-substituent. Suitable 2'-substituents include 2'- _OCH3 , 2'- _{OCH2CH2OCH3 ,} and 2'-F.

In some embodiments, the modified oligonucleotide contains one or more inter-nucleotide modifications. In some such embodiments, the inter-nucleotide links of the modified oligonucleotide are modified inter-nucleotide links. In some embodiments, the modified inter-nucleotide links contain a phosphorus atom.

In some embodiments, the modified oligonucleotide contains at least one phosphate thioester nucleoside linker. In some embodiments, the nucleoside links of the modified oligonucleotide are phosphate thioester nucleoside links.

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

In some embodiments, the modified nucleobase comprises a polycyclic heterocycle. In some embodiments, the modified nucleobase comprises a tricyclic heterocycle. In some embodiments, the modified nucleobase comprises a phenoxazine derivative. In some embodiments, the phenoxazine may be further modified to form a nucleobase referred to in this art as G-clipper.

In some embodiments, the modified oligonucleotide is bound to one or more moieties that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. In some such embodiments, the moieties are cholesterol moieties. In some embodiments, the moieties are lipid moieties. Additional moieties used for binding include carbohydrates, peptides, antibodies or antibody fragments, phospholipids, biotin, phenazine, folic acid, phenazine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In some embodiments, the carbohydrate moieties are N-acetylglucosamine (GalNac). In some embodiments, the binding group is directly attached to the oligonucleotide. In some embodiments, the binding group is attached to the modified oligonucleotide by means of a linker selected from the following: amino, azido, hydroxyl, carboxylic acid, thiol, unsaturated (e.g., double or triple bond), 8-amino-3,6-dioxooctanoic acid (ADO), 4-(N-cis-butenediaminomethyl)cyclohexane-1-carboxylic acid succinimino ester (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl and substituted or unsubstituted C2-C10 alkynyl. In some of these embodiments, the substituents are selected from hydroxyl, amino, alkoxy, azido, carboxyl, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In some of these embodiments, the compound comprises a modified oligonucleotide having one or more stabilizing groups attached to one or both ends of the modified oligonucleotide to enhance properties such as nuclease stability. The stabilizing groups include a cap structure. These end modifications protect the modified oligonucleotide from exonuclease degradation and aid in delivery and/or localization within the cell. The cap may be present at the 5' end (5'-cap) or the 3' end (3'-cap), or may be present at both ends. The cap structure includes, for example, a reverse deoxyalkaline cap.

Certain pharmaceutical compositions are provided herein that comprise the compounds or modified oligonucleotides provided herein and a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is an aqueous solution. In some embodiments, the aqueous solution is a saline solution. As used herein, a pharmaceutically acceptable diluent should be understood as a sterile diluent. Suitable routes of administration include, but are not limited to, intravenous and subcutaneous administration. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. In some embodiments, administration is oral.

In some embodiments, the pharmaceutical composition is administered in the form of a dosage unit. For example, in some embodiments, the dosage unit is in the form of a tablet, capsule, or large-dose injection.

In some embodiments, the drug is a modified oligonucleotide prepared in a suitable diluent, adjusted to pH 7.0-9.0 with acid or alkali during preparation, and then freeze-dried under sterile conditions. The freeze-dried modified oligonucleotide is then reconstituted with a suitable diluent, such as an aqueous solution (e.g., water) or a physiologically compatible buffer (e.g., saline solution, Hanks' solution, or Ringer's solution). The reconstituted product is administered subcutaneously or intravenously. The freeze-dried drug can be packaged in a 2 mL Type I clear glass vial (ammonium sulfate treated), sealed with a bromobutyl rubber stopper and an aluminum outer seal.

In some embodiments, the pharmaceutical compositions provided herein may additionally contain other adjunct components known in pharmaceutical compositions, to the level of use established in their respective fields. Thus, for example, the composition may contain additional compatible pharmaceutically active materials, such as antipruritics, astringents, local anesthetics, or anti-inflammatory agents.

In some embodiments, the pharmaceutical compositions provided herein may contain additional materials that can be used to physically formulate various dosage forms of the compositions provided herein, such as dyes, 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, silica, viscous paraffin, hydroxymethyl cellulose, and polyvinylpyrrolidone. In all embodiments, the addition of such materials should not excessively interfere with the biological activity of the components of the compositions provided herein. The formulation can be sterilized and, if necessary, mixed with excipients such as lubricants, preservatives, stabilizers, humectants, emulsifiers, salts to affect osmotic pressure, buffers, colorants, flavorings, and/or aromatic substances and their analogues, which do not adversely interact with the oligonucleotides of the formulation. Some injectable pharmaceutical compositions are suspensions, solutions, or emulsions in oily or aqueous media and may contain formulation agents such as suspending agents, stabilizers, and/or dispersants. Certain solvents suitable for use in injectable pharmaceutical compositions include (but are not limited to) lipophilic solvents and fatty oils (such as sesame oil), synthetic fatty acid esters (such as ethyl oleate or triglycerides), and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or polydextrose. Depending on the circumstances, such suspensions may also contain suitable stabilizers or reagents that increase the solubility of the drug to allow for the preparation of high-concentration solutions.

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

In some embodiments, the pharmaceutical compositions provided herein comprise a polyamine compound or lipid moiety complexed with a nucleic acid. In some embodiments, such formulations comprise one or more compounds having individually the structure defined by formula (Z) or a pharmaceutically acceptable salt thereof. In this formulation, ^Xa and ^Xb are each independently _C1-6 alkyl groups in each occurrence; n is 0, 1, 2, 3, 4, or 5; each R is independently H, wherein at least about 80% of the compounds of formula (Z) in the formulation have at least n + 2 R moieties that are not H; m is 1, 2, 3, or 4; Y is O, ^NR2 , or S; ^R1 is alkyl, alkenyl, or alkynyl, each substituted with one or more substituents as appropriate; and ^R2 is H, alkyl, alkenyl, or alkynyl, each substituted as appropriate, each substituted with one or more substituents as appropriate; provided that if n = 0, at least n + 3 R moieties are not H. Such formulations are described in PCT Publication WO/2008/042973, which is incorporated herein by reference in its entirety for the purpose of disclosing lipid formulations. Some additional formulations are described in Akinc et al., Nature Biotechnology 26 , 561-569 (May 1, 2008), which is incorporated herein by reference in its entirety for the purpose of revealing lipid formulations.

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

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

In some embodiments, the pharmaceutical compositions provided herein are formulated as storage preparations. Some of these storage preparations typically have a longer duration of action than non-storage preparations. In some embodiments, such preparations are administered by implantation (e.g., subcutaneous or intramuscular) or by intramuscular injection. In some embodiments, storage preparations are prepared using suitable polymers or hydrophobic materials (e.g., emulsions in acceptable oils) or ion-exchange resins or as microsoluble derivatives, such as as microsoluble salts.

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

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

In some embodiments, the pharmaceutical compositions provided herein include a sustained-release system. Non-limiting examples of such sustained-release systems are semi-permeable matrices of solid hydrophobic polymers. In some embodiments, the sustained-release system may release the drug over time periods of hours, days, weeks, or months, depending on its chemical properties.

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

In some embodiments, the pharmaceutical compositions provided herein contain a therapeutically effective amount of modified oligonucleotides. In some embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or improve symptoms of the disease or prolong the survival of the treated individual.

In some embodiments, one or more of the modified oligonucleotides provided herein are formulated as prodrugs. In some embodiments, after in vivo administration, the prodrug is chemically converted into the biological, pharmaceutical, or therapeutically active form of the oligonucleotide. In some embodiments, the prodrug is useful because it is easier to administer than the corresponding active form. For example, in some cases, the prodrug may be more bioavailable than the corresponding active form (e.g., via oral administration). In some cases, the prodrug may have improved solubility compared to the corresponding active form. In some embodiments, the prodrug is less water-soluble than the corresponding active form. In some cases, such prodrugs have excellent transmembrane transport, where water solubility is detrimental to migration rate. In some embodiments, the prodrug is an ester. In some of these embodiments, the ester is metabolically hydrolyzed to a carboxylic acid after administration. In some cases, the carboxylic acid compound is the corresponding active form. In some embodiments, the prodrug comprises a short peptide (polyamino acid) bound to an acid group. In some of these embodiments, the peptide is cleaved post-administration to form the corresponding active form.

In some embodiments, prodrugs are produced by modifying pharmaceutical active compounds to regenerate them in vivo. Prodrugs can be designed to alter the metabolic stability or transport characteristics of a drug, mask side effects or toxicity, improve the taste of a drug, or change other characteristics or properties of a drug. With knowledge of in vivo pharmacodynamics and drug metabolism, skilled practitioners can design prodrugs of pharmaceutical active compounds once the active compound is known (see, for example, Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392).

Additional routes of administration include, but are not limited to, oral, rectal, mucosal, intraenteral, enteric, local, suppository, inhalation, intrathecal, intracardiac, intraventricular, intraperitoneal, intranasal, intraocular, intratumoral, intramuscular, and intramedullary administration. In some embodiments, intrathecal administration of medicines achieves local rather than systemic exposure. For example, a pharmaceutical composition may be injected directly into the area of desired action (e.g., into the kidney).

Some kits are also provided as kits. In some embodiments, the kit contains one or more compounds, which include the modified oligonucleotides disclosed herein. In some embodiments, the kit can be used to administer the compound to an individual.

In some embodiments, the kit contains a pharmaceutical composition ready for administration. In some embodiments, the pharmaceutical composition is contained in vials. A plurality of vials (e.g., 10) may be contained, for example, in a dispensing kit. In some embodiments, the vials are manufactured for administration via a syringe. The kit may also contain instructions for use of the compound.

In some embodiments, the kit contains a pharmaceutical composition in a pre-filled syringe (such as a single-dose syringe with, for example, a 27.5-gauge needle with a needle guard) rather than in a vial. A plurality of pre-filled syringes (e.g., 10) may be contained, for example, in a dispensing kit. The kit may also contain instructions for administering the compounds comprising modified oligonucleotides disclosed herein.

In some embodiments, the kit includes the modified oligonucleotides provided herein as a freeze-dried pharmaceutical product and a pharmaceutically acceptable diluent. When prepared for administration to an individual, the freeze-dried pharmaceutical product is reconstituted in a pharmaceutically acceptable diluent.

In some embodiments, in addition to the compounds containing modified oligonucleotides disclosed herein, the kit may further include one or more of the following: syringes, alcohol swabs, cotton balls and/or gauze pads.

Some experimental models , in certain embodiments, provide methods for using and/or testing the modified oligonucleotides provided herein. Those skilled in the art can select and modify protocols of such experimental models to evaluate the reagents provided herein.

Generally, the modified oligonucleotides are first tested in cultured cells. Suitable cell types include those related to the cell types from which the delivery of the modified oligonucleotides in vivo is desired. For example, cell types suitable for studying the methods described herein include primary cells or cultured cells.

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

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

Some quantitative analyses , in certain embodiments, quantify microRNA levels in cells or tissues, either in vitro or in vivo. In some embodiments, changes in microRNA levels are measured using microarray analysis. In some embodiments, changes in microRNA levels are measured using one of several commercially available PCR assays, such as the TaqMan® microRNA assay (Applied Biosystems).

The regulation of microRNA activity by anti-miR or microRNA mimics can be assessed using mRNA microarray analysis. A search is conducted on microRNA seed sequences for mRNA sequences regulated (increased or decreased) by anti-miR or microRNA mimics to compare the regulation of mRNAs that are microRNA targets with the regulation of mRNAs that are not microRNA targets. This allows for the assessment of interactions between anti-miR and its target microRNA, or between microRNA mimics and their targets. In the case of anti-miR, mRNAs with increased expression levels are screened for seed sequences containing microRNAs complementary to the anti-miR.

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

Examples are presented below to illustrate some embodiments of the invention more fully. However, they should not be construed as limiting the broad scope of the invention.

Without departing from the spirit of this invention, those generally familiar with this technology will easily adopt the basic principles of this discovery to design various compounds.

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

The miR-17-92 cluster includes two microRNAs, miR-17 and miR-20a, which are members of the miR-17 family of microRNAs. Members of this family share 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 in the miR-106a-363 cluster on human chromosome X, while miR-93 and miR-106b are located in the miR-106b-25 cluster on human chromosome 7. Table 1 shows the sequences of the miR-17 family members.

Table 1: miR-17 family of microRNAs microRNA Sequence (5' to 3') Seed region in bold SEQ ID NO: miR-17 c aaagug cuuacagugcagguag 1 miR-20a U AAAGUG CUUAUAGUGCAGGUAG 2 miR-20b C AAAGUG CUCAUAGUGCAGGUAG 3 miR-93 C AAAGUG CUGUUCGUGCAGGUAG 4 miR-106a A AAAGUG CUUACAGUGCAGGUAG 5 miR-106b U AAAGUG CUGACAGUGCAGAU 6

The anti-miR-17 compound RGLS4326 was discovered by screening a chemically diverse and rationally designed anti-miR-17 oligonucleotide library for optimal pharmaceutical properties. RGLS4326 preferentially distributes to renal and collecting duct-derived cysts, induces miR-17 autotransformation-active polyribosome shift, and de-represses multiple miR-17 mRNA targets, including Pkd1 and Pkd2. Importantly, RGLS4326 attenuates cyst growth in a human in vitro ADPKD model and several PKD mouse models after subcutaneous administration. The Phase 1 single-dose (SAD) clinical trial of RGLS4326 in healthy volunteers began in December 2017, followed by the Phase 1 multiple-dose (MAD) study in healthy volunteers in May 2018. The Phase 1b clinical trial of RGLS4326 for the treatment of patients with autosomal dominant polycystic kidney disease (ADPKD) began in October 2020.

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

Example 2: Screening for the antimiR-17 compound RGLS4326 with reduced AMPA receptor binding. RGLS4326 has the following sequence and chemical modification pattern: AS _{G S} C _M A _F C _F U _F U _M U _S G _S , where 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, all cytosines are unmethylated, and all bonds are phosphate thioester bonds. Chemical modifications and length variants of RGLS4326 were designed and screened to identify compounds that retain the potency and pharmacokinetic characteristics of RGLS4326 and exhibit reduced binding to the AMPA receptor (AMPA-R).

A compound library with different chemical modifications, nucleobase sequences and lengths relative to RGLS4326 was designed.

Table 2: Anti-miR-17 Library Compound number Chemical symbols nucleobase sequence SEQ ID NO Length RG-NG-1001 A _S G _S C _S A _S C _S U _S U _S U _S G _S AGCACUUUG 9 RG-NG-1002 A _S G _S C _S A _S C _S U _S U _S U _S AGCACUUU- 8 RG-NG-1003 A _S G _S C _M A _S C _M U _S U _M U _S G _S AGCACUUUG 9 RG-NG-1004 U _S A _S A _S G _{S C} S A _S C _S U _S U _S U _S G _S UAAGCACUUUG 12 11 RG-NG-1005 U _S A _S A _M G C _S A _S C _S U _M U _M U _S G _S UAAGCACUUUG 13 11 RG-NG-1006 A _L G _L C _L A _L C _L T _L T _L T _{L G L} AGCACTTTG 9 RG-NG-1007 A _S G _S C _F A _F C _F U _F U _F U _S G _S AGCACUUUG 9 RG-NG-1008 A _S G _E C _M A _F C _F U _F U _M T _E G _S AGCACUUTG 9 RG-NG-1009 A _E G _E C _M A _F C _F U _F U _M T _E G _E AGCACUUTG 9 RG-NG-1010 A _E G _E C _M A _F C _F U _F U _M U _S G _S AGCACUUUG 9 RG-NG-1011 A _L G _L C _M A _F C _F U _F U _M U _S G _S AGCACUUUG 9 RG-NG-1012 A _S G _L C _M A _F C _F U _F U _M T _L G _S AGCACUUTG 9 RG-NG-1013 A _S A _S G _S C _M A _F C _F U _F U _M U _S AAGCACUUU- 9 RG-NG-1014 A _S G _S C _M A _F C _F U _F U _M U _S AGCACUUU- 8 RG-NG-1015 A _S G _S C _M A _F C _F U _F U _M U _S A _S AGCACUUUA 9 RG-NG-1016 A _S G _S C _M A _F C _F U _F U _M U _S C _S AGCACUUUC 9 RG-NG-1017 A _S G _S C _M A _F C _F U _F U _M U _S U _S AGCACUUUU 9 RG-NG-1018 A _E C _E T _E G _S T _E AA _S G _S C _M A _F C _F U _F U _{M U S} C _S ACTGTAAGCACUUUC 14 15 RG-NG-1019 A _S C _S TG _S U _S AA _S G _S C _M A _F C _F U _F U _{M U S} C _S ACTGUAAGCACUUUC 15 15 RG-NG-1020 T _E G _S T _E AA _S G _S C _M A _F C _F U _{F U M U S} C _S TGTAAGCACUUUC 16 13 RG-NG-1021 T _E AA _S G _S C _M A _F C _F U _F U _M U _S C _S TAAGCACUUUC 17 11 RG-NG-1022 T _E A _E A _S G _S C _M A _F C _F U _F U _{M U S} C _S TAAGCACUUUC 18 11 RG-NG-1023 A _E G _S C _M A _F C _F U _F U _M U _S G _E AGCACUUUG 9 RG-NG-1024 A _S G _S C _M A _F C _F U _F U _M T _E G _E AGCACUUTG 9 RG-NG-1025 A _S G _S C _M A _F C _F U _F U _M U _S G _E AGCACUUUG 9 RG-NG-1026 A _S G _S C _M A _L C _F U _F U _M U _S C _S AGCACUUUC 9 RG-NG-1027 A _S G _S C _S A _F C _F U _F U _M U _S C _S AGCACUUUC 9 RG-NG-1028 A _S G _S C _S A _L C _F U _F U _M U _S C _S AGCACUUUC 9 RG-NG-1029 A _M G _L C _L A _L C _L U _M T _L U C _M AGCACUTUC 9 RG-NG-1030 A _M A _M G _L C _L A _L C _L U _M T _L U C _M AAGCACUTUC 19 10 RG-NG-1031 A _M A _M G _L C _L A _L C _L U _M T _L U _M AAGCACUTU- 9 The nucleoside followed by the subscript "M" is 2'-O-methyl nucleoside; the nucleoside followed by the subscript "F" is 2'-fluoro nucleoside; the nucleoside followed by the subscript "S" is S-cEt nucleoside; the nucleoside followed by the subscript "E" is 2'-O-methoxyethyl (2'-MOE) nucleoside; and the nucleoside followed by the subscript "L" is LNA nucleoside.

The activity of antimiR-17 compounds was evaluated in a radioligand binding assay, which measured the binding of the [ ^3H ]AMPA ligand to AMPA-R on the synaptic membrane of rat brain in the presence of increased concentrations of the antimiR-17 compound. The antimiR-17 compound with affinity for AMPA-R will bind and competitively bind to the [ ^3H ]AMPA ligand.

Analysis 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). AntimiR compounds at concentrations of 5.0 nM ligand [ ^3H ]AMPA, 1.0 mM nonspecific ligand L-glutamic acid, and μM were incubated with synaptic membranes prepared from the cerebral cortex of Wistar rats for 90 minutes. The compounds shown in Table 2 were tested in three experiments. AntimiR compounds targeting microRNAs other than miR-17 were used as control compounds (RG5124 targeting miR-33a; RG5365 targeting let-7a; RG8093 targeting miR-214). RGLS4326 and RG-NG-1001 were also tested in various experiments, demonstrating their ability to bind AMPA-R and inhibit AMPA-R activity. The amount of [ ^3H ]AMPA ligands was quantified by radioligand binding and is shown in Tables 3, 4 and 5. As the data indicate, the compounds exhibit different abilities to inhibit the binding of radiolabeled ligands to AMPA-R.

Table 3: Inhibition of ligand-AMPA-R binding experiment No. 1 SEQ ID NO Inhibition under uM % Compound number nucleobase sequence and chemistry (5 ' Up to 3 ') 100 10 1 0.1 IC ₅₀ (uM) RG-NG-1001 A _S G _S C _S A _S C _S U _S U _S U _S G _S 91.3 89.1 76.2 41.1 0.17 RGLS4326 A _S G _S C _M A _F C _F U _F U _M U _S G _S 104.1 93 55.5 17.6 0.70 RG-NG-1002 A _S G _S C _S A _S C _S U _S U _S U _S 24.0 -16.9 -2.0 0 >100 RG-NG-1003 A _S G _S C _M A _S C _M U _S U _M U _S G _S 77.8 56.9 13.9 5.7 9.26 RG-NG-1004 20 U _S A _S A _S G _{S C} S A _S C _S U _S U _S U _S G _S 76.1 44.2 4.7 -4.7 17.28 RG-NG-1005 twenty one U _S A _S A _M G C _S A _S C _S U _M U _M U _S G _S 28.3 0.8 -1.3 10.8 >100 RG-NG-1006 A _L G _L C _L A _L C _L T _L T _L T _{L G L} 93.9 68.9 35.3 6.7 2.77 RG5124 A _S C _S A _M A _F U _F G _F C _M A _S C _S (anti-miR-33a) -5.1 -3.7 12.9 0.4 >100 RG5365 Anti-let7a -4 6.5 -6.3 2.4 >100 RG8093 Anti-miR-214 -9.8 -2 -11.5 -6.9 >100

Table 4: Inhibition of ligand-AMPA-R binding - Experiment No. 2 SEQ ID NO Inhibition under uM % Compound number Chemical symbols 100 10 1 0.1 IC ₅₀ (uM) RG-NG-1001 A _S G _S C _S A _S C _S U _S U _S U _S G _S 93.5 90.1 68.3 42.2 0.19 RGLS4326 A _S G _S C _M A _F C _F U _F U _M U _S G _S 96.8 93.6 73.1 31.5 0.27 RG-NG-1002 A _S G _S C _S A _S C _S U _S U _S U _S 24.6 -2.1 4.6 -5.5 >100 RG-NG-1007 A _S G _S C _F A _F C _F U _F U _F U _S G _S 94.3 83.2 39.7 3.1 1.69 RG-NG-1008 A _S G _E C _M A _F C _F U _F U _M T _E G _S 59.6 22.1 13.6 -22.4 56.58 RG-NG-1009 A _E G _E C _M A _F C _F U _F U _M T _E G _E 32.6 13 2.7 -11.8 >100 RG-NG-1010 A _E G _E C _M A _F C _F U _F U _M U _S G _S 86.4 70.1 31.3 0.7 3.44 RG-NG-1011 A _L G _L C _M A _F C _F U _F U _M U _S G _S 94 78.5 30.6 -2.2 2.56 RG-NG-1012 A _S G _L C _M A _F C _F U _F U _M T _L G _S 88.3 69 38.5 9.9 2.56 RG-NG-1013 A _S A _S G _S C _M A _F C _F U _F U _M U _S 37.2 3.7 -5.6 -3.8 >100 RG-NG-1014 A _S G _S C _M A _F C _F U _F U _M U _S 20.4 8.6 -5.5 14.9 >100 RG-NG-1015 A _S G _S C _M A _F C _F U _F U _M U _S A _S 19.9 6.5 11 2.3 >100 RG-NG-1016 A _S G _S C _M A _F C _F U _F U _M U _S C _S 5.9 12.3 10.4 4.7 >100 RG-NG-1017 A _S G _S C _M A _F C _F U _F U _M U _S U _S 8.1 4.3 -1.3 0.2 >100 RG-NG-1026 A _S G _S C _M A _L C _F U _F U _M U _S C _S 2.3 -3.5 -1.1 -3.3 >100 RG-NG-1027 A _S G _S C _S A _F C _F U _F U _M U _S C _S 3 -2.2 2.3 -2.2 >100 RG-NG-1028 A _S G _S C _S A _L C _F U _F U _M U _S C _S 2.7 2 2.1 15.7 >100 RG-NG-1029 A _M G _L C _L A _L C _L U _M T _L U C _M 38.4 7 15.9 0.9 >100 RG-NG-1030 19 A _M A _M G _L C _L A _L C _L U _M T _L U C _M 1.9 15 6.5 2.9 >100 RG-NG-1031 A _M A _M G _L C _L A _L C _L U _M T _L U _M 16.7 2.7 1 -4.3 >100 RG5124 A _S C _S A _M A _F U _F G _F C _M A _S C _S (anti-miR-33a) 7.2 -4.2 1.6 -1.2 >100

Table 5: Inhibition of AMPA-R binding by ligands (Experiment No. 3) SEQ ID NO Inhibition under uM % Compound number Chemical symbols 100 10 1 0.1 IC ₅₀ (uM) RG-NG-1001 A _S G _S C _S A _S C _S U _S U _S U _S G _S 96.6 94.6 84.8 59.4 0.10 RGLS4326 A _S G _S C _M A _F C _F U _F U _M U _S G _S 97.2 89 55.8 14.6 0.78 RG-NG-1018 twenty two A _E C _E T _E G _S T _E AA _S G _S C _M A _F C _F U _F U _{M U S} C _S 12.3 1 8.1 3 >100 RG-NG-1019 twenty three A _S C _S TG _S U _S AA _S G _S C _M A _F C _F U _F U _{M U S} C _S twenty one 15.6 17.2 12.9 >100 RG-NG-1020 twenty four T _E G _S T _E AA _S G _S C _M A _F C _F U _{F U M U S} C _S -7.9 -23.3 6.6 17 >100 RG-NG-1021 25 T _E AA _S G _S C _M A _F C _F U _F U _M U _S C _S -1.5 -11.5 -10.9 -2 >100 RG-NG-1022 26 T _E A _E A _S G _S C _M A _F C _F U _F U _{M U S} C _S -10.9 -7 -5.4 -23.5 >100 RG-NG-1023 A _E G _S C _M A _F C _F U _F U _M U _S G _E 95.3 75.2 25.6 3.1 3.12 RG-NG-1024 A _S G _S C _M A _F C _F U _F U _M T _E G _E 35.2 6.5 -4.7 -17 >100 RG-NG-1025 A _S G _S C _M A _F C _F U _F U _M U _S G _E 96.4 84.1 36.5 -3.8 1.85 RG5124 A _S C _S A _M A _F U _F G _F C _M A _S C _S (anti-miR-33a) twenty three 16.1 9.3 5.1 >100

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

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

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

Table 6: Functional antagonistic effect of AMPA-R in whole-cell membrane patch studies Compound ID Sequence and Chemistry (5'-3') Target Membrane current induced by 10 μM (s)-AMPA of inhibition% (Average ± SD) 0.3 uM 3 uM RG-NG-1001 A _S G _S C _S A _S C _S U _S U _S U _S G _S miR-17 80.5 ± 6.3 88.8 ± 4.5 RGLS4326 A _S G _S C _M A _F C _F U _F U _M U _S G _S miR-17 46.9 ± 9.8 87.4 ± 7.9 RG-NG-1015 A _S G _S C _M A _F C _F U _F U _M U _S A _S miR-17 16.0 ± 5.0 39.5 ± 5.4 RG-NG-1016 A _S G _S C _M A _F C _F U _F U _M U _S C _S miR-17 16.0 ± 6.2 27.1 ± 9.8 RG-NG-1017 A _S G _S C _M A _F C _F U _F U _M U _S U _S miR-17 20.0 ± 6.3 28.2 ± 5.5 RG5124 A _S C _S A _M A _F U _F G _F C _M A _S C _S miR-33a 14.1 ± 7.2 42.1 ± 9.4

Example 3: Relationship between nucleobase properties and AMPA-R binding. As demonstrated by AMPA-R binding and whole-cell membrane patch studies, the presence of guanosine at the 3' end of the anti-miR-17 oligonucleotide, where it complements the first nucleotide of miR-17, affects the functional antagonism of AMPA-R. Like guanosine, adenosine is a purine; however, adenosine does not inhibit AMPA-R. Guanosine and adenosine are similar in several properties except for hydrogen bonding; therefore, the differences in hydrogen bonding at positions 1, 2, and 6 of the purine bases were evaluated. The tested purine nucleobases are shown in Figure 1 and Table 7. In the "Purine Positions" row of Table 7, "A" indicates the position of the purine acting as a hydrogen acceptor, and "D" indicates the position of the purine acting as a hydrogen donor. In the "Purine Position" row of Table 7, "N" indicates a neutral position that is neither a hydrogen acceptor nor a hydrogen donor. Different 2'-sugar moieties on the purine nucleotide base were also tested to evaluate the effect of 2'-sugar moieties on the ability of the purine nucleotide base to inhibit AMPA-R.

Table 7: AntimiR-17 compounds with different nucleobase and sugar moieties Compound ID 3 terminal nucleobase ( Single-letter notation) 3' terminal 2'-sugar portion SEQ ID NO Sequence and Chemistry (5'-3') Purine position #6 #1 #2 RG-NG-1001 Guinea (G) S-cEt A _S G _S C _S A _S C _S U _S U _S U _S G _S A D D RG-NG-1040 Guinea (G) S-cEt 27 C _E T _E G _S C _{E A E} C _E T _E G _S T _E A _D A _S G _{S C} M A _F C _F U _F U _M U _S G _S A D D RG-NG-1032 Guinea (G) S-cEt 28 G _S C _E A _E C _{E T E} G _S T _E A _D A _S G _S C _{M A F} C _F U _F U _M U _S G _S A D D RG4326 Guinea (G) S-cEt A _S G _S C _M A _F C _F U _F U _M U _S G _S A D D RG-NG-1033 Guinea (G) DNA A _S G _S C _M A _F C _F U _F U _M U _S G _D A D D RG-NG-1034 Guinea (G) 2'-O-methyl A _S G _S C _M A _F C _F U _F U _M U _S G _M A D D RG-NG-1035 Inosine (I) S-cEt A _S G _S C _M A _F C _F U _F U _M U _S I _M A D N RG-NG-1036 2-Aminopurine (N) 2'-O-methyl A _S G _S C _M A _F C _F U _F U _M U _S N _M N A D RG-NG-1037 2,6-Diaminopurine (D) 2'-O-methyl A _S G _S C _M A _F C _F U _F U _M U _S D _M D A D RG-NG-1039 Heteropurin (F) DNA A _S G _S C _M A _F C _F U _F U _M U _S F _D D D A RG-NG-1038 Adenosine (A) 2'-O-methyl A _S G _S C _M A _F C _F U _F U _M U _S A _M D A N RG-NG-1015 Adenosine (A) S-cEt A _S G _S C _M A _F C _F U _F U _M U _S A _S D A N RG5124 Cytidine (C) S-cEt A _S C _S A _M A _F U _F G _F C _M A _S C _S

Compounds were tested in the radioligand binding assays described herein to determine the ability of anti-miR-17 compounds to competitively bind to the [ ^3H ]AMPA ligand. As shown in Table 8, a correlation was observed between inhibition of ligand binding to +AMPA-R and the presence of a hydrogen-bonded acceptor at the purine position #6 of the 3' end nucleobase of the oligonucleotide. For example, compounds with guanosine or inosine at the 3' end inhibited ligand binding to AMPA-R. Compounds with a 3' end nucleobase containing a hydrogen-bonded acceptor at the purine position #6 (e.g., RG-NG-1037 and RG-NG-1039) were unlikely to inhibit ligand binding to AMPA-R.

Table 8: Inhibition of ligand-AMPA-R binding ID 3-terminal nucleobase 3' terminal 2'-sugar portion purine Location the following Inhibition at concentration % IC50 (uM) #6 #1 #2 100 uM 10 uM 1 uM 0.1 uM RG-NG-1001 guanosine S-cEt A D D 102.9 99.3 81.3 41.9 0.15 RG-NG-1040 guanosine S-cEt A D D 64.1 38.9 14.4 10.4 28.61 RG-NG-1032 guanosine S-cEt A D D 92 78.3 43.8 24.3 1.17 RG-NG-4326 guanosine S-cEt A D D 99.7 83.5 40.8 -0.1 1.61 RG-NG-1033 guanosine DNA A D D 97.3 96.5 80.9 44.9 0.13 RG-NG-1034 guanosine 2'-O-methyl A D D 99.7 85.2 44.8 14.3 1.20 RG-NG-1035 Inosine S-cEt A D N 98.5 91.3 46.3 27.2 0.76 RG-NG-1036 2-Aminopurine 2'-O-methyl N A D 78.1 36.2 11.3 19.2 18.61 RG-NG-1037 2,6-Diaminopurine 2'-O-methyl D A D 10.7 2.4 9.2 11.1 100.00 RG-NG-1039 purine DNA D D A 30.2 35.8 27 27.3 100.00 RG-NG-1038 adenosine 2'-O-methyl D A N 81.3 47.4 25.4 17.9 8.23 RG-NG-1015 adenosine S-cEt D A N 5.6 -7 -10.3 -0.8 100.00 RG5124 cytidine S-cEt 10.1 4.4 -0.6 -8.6 100.00

Example 4: Anti-miR-17 compounds that reduce AMPA-R binding and inhibit AMPA-R did not show 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 at a single dose of 2000 mg/kg and at incremental doses (100, 450, and 2000 mg/kg). As shown in Table 9, although incremental doses of RG-NG-1001 and RGLS4326 caused ataxia and somnolence, and in the case of RGLS4326, unconsciousness was observed at the highest dose, no CNS toxicity was observed in RG-NG-1015, RG-NG-1016, or RG-NG-1017.

Table 9: Anti-miR-17 compounds and related findings in CNS Mice/group SC dosage (mg/kg/dose) schedule Note the relevant findings in CNS: RG-NG-1001 8 100, 450, 2000 QD×3 Mild body scratching and ataxia were observed at 100 mg/kg, increasing at 450 mg/kg; somnolence was observed at 2000 mg/kg. RGLS4326 10 100, 450, 2000 QD×4 Ataxia and/or somnolence were observed at 450 mg/kg; ataxia, somnolence, and/or unconsciousness were observed at 2000 mg/kg. RG-NG-1015 7 2000 Single no 6 100, 450, 2000 QD×4 no RG-NG-1016 10 2000 Single no 6 100, 450, 2000 QD×4 no RG-NG-1017 10 2000 Single no 6 100, 450, 2000 QD×4 no RG5124 7-10 100, 450, 2000 QD×3 Mild ataxia was observed at 2000 mg/kg.

Example 5: Maximum tolerated dose (MTD) studies and comparative dose evaluations of different compounds. Data from the following studies further support the claim that AMPA-R antagonism is the cause of CNS toxicity and mortality observed in previous RGLS4326 toxicity studies.

Study 1: Maximum Tolerated Dose (MTD) Study and Comparative Dose Evaluation of RG-NG-1017, RGLS4326, and RG-NG-1001 Compounds (RG-NG-1017, RGLS4326, and RG-NG-1001) were evaluated in a pilot maximum tolerated dose (MTD) study (discussed below). RG-NG-1017, RGLS4326, and RG-NG-1001 were initially evaluated at four dose levels. RG-NG-1017 was included as a non-AMPA-R bound compound for evaluation and compared with AMPA-R bound RGLS4326 and RG-NG-1001. Six- to seven-week-old male C57Bl/6J mice (Jackson Laboratories) were used in this study. Mice were randomly assigned to the treatment group, and the study was blinded. Animals were acclimatized for at least 5 days and kept in captivity during a 12-hour light/dark cycle (lights were turned on at 7:00 AM). No more than 4 mice were housed in each cage in a ventilated cage system. The diet consisted of standard rodent food and free access to water.

The following parameters were used in this MTD pilot study: 1. Administration route: Intraventricular (ICV) administration of RG-NG-1017, RG-NG-1001, and RGLS4326; 2. Dosage volume: 4 μL; 3. Preparation: Mediator, ^Ca²⁺ and ^Mg²⁺ -free dPBS; 4. Dosage frequency: Once; 5. Study duration: 8 days; 6. Number of groups: 3; 7. Number of animals per group: (2-4 animals per group); 8. Total number of animals: 54

For ICV administration, mice were anesthetized and positioned for injection. The skin above the skull was incised, and a small hole was created in the skull above the target using a microdrill. The stereotactic coordinates were: anterior-posterior (AP) -0.4 mm; medial-lateral (ML) +/- 1.0–1.5 mm; dorsoventral (DV) -3.0 mm from the anterior fontanelle, for injection into the right and left ventricles (Hironaka et al., 2015). Four μl was injected unilaterally into the right ventricle. The compound was injected over 1–2 minutes, and the needle was left in place for 0.5–1 minute before withdrawal. The incision was closed with sutures, wound clips, or VetBond.

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

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 injection. Mice injected with 2.5 μg RGLS4326 showed some direct signs of respiratory distress, even after being provided with a heating pad. RG-NG-1017 (a non-AMPA-R conjugate) was well tolerated at high doses, and no MTD was established for this compound (0 deaths at 600 μg, 100 μg, or 50 μg; 1 death at 300 μg). In addition to the two AMPA-R conjugates showing 100% mortality at 50 μg and 25 μg, RGLS4326 and RG-NG-1001 also showed 100% mortality at high doses (e.g., 600, 300, 100 μg). The MTD of RG-NG-1001 was not obtained in this study and was predicted to be less than 2.5 μg. The MTD of RGLS4326 was predicted to be approximately 2.5 < 5.0 μg using ICV. All animals reportedly fully recovered by day 2 of observation.

Table 10: Summary Results of the 7-Day MTD Study Mortality rate / Total number of mice Dosage (μg) RG-NG-1017 RGLS4326 RG-NG-1001 600 0/2 2/2 2/2 300 1/2 2/2 2/2 100 0/2 2/2 2/2 50 0/2 3/3 3/3 25 3/3 3/3 10 3/4 3/4 5 2/4 4/4 2.5 0/3 1/3

The maximum tolerated dose (MTD) study of RGLS4326 was conducted as a second MTD study of RGLS4326 by ICV to evaluate the dosage selection of the compound for evaluation in disease models (Table 11). In this study, different mouse strains (Swiss:Rjorl male mice, 5 weeks old, from Janvier) were evaluated. Mice were anesthetized with isoflurane (5% for induction and 2% for maintenance, at 100% _O2 ) and subcutaneously administered 5 mg/kg 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 (0.51 mm outer diameter) was stereotactically placed in the left ventricle at the following coordinates: +0.5 mm posterior to the anterior fontanelle, L ± 0.7 mm, V = -2.7 mm. After a 2-minute delay to allow brain tissue to slide along the cannula, 4 μL of a solution containing 0.625 mg/mL RGLS4326 was slowly infused over 2 minutes. Following infusion, the cannula was left in place for another 5 minutes to prevent backflow along the cannula's tracks. Mice were subcutaneously administered 5 mg/kg carbofenone (Rimadyl®) 24 and 48 hours post-surgery. Mice were monitored for health status daily for 3–7 days post-surgery (starting 24 hours after ICV administration). For mice monitored for 7 days, their weight was measured on day 1 and day 7 post-surgery to check their health status.

Table 11: Design of MTD Study for RGLS4326 Group animal numbers Processing (RG4326) Dosage level Concentration (mg/mL) Projection volume 1 4 males RGLS4326 (i.c.v.) 3 mg/mouse 0.75 mg/mL 4 mL/mouse 2 4 males RGLS4326 (i.c.v.) 4 mg/mouse 1 mg/mL 4 mL/mouse 3 4 males RGLS4326 (i.c.v.) 5 mg/mouse 1.25 mg/mL 4 mL/mouse 4 4 males RGLS4326 (i.c.v.) 7.5 mg/mouse 1.875 mg/mL 4 mL/mouse

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

The combined results of Studies 1 and 2 indicate that RGLS4326 is generally well tolerated in test individuals, but only at doses significantly lower than RG-NG-1017 (see Figure 3).

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

Table 12: 7-day survival data from MTD studies 1 and 2 Death/Total Mice Dosage (μg) RGLS4326 Study 1 Study 2 600 2/2 300 2/2 100 2/2 50 3/3 25 3/3 10 3/4 7.5 - 1/4 5 2/4 0/4 4 0/4 3 0/4 2.5 0/3 0/6

In summary, the two MTD studies evaluating compounds RG-NG-1017, RGLS4326, and RG-NG-1001 demonstrated a significant difference in tolerability between the non-AMPA-R bound compound (RG-NG-1017) and the AMPA-R bound compounds (RGLS4326, RG-NG-1001) (see Figure 3). Although one death occurred at a dose of 300 μg of ICV, the MTD of RG-NG-1017 was not established because no death occurred at the higher test dose of 600 μg. Furthermore, no effect on mortality was observed at doses of 100 μg and 50 μg for RG-NG-1017. In contrast, the AMPA-R binding compounds RGLS4326 and RG-NG-1001 had a significant effect on mortality, with no surviving animals in the test dose range of 25 μg to 600 μg. A trend towards improved survival was observed at lower doses of RGLS4326 (10 μg), with 50% survival at 5 μg and 100% at 2.5 μg. Similarly, with RG-NG-1001 (which showed stronger AMPA-R binding than RGLS4326), 100% mortality was evident at a low dose of 5 μg, with a trend towards improved survival at 2.5 μg. The results from Study 1 regarding RGLS43426 were further confirmed in a second MTD study using different mouse strains (Study 2). This study found that there may be slight differences in tolerance to RGLS4326 between strains. In Study 1 using C57/Bl/6J, survival was observed to be affected only at the maximum dose of 7.5 μg, compared to a control of 5 μg. However, these results still support the MTD of AMPA-R-bound RGLS4326 occurring between approximately 2.5 μg and 5–7.5 μg (depending on the strain) compared to the significantly higher MTD (at least >40-fold) of non-AMPA-R-bound RG-NG-1017 (Figure 3).

Example 6: In vitro and in vivo potency of anti-miR-17 compounds. The in vitro potency of certain compounds was evaluated using a miR-17 luciferase sensor assay. This assay employed a miR-17 luciferase reporter vector in which two perfectly complementary miR-17 binding sites are tandemly linked to the 3'-UTR of the luciferase gene. HeLa cells were co-transfected with the luciferase reporter vector and an exogenous miR-17 expression vector for repressing 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 inhibited miR-17 function and de-inhibited miR-17 luciferase reporter activity, with _EC50 values similar to those of in vitro RGLS4326.

Table 13: Inhibition of miR-17 in luciferase analysis Compound ID EC50 (nM) Log2FC (37.5 nM) RGLS4326 18.50 2.70 RG-NG-1032 < 1 3.85 RG-NG-1015 22.40 2.51 RG-NG-1016 20.30 2.32 RG-NG-1017 15.00 2.67 RG5124 n/a 0.40

As shown in Figure 4, in the luciferase analysis in HeLa cells, RG-NG-1015 inhibited miR-17 as well as miR-20a, miR-106a and miR-93, with similar _EC50 values compared to in vitro RGLS4326.

RG-NG-1015 also derepresses luciferase sensors containing the full-length 3' untranslated regions (UTRs) of miR-17 direct target genes PKD1 and PKD2, with similar _EC50 values to in vitro RGLS4326.

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

As shown in Table 13, the tested oligonucleotides inhibited miR-17 function and unblocked the expression of multiple direct miR-17 target genes in normal and PKD renal cell lines (mouse and human) (measured by miR-17 PD-characterization), with _EC50 values similar to those of RGLS4326 in vivo. No PD-Sig (77.2, indicated by "*") of RGLS4326 was generated in mIMCD3 cells in this experiment; the values in Table 14 are reported by Lee et al., Nat. Commun ., 2019, 10, 4148. Blank cells in the tables indicate compounds that were not tested in the specific cell lines.

Table 14: miR-17 PD-Sig in normal and PKD cell lines cell line species Origin Anti-miR RGLS4326 RG-NG-1015 RG-NG-1016 RG-NG-1017 RG5124 mIMCD3 mice normal 77.2* 168 146.3 134.5 n/a D52B5 mice PKD 92.8 82.4 74.1 506.6 HK-2 human normal 47.8 55.6 49.2 WT9-7 human PKD 17.3 21.2 20.1

In vivo potency was assessed using microRNA polysome transfer assay (miPSA). This assay was used to determine the extent to which compounds directly bind to the miR-17 target in the kidney in normal and PKD mice. miPSA relies on the principle that active miRNAs bind to their mRNA targets in high molecular weight (HMW) polysomes, while repressed miRNAs are present in low molecular weight (LMW) polysomes. Treatment with antimiRs causes microRNAs to transfer from HMW polysomes to LMW polysomes. Therefore, miPSA directly measures the binding of complementary antimiRs to microRNA targets (Androsavich et al., Nucleic Acids Research , 2015, 44: e13).

Wild-type mice were administered a single dose of 0.3 mg/kg, 3 mg/kg, or 30 mg/kg. Kidney tissue was collected seven days later for miPSA analysis. Mean shift scores for each treatment are shown in Table 15 (PBS, n = 17; RGLS4326 30 mg/kg, n = 10; all other treatments, n = 4–5). In normal mouse kidneys, the tested oligonucleotides shifted miR-17 autotransformationally active polyribosomes (measured by miPSA).

Table 15: miPSA Displacement Assessment Anti-miR dosage 0.3 m/kg 3 mg/kg 30 mg/kg RG-NG-1015 Average 2.238 2.562 2.718 SEM 0.1242 0.1303 0.118 RG-NG-1016 Average 1.892 2.263 1.134 SEM 0.1088 0.1716 0.5324 RG-NG-1017 Average 2.077 2.292 2.793 SEM 0.1513 0.08974 0.09685 RGLS4326 Average 1.846 2.247 2.362 SEM 0.1234 0.0478 0.1439 Pharmacy Propagant (PBS) Average 0 SEM 0.06318 RG5124 Average 0.5935 SEM 0.1784

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

Table 16: Overview of Pharmacokinetics and Target Binding organization Test items Dosage T _max C _max AUC _last T _{1/2 Finally} , the ratio of kidney to liver is determined by AUC. plasma RGLS4326 26.0 mg/kg 1 h 32 mg/mL 95.3 (mg*h)/mL 2.9 h - RG-NG-1015 26.9 mg/kg 1 h 26 mg/mL 86.9 (mg*h)/mL 2.6 h - kidney RGLS4326 26.0 mg/kg 8 h 54 mg/g 395 (mg*d)/g 9.4 d 23.9 RG-NG-1015 26.9 mg/kg 8 h 53 mg/g 447 (mg*d)/g 9.9 d 22.6 liver RGLS4326 26.0 mg/kg 1 h 4.8 mg/g 16.5 (mg*d)/g 4.7 d - RG-NG-1015 26.9 mg/kg 1 h 5.2 mg/g 19.8 (mg*d)/g 6.2 d -

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. The Pkd1 -F/RC lineage is an orthologous ADPKD model containing a germline attenuated Pkd1 mutation (the mouse equivalent of human PKD1-R3277C (RC mutation)) in one pair of genes and loxP sites laterally attached to exons 2 and 4 of Pkd1 in the other pair of genes. KspCre-mediated recombination resulted in the deletion of the flux Pkd1 exon, producing a complex mutant mouse with a renal tubule-specific somatic ineffective mutation in one pair of genes and a germline attenuated mutation in the other pair of genes. This is a model of the aggressive but long-term nature of ADPKD (Hajarnis et al., Nat. Commun ., 2017, 8, 14395).

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

The results are shown in Table 17 and Figure 2 (****=p<0.0001;***=p<0.001;**=p<0.01; ns=not significant). The efficacy of RG-NG-1015 was similar to that of RGLS4326. The mean ratio of kidney weight to body weight (KW/BW ratio) in Pkd1 -F/RC mice treated with RGLS4326 and RG-NG-1015 was significantly lower than that in Pkd1 -F/RC mice administered PBS (Figure 2A). Compared with mice treated with PBS, the mean BUN levels in Pkd1 -F/RC mice treated with RGLS4326 and RG-NG-1015 were significantly reduced (Figure 2B). In Pkd1 -F/RC mice, the mean serum creatinine levels were reduced in mice treated with RGLS4326 and RG-NG-1015, respectively, compared to mice treated with PBS; however, this 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 due to miR-17-specific inhibition.

Table 17: Efficacy of RG-NG-1015 in ADPKD mouse model PBS RGLS4326 RG-NG-1015 RG5124 average BW/KW ratio 65.61 16.81 16.83 91.75 Average difference from PBS -48.8 -48.79 26.14 Adjusted P-value <0.0001 <0.0001 0.0045 average Serum BUN 42.48 22.41 22.26 50.61 Average difference from PBS -20.06 -20.21 +8.138 Adjusted P-value <0.0001 <0.0001 0.1142 average Serum creatine 0.1619 0.1273 0.1279 0.2403 Average difference from PBS -0.03463 -0.03400 +0.07838 Adjusted P-value 0.1456 0.1557 0.0004

The efficacy of RG-NG-1015, alone and in combination with tolvaptan, was also evaluated in a Pcy /DBA mouse PKD model. Pcy/DBA mice exhibit slowly progressive PKD caused by missense mutations in the Nphp3 gene, which lead to juvenile nephropathy 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 in the distal tubules, and by 30 weeks of age, with disease progression, the entire renal unit is diffusely occupied by cysts, often accompanied by ESRD (Nagao et al., Exp Anim 2012, 61:477-488). Specifically, male Pcy /DBA mice have been used to characterize the pharmacological features of many investigational products used to treat ADPKD, including tolvaptan and RGLS4326, first-generation anti-miR-17 (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 treatment starting at approximately 5 weeks of age and continuing until 15-30 weeks of age.

As illustrated in Figures 6A and 6B, five groups of male Pcy/DBA mice (n=13/treatment group) were subcutaneously treated with RG-NG-1015 every two weeks using PBS or at doses of 25 mg/kg, 5 mg/kg, 1 mg/kg, or 0.2 mg/kg (Q2W). Two groups of male Pcy/DBA mice (n=13/group) were also treated with RG-NG-1015 at doses of 50 mg/kg every four weeks (Q4W) or 12.5 mg/kg every week (QW). Four additional groups of male Pcy/DBA mice (n=13/group) were subcutaneously treated with RG-NG-1015 in combination with PBS or at doses of 25 mg/kg, 5 mg/kg, or 1 mg/kg (Q2W) and 0.3% (w/w food) tolvaptan (unfeedable). Male WT-BDA/2J mice that received subcutaneous PBS Q2W were included in the study as a normal range reference. Mice were randomly assigned to the treatment group at 5 weeks of age, and treatment began at 6 weeks of age and lasted for 17 weeks, ending 7 days after final treatment. Kidney weight, body weight, renal cyst index, and urinary Ngal/Cr ratio were measured. Urinary Ngal/Cr is a marker of kidney damage.

As shown in Figures 6C-6E and Tables 18-20, RG-NG-1015 was effective in the Pcy/DBA mouse PKD model at various doses and regimens, and also provided additive or synergistic effects when used in combination with tolvaptan. Specifically, 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). Furthermore, RG-NG-1015 treatment with similar total doses (212.5–250 mg per mouse over the study duration) but different administration regimens (including QW, Q2W, and Q4W) reduced mean KW/BW, urinary Ngal/Cr, and renal cyst index in Pcy/DBA mice at similar levels (Table 19; Figures 6C–6E). Tolvaptan alone reduced mean KW/BW, urinary Ngal/Cr, and renal cyst index in Pcy/DBA mice, and the combination of RG-NG-1015 and tolvaptan further reduced mean KW/BW, urinary Ngal/Cr, and renal cyst index (Table 20; Figures 6C–6E). As indicated by Bliss additivity analysis, the observed effects of drug combinations on KW/BW, urinary Ngal/Cr, and renal cyst index were synergistic, primarily additive, and less additive, respectively (Table 20).

Table 18: Effects of Dosage Test Item 1 SC Medication Test item 2: Supplemental food Dosage plan Average KW/BW Average urinary Ngal/Cr Average cyst area mg/kg Inhibit % mg/mg Inhibit % % Inhibit % Mediator - - Q2W 91.3 0 21.07 0 60.62 0 RG-NG-1015 - 25 mg/kg Q2W 66.87 34.3 11.36 48.8 46.46 26.0 RG-NG-1015 - 5 mg/kg Q2W 70.02 29.9 14.65 32.3 44.58 29.4 RG-NG-1015 - 1 mg/kg Q2W 82.43 12.5 15.18 29.6 50.58 18.4 RG-NG-1015 - 0.2 mg/kg Q2W 78.02 18.7 18.38 13.5 47.09 24.8

Table 19: Effects of the Plan Test Item 1 SC Medication Test item 2: Supplemental food Dosage plan Average KW/BW Average urinary Ngal/Cr Average cyst area Dosage number Total dose (mg) mg/kg Inhibit % mg/mg Inhibit % % Inhibit % Mediator - - Q2W 91.3 0 21.07 0 60.62 0 17 RG-NG-1015 - 25 mg/kg Q2W 66.87 34.3 11.36 48.8 46.46 26.0 9 225 RG-NG-1015 - 50 mg/kg Q4W 67.09 34.0 9.32 59.1 47.35 24.3 5 250 RG-NG-1015 - 12.5 mg/kg QW 74.52 23.6 8.82 61.6 44.99 28.7 17 212.5

Table 20: Effects of Combinations Test Item 1 SC Medication Test item 2: Supplemental food Dosage plan Average KW/BW Average urinary Ngal/Cr Average cyst area mg/kg Inhibit % Briss Analysis mg/mg Inhibit % Briss Analysis % Inhibit % Briss Analysis Mediator - - Q2W 91.3 0 - 21.07 0 - 60.62 0 - Mediator 0.3% Tolvaptan - - 59.26 44.9 - 8.73 62.1 - 44.68 29.2 - RG-NG-1015 - 25 mg/kg Q2W 66.87 34.3 - 11.36 48.8 - 46.46 26.0 - RG-NG-1015 0.3% Tolvaptan 25 mg/kg Q2W 42.21 68.9 Collaboration 5.04 80.6 Accumulation 38.61 40.4 <Accumulation RG-NG-1015 - 5 mg/kg Q2W 70.02 29.9 - 14.65 32.3 - 44.58 29.4 - RG-NG-1015 0.3% Tolvaptan 5 mg/kg Q2W 45.62 64.1 Collaboration 5.84 76.6 Accumulation 37.47 42.5 <Accumulation RG-NG-1015 - 1 mg/kg Q2W 82.43 12.5 - 15.18 29.6 - 50.58 18.4 - RG-NG-1015 0.3% Tolvaptan 1 mg/kg Q2W 47.11 62.0 Collaboration 11.16 49.8 <Accumulation 41.91 34.3 <Accumulation

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

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

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

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

Table 21: Sequences, precise mass, m/z, and charge state of RG-NG-1015 and its potential metabolites Compound Name SEQ ID NO Compound sequence (5'->3')* Precise quality of neutral molecules Mass-to-charge ratio (m/z) State of charge Comments RG-NG-1015 A _S G _S C _M A _F C _F U _F U _M U _S A _S 3064.31 1531.15 -2 parent molecule 3'N-1 A _S G _S C _M A _F C _F U _F U _M U _S 2693.26 1345.63 -2 Metabolites 3'N-2 A _S G _S C _M A _F C _F U _F U _M 2345.24 1171.62 -2 Metabolites 3'N-3 A _S G _S C _M A _F C _F U _F 2009.22 1003.61 -2 Metabolites 3'N-4 A _S G _S C _M A _F C _F 1685.23 841.61 -2 Metabolites 3'N-5 A _S G _S C _M A _F 1362.21 680.11 -2 Metabolites 3'N-6 A _S G _S C _M 1015.19 506.59 -2 Not observed 3'N-7 A _S G _S 680.15 339.08 -2 Not observed 3'N-8 A _S 293.11 145.56 -2 Not observed 5'N-1 G _S C _M A _F C _F U _F U _M U _S A _S 2693.26 1345.63 -2 Metabolites 5'N-2 C _M A _F C _F U _F U _M U _S A _S 2306.22 1152.11 -2 Metabolites 5'N-3 A _F C _F U _F U _M U _S A _S 1971.19 984.59 -2 Metabolites 5'N-4 C _F U _F U _M U _S A _S 1624.16 811.08 -2 Metabolites 5'N-5 U _F U _M U _S A _S 1301.15 649.57 -2 Not observed 5'N-6 U _M U _S A _S 977.15 487.57 -2 Not observed 5'N-7 U _S A _S 641.13 319.57 -2 Not observed 5'N-8 A _S 293.11 145.56 -2 Not observed 5'N+1 29 A _S A _S G _S C _M A _F C _F U _F U _M U _S A _S 3435.35 1717.17 -2 Synthetic impurities

Example 9: A clinical study evaluating the safety, tolerability, pharmacodynamics, and pharmacokinetics of RG-NG-1015 (RGLS8429) in patients with in vivo chromosomal dominant polycystic kidney disease. A. Study Design Overview This study consists of Part A and Part B. The drug names "RG-NG-1015" and "RGLS8429" are used interchangeably in this document.

Part A is a phase Ib, double-blind, placebo-controlled, multiple escalation-dose (MAD) study in which approximately 36 individuals diagnosed with Mayo Cognitive Classification (MIC) 1C, 1D, or 1E chromosomal dominant polycystic kidney disease (ADPKD) (based on MRI obtained during the screening period, or a previous MRI obtained within 5 years of screening using the recorded Mayo Cognitive Classification) were administered RG-NG-1015 (RGLS8429) or a placebo via subcutaneous (SC) injection. Informed consent (ICF) was required from individuals, and assessments were conducted for inclusion/exclusion criteria during the screening period.

Individuals meeting all inclusion/exclusion criteria were randomly assigned in a 3:1 pool to receive RG-NG-1015 (RGLS8429) or placebo via subcutaneous (SC) injection every two weeks (Q2W) for seven doses: Team 1 (approximately 12 individuals): 1 mg/kg RG-NG-1015 (RGLS8429) or placebo; Team 2 (approximately 12 individuals): 2 mg/kg RG-NG-1015 (RGLS8429) or placebo; Team 3 (approximately 12 individuals): 3 mg/kg RG-NG-1015 (RGLS8429) or placebo.

Part B is an open-label, fixed-dose study. Part B consists of one cohort of up to 30 individuals who met all inclusion/exclusion criteria and received RG-NG-1015 (RGLS8429) via SC injection at Q2W×7 doses: Cohort 4 (up to 30 individuals): 300 mg RG-NG-1015 (RGLS8429).

The investigational drug is administered via SC injection at the clinic by the investigator or another qualified and trained field staff member, with the field staff monitoring safety for at least 4 hours.

Individual participation in the study lasted 141 days. The study consisted of a maximum 28-day screening period (day -28 to day -1), followed by an 85-day treatment period (day 1 to day 86), and a 28-day follow-up period (day 92 to day 113). During the treatment period, clinic visits were conducted on days 1 and/or 2 (day 1, dose 1), day 15 (dose 2), day 29 (dose 3), day 43 (dose 4), day 57 (dose 5), day 71 (dose 6), and day 85 and/or day 86 (day 85, dose 7). During the follow-up period, clinic visits were conducted on days 92, 99, and 113 (the end of the study visits).

B. Individual Population This study consisted of the following: Part A, three consecutive cohorts of approximately 12 individuals, each randomly assigned in a 3:1 ratio, to receive RG-NG-1015 (RGLS8429) or a placebo via SC injection at 1 mg/kg, 2 mg/kg, or 3 mg/kg every two weeks (Q2W) × 7 doses (totaling 36 individuals); and Part B, one cohort of up to 30 individuals, to receive a fixed dose of RG-NG-1015 (RGLS8429) via SC injection at 30 mg/kg every two weeks × 7 doses.

Individuals eligible for inclusion must meet all of the following inclusion criteria to participate in the study: 1) Be between 18 and 70 years of age (inclusive) at the time of signing the informed consent form; 2) Be diagnosed with ADPKD (according to the Mayo Cognitive Classification, 1C, 1D, or 1E, based on magnetic resonance imaging (MRI) obtained during the screening period, or a previous MRI obtained within 5 years of screening using the recorded Mayo Cognitive Classification); 3) Have an estimated glomerular filtration rate (eGFR) between 30 and 90 mL/min/1.73 ^M² ; 4) Have a body mass index (BMI) between 18 and 35 kg/ ^m² ; If an individual has hypertension, the antihypertensive regimen must be stable for at least 28 days prior to randomization (Part A) or Day 1 (Part B), and blood pressure must be adequately controlled prior to randomization (Part A) or Day 1 (Part B); 5) Screen for the following hematological and clinical chemistry: a) Platelet count within the normal range, b) Total bilirubin and direct bilirubin <1.5 × upper limit of normal (ULN), unless the bilirubin elevation is associated with a known benign condition (e.g., Gilbert's syndrome), c) Alanine aminotransferase (ALT) <1.5 × ULN, d) Aspartate aminotransferase (AST) <1.5 × ULN, e) Alkaline phosphatase (ALP) <1.5 × ULN, f) 6) Gamma-glutamyl transferase (GGT) <1.5×ULN. 7) Applicants must understand and agree to the study procedures as explained in the Informed Consent Form (ICF) and be willing and able to comply with the protocol. 8) Female individuals of reproductive potential must not be breastfeeding and must not plan to become pregnant during the study until 28 days after the last dose of the study drug. Heterosexual women of reproductive potential must agree to use one of the following methods of contraception considered highly effective (i.e., with a failure rate of <1% when used consistently and correctly) for 28 days from the date of selection to the last dose of the study drug: a) an intrauterine device (IUD) or intrauterine system (IUS) in proper position for at least 3 months prior to random assignment (Part A) or day 1 (Part B); b) the partner has undergone vasectomy. Vasectomy in the partner is considered highly effective only when the partner is the sole sexual partner of a woman of fertility potential, and the vasectomy must have been performed 6 months or longer prior to randomization (Part A) or Day 1 (Part B). c. Stable hormonal contraception associated with ovulation suppression (approved oral, percutaneous, or accumulatory regimens) must have been maintained for at least 3 months prior to randomization (Part A) or Day 1 (Part B). 8) A woman of infertility potential must have undergone one of the following sterilization procedures at least 6 months prior to the first dose of the study drug: a. Hysterectomy b. Bilateral oophorectomy c. Bilateral tubal obstruction d. 9) Bilateral salpingectomy or being postmenopausal and without menstruation for at least one year prior to the first dose of the study drug. Any heterosexual male individual who has not undergone vasectomy must agree to use condoms containing spermicide (no restrictions apply to heterosexual males who have undergone vasectomy, provided the vasectomy was performed 6 months or longer prior to the start of the study. Heterosexual males who underwent vasectomy within 6 months prior to the start of the study must follow the same restrictions as heterosexual males who have not undergone vasectomy). 10) From day 1 until 28 days after the last dose of the study drug, both male and female individuals must agree not to donate sperm or not to preserve eggs (ovaries). 11) You must agree not to donate blood 28 days before random allocation (Part A) or day 1 (Part B) until the end of the study visit (EOS), or not to donate plasma 7 days before random allocation (Part A) or day 1 (Part B) until the end of the study visit (EOS).

Exclusion Criteria Individuals meeting any of the following criteria will be excluded from the study: 1) Administered tolvaptan 28 days prior to random allocation (Part A) or day 1 (Part B); 2) Individuals are mentally incapacitated or have significant emotional problems; 3) Any medical condition or social environment that the investigator believes may make it unlikely that the individual will complete the study or comply with the study procedures and requirements, or may pose a risk to the individual's safety; 4) History of alcohol or drug abuse within 2 years prior to screening, or presence of alcohol or drug abuse; 5) Active infection of the urinary tract (e.g., kidneys, bladder, etc.); 6) Known infection with hepatitis B, hepatitis C, or human immunodeficiency virus (HIV); 7) Only one kidney or kidney transplant recipient; 8) 9) History of malignant disease, excluding squamous cell carcinoma or basal cell carcinoma of the skin that has been successfully treated; 10) History of clinically significant response to oligonucleotide compounds, as considered by the investigator; 11) Tattoos or scars at or near the SC injection site, or any other condition that the investigator believes may interfere with examination of the injection site; 12) Prior to administration, participation in another clinical trial and/or exposure to any investigational drug or approved therapy for investigational purposes within 28 days or 5 half-lives (whichever is longer) of administration of the investigational drug. The 28-day or 5-half-life window will be calculated from the date of the last administration in the previous study to Day 1 of the current study.

C. The drug RG-NG-1015 (RGLS8429) is provided in a 2 mL clear glass vial containing sufficient volume to dispense 1 mL labeled volume of 0.3% saline solution containing 150 mg/mL RG-NG-1015. The placebo injection is provided in a 2 mL clear glass vial containing sufficient volume to dispense 1 mL labeled volume of 0.9% sodium chloride containing 1.5 μg/mL riboflavin. Both RG-NG-1015 (RGLS8429) and the placebo solution are clear and colorless to pale yellow.

D. Administration: Following standard care procedures for the anterior abdominal wall, RG-NG-1015 (RGLS8429) and a placebo are administered via subcutaneous (SC) injection at that site in a large dose. The injection site is rotated to a different quadrant of the abdomen on each administration day. The study drug is administered by qualified and trained field personnel. In Part A, because the volume of the study drug varies significantly with dose levels, the following guidelines must be followed: the maximum volume of each injection must not exceed 2 mL (e.g., a 6 mL dose would require three 2 mL injections in the same quadrant of the abdomen). In Part B, the volume must not exceed 2 mL. Team 4 will receive a fixed dose of the active drug, and therefore the volume will not change.

E. Endpoint The main objectives and endpoints of this study are: Main objectives Main Terminal Evaluation of the safety and tolerability of RG-NG-1015 (RGLS8429) Incidence of adverse events (AEs), laboratory test results, vital signs, ataxia assessment and rating scale [SARA], and 12-lead electrocardiogram (ECG) over time. Evaluate the impact of RG-NG-1015 (RGLS8429) on ADPKD biomarkers Changes from baseline in polycystic protein-1 (PC1), polycystic protein-2 (PC2), neutrophil gelatinase-associated lipotransferase (NGAL), and renal injury molecule-1 (KIM-1) biomarkers.

The secondary objectives and endpoints of this study are: Secondary objectives Secondary endpoints Evaluate the effect of RG-NG-1015 on height-adjusted total renal volume (htTKV). htTKV from the base line change Characterizing the pharmacokinetic (PK) properties of RG-NG-1015 (RGLS8429) The pharmacokinetic parameters of RG-NG-1015 include: • Maximum observed concentration ( _Cmax ) • Time to reach maximum observed concentration ( _Tmax ) • Concentration-time under-curve up to 24 hours after administration (AUC _0-24 ) • Concentration-time under-curve up to the final quantifiable concentration (AUC _0-t ) • Concentration-time under-curve up to the dosing interval (AUC _τ ) • Concentration-time under-curve up to infinity (AUCinf) • Half-life ( _t½ ) • Apparent clearance (CL/F) • Apparent volume of distribution ( _Vz /F) • Percentage of unmodified RG-NG-1015 excreted in urine (fe) Total unaltered RG-NG-1015 excreted in urine (Ae) Presence of antidrug antibodies (ADA) Evaluation of the effects of RG-NG-1015 (RGLS8429) on kidney function Changes in eGFR, urinary albumin-to-creatinine ratio (UACR), serum creatinine (SCr), and BUN from baseline

The exploratory goals and endpoints of this study are: Exploratory goals Exploratory endpoint Characterizing the effects of RG-NG-1015 on potential renal biomarkers • Changes in urinary mononuclear globulin-1 (MCP-1) and β-2-microglobulin (B2M) renal biomarkers from baseline. • Changes in serum latent renal biomarkers (e.g., insulin-like growth factor-binding protein acid-instable subunit [IGFALS], and CT-proAVP, N-acetylglucosamine, and others) from baseline. Characterizing the effects of RG-NG-1015 (RGLS8429) on image-based potential biomarkers Additional biomarkers based on image variation from baseline (e.g., total cyst volume, number, and/or size distribution). Characterizing the relationship between ADPKD gene mutations and biomarker responses The relationship between ADPKD gene mutations (such as PKD1 or PKD2 mutations, truncated or missense mutations, etc.) and biomarker responses (such as PC1 and PC2 levels).

F. Evaluation of clinical and safety assessments performed at screening and at predetermined time points during the study period includes: • Demographic data, medical history and concomitant medications; • Height and weight measurements; • Vital signs (temperature, sitting systolic and diastolic blood pressure, BP, HR, and RR); • Physical examination (comprehensive and limited); • SARA assessment to detect potential CNS damage caused by the investigational drug; • Safety laboratory tests (hematological complete blood count, chemometabolic analysis, urinalysis, coagulation and lipid profile); • 12-lead ECG; • ADPKD gene testing; • Plasma sample testing for C3a and Bb complement; • Plasma sample testing for antidrug antibodies; Urine biomarker testing (PC1, PC2, NGAL, KIM-1), • Kidney function testing (eGFR calculation and UACR, SCr and BUN testing), o eGFR was calculated during the screening period using the 2021 CKD-EPI creatinine-cystin C age-sex equation (Inker, 2021). • Plasma and Urine Pharmacokinetic Testing o Plasma and urine concentrations over time are used to derive the following PK parameters: _Cmax , _Tmax , AUC _0-24 , AUC _inf (where calculable), AUC _τ , _t½ , CL/F, _Vz /F, fe, and Ae, and additional PK parameters as appropriate. o Pre-administration plasma PK samples on days 1 and 85 should be obtained within 60 minutes prior to administration. Post-administration plasma samples on days 1, 2, 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. o Collect PK samples before drug administration on days 15, 43, and 71. Collect samples before drug administration and 4 hours ± 15 minutes after drug administration on days 29 and 57. Collect PK samples at the EOS visit on days 99 and 113. o Begin 24-hour urine collection for PK analysis immediately after drug administration on days 1 (0-24 hours) and 71 (0-24 hours). Individuals should empty their bladder immediately before drug administration (before the start of 24-hour urine collection). The last invalid 24-hour collection is 24 hours after drug administration. • MRI to determine htTKV changes from baseline. • Exploratory renal biomarker testing in residual urine (MCP-1 and B2M) and residual serum (e.g., IGFALS, CT-proAVP, N-acetylglucosamine, and others); and image-based exploratory biomarker testing (e.g., total cyst volume, number, and/or size distribution). • Acute phase response (Alb, cellulose, and high-sensitivity C-reactive protein) testing in plasma samples.

G. Data Analysis/Statistical Methods: The results will be tabulated and analyzed based on the dosage levels of RG-NG-1015 and placebo (aggregated across all cohorts). Descriptive statistics by dosage level and treatment group will be tabulated based on interviews.

The following analyses were performed: Safety analysis: Safety data (frequency of TEAEs (treatment emergency adverse events) and SAEs (serious adverse events), safety laboratory tests, vital signs, ECG, and SARA test scores) were descriptively summarized as appropriate based on dosage level and treatment group. Analysis of safety laboratory tests, vital signs, and ECG included time-varying statistical data and descriptive summaries of changes from baseline. Descriptive summaries of changes from baseline were performed for SARA test scores over time for each category and the overall score; the SARA test has eight categories, with a cumulative score range of 0 (no ataxia) to 40 (most severe ataxia). When the outcome measures are completed, each category is assessed and scored accordingly. The scoring ranges for the eight items are as follows: 1. Gait (0-8 points), 2. Posture (0-6 points), 3. Sitting (0-4 points), 4. Speech disorder (0-6 points), 5. Finger tracking (0-4 points), 6. Nose-finger test (0-4 points), 7. Rapid alternating hand movements (0-4 points), 8. Heel-to-shoulder gliding (0-4 points). Once each of the eight categories has been assessed, a total score is calculated to determine the severity of the ataxia.

Pharmacodynamic (biomarker) analysis: characterizing urinary biomarker (PC1, PC2, NGAL, KIM-1) responses. In Part A, covariance analysis was used, adjusted for baseline biomarker values and treatment groups, to compare changes in urinary biomarkers (i.e., PC1, PC2, NGAL, KIM-1) over time with baseline (samples at screening and on day 1), and to compare each RG-NG-1015 (RGLS8429) dose level with placebo (placebo aggregated in cohorts 1-3). Data collected on days 29, 57, 85, 86, 92, 99, and 113 were analyzed, but primary comparisons were performed on day 86 and 113. In Part B, two-sided paired t-tests were used to compare changes in urinary biomarker values (PC1, PC2, NGAL, KIM-1) from baseline. Data collected on days 29, 57, 85, 86, 92, 99, and 113 were analyzed, but primary comparisons were performed on day 86 and 113. A p-value < 0.05 was considered statistically significant, and no adjustments were made for multiplicity. The graph will be used to evaluate the relationship between changes in biomarkers (PC1, PC2, NGAL, KIM-1) and htTKV from baseline and plasma exposure (e.g., AUC _τ , _Cmax , _Cmin ) and dose levels.

MRI Analysis: Absolute values of htTKV and percentage changes from baseline will be summarized using descriptive statistics. In Part A, comorbidities were analyzed and adjusted for baseline htTKV values, baseline Mayo imaging classification (MIC) (1C, 1D, or 1E), and treatment groups to compare htTKV values from baseline between treatment groups at each dose level and between all individuals receiving RG-NG-1015 (RGLS8429) and placebo (placebo in cross-cohort 1–3 pools). In Part B, two-sided paired t-tests will be used to compare htTKV changes from baseline. P values < 0.05 will be considered statistically significant, without adjustment for multiplicity.

Renal function analysis: Changes in eGRF (calculated using the CKD-EPI equation with creatinine and cystin-C without race), UACR, SCr, and BUN were analyzed and descriptively summarized from baseline.

Pharmacokinetic Analysis: The laboratories analyzing the PK samples will be non-blinded; therefore, PK analysis will only be performed on individuals receiving RG-NG-1015 (RGLS8429). Plasma and urine concentrations over time were used to derive the following PK parameters: _Cmax , _Tmax , AUC _0-24 , AUC _inf (where calculable), AUC _τ , _t½ , CL/F, _Vz /F, fe, and Ae. Descriptive statistics of total plasma concentrations were summarized by time point and cohort. Using a non-compartmental method, plasma and urine concentrations over time were used to derive the following PK parameters: Cmax, Tmax, AUC 0-24, AUC inf (where calculable), AUC _τ , t½, CL/F, Vz/F, fe, and Ae. Where data allows, efficacy models using _Cmax , AUC _0-24 , AUC _inf , and AUC _τ were used to explore dosage ratios.

ADA analysis: The incidence and titer of ADA in individuals who developed ADA at any time during the study period are tabulated by cohort. The effect of ADA on PK parameters will be assessed by subgroup analysis (e.g., calculating PK parameters in individuals with and without ADA).

Exploratory analysis: The following descriptive statistics are tabulated on a per-visit basis: exploratory renal biomarkers in urine (MCP-1 and B2M) and serum (e.g., IGFALS, CT-proAVP, N-acetylglucosamine, and others); and imaging-based exploratory biomarkers (e.g., total cyst volume, number, and/or size distribution). The relationship between ADPKD gene mutations (e.g., PKD1 or PKD2 mutations, truncated or missense mutations) and biomarker responses (e.g., PC1 and PC2 levels) is being explored.

Interim Analysis: Part A: After individuals in each cohort complete drug administration and EOS (Effective Orthopaedic Study) visits, the trial administrators will analyze unblinded safety, biomarkers, renal function, and pharmacokinetic (PK) data. Part B: After individuals in cohort 4 complete RG-NG-1015 administration and EOS visits, the trial administrators will analyze all available unblinded safety, biomarkers, efficacy, and PK data.

Clinical research organization (CRO) medical monitors continuously review safety data. The trial sponsor and CRO medical monitors will conduct a blinded safety review of adverse events (AEs) and safety laboratory test results monthly to monitor safety throughout the study. At least four weeks after the last individual in the enrolled cohort receives their first dose (Day 1), a cohort dose escalation meeting will be held. The trial sponsor will review available safety data to make a dose escalation decision for Cohort 2, and will repeat this process to make a dose escalation decision for Cohort 3.

H. Partial A results achieved with RG-NG-1015 treatment of one or more of the primary, secondary and/or exploratory endpoints, with acceptable safety and tolerability.

Table 22 provides the baseline features for queue 1 and queue 2: Baseline features RG-NG-1015 (2 mg/kg) N=11 RG-NG-1015 (1 mg/kg) N=9 Placebo (queues 1 and 2) N=6 Average age (SD) 46 (12) 52 (12) 45 (12) female n (%) 5 (46%) 5 (56%) 3 (50%) White people n (%) 10 (91%) 9 (100%) 4 (67%) Average BMI (SD) 26 (3) 30 (5) 28 (5) Tolvaptan usage in the first 3 months (n (%)) 1 (10%) 2 (22%) 1 (17%) Average eGFR (mL/min/1.73m2) (SD) 68 (19) 47 (20) 52 (18) htTKV (mL/m) average value (SD) 1264 (567) 1698 (737) 1684 (575) Mayo Imaging Classification 1C/1D/1E (%) 46%/36%/18% 56%/33%/11% 17%/67%/17% Gene mutation PKD1/PKD2 (%) 82%/18% 56%/33% 67%/0%

The selected population represents the disease burden based on kidney size and a significant reduction in eGFR.

Studies showed that administration of 1 mg/kg and 2 mg/kg RG-NG-1015 (RGLS8429) every two weeks for 12 weeks was well tolerated and no significant safety findings were observed.

Table 23 provides the adverse events observed in queues 1 and 2: RG-NG-1015 (2 mg/kg) N=11 RG-NG-1015 (1 mg/kg) N=9 Placebo (queues 1 and 2) N=6 Any treatment emergency adverse event (TEAE) 7 (64%) 7 (78%) 3 (50%) Any treatment-related TEAEs 5 (46%) 1 (11%) 0 Any treatment emergency serious adverse event (TESAE) 0 1 (11%)* 0 Any treatment-related TESAE 0 0 0 Any TEAE that leads to early withdrawal** 1 (9%)** 0 0 Any TEAE that leads to death 0 0 0

Repeated weekly administration resulted in no accumulation of RG-NG-1015 in plasma or urine. Patients administered 1 mg/kg RG-NG-1015 showed nearly twice the AUC plasma exposure of healthy volunteers, consistent with a 35% reduction in renal excretion. AUC plasma exposure increased at 2 mg/kg relative to 1 mg/kg.

Polycystic protein (PC) measurement in urine: Measurements of PC1 and PC2 in urinary cytosolic somes have shown a clear difference between healthy individuals and ADPKD patients, and are negatively correlated with disease severity. See Figures 7A and 7C.

Multiple analytical methods were used to assess urinary PC levels: • Absolute changes in urinary PC1 and PC2 levels from baseline, including the best-fit regression model over the duration of treatment (see Figures 7B and 7D, 8A-8C, 9A-9C, and 10A-10B); • Percentage changes in urinary PC1 and PC2 levels from baseline (see Figures 11A and 11B); • Mean changes in urinary PC1 and PC2 levels from baseline after three months of RG-NG-1015 administration (see Figures 12A-12B and 13A-13B).

Studies showed that treatment with 1 mg/kg and 2 mg/kg RG-NG-1015 (RGLS8429) increased the absolute and percentage changes in urinary polycystic protein (PC1 and PC2) levels from baseline. See Figures 7B and 7D, 8A-C, 9A-9C, 10A-10B, and 11A-11B. As shown in Figure 11A, for 1 mg/kg RG-NG-1015, a statistically significant increase in urinary PC1 from baseline (the average of three separate samples taken before treatment) was observed at 12 weeks of treatment (36% and 41% on days 85 and 86, respectively). As shown in Figure 11B, an increase in urinary PC2 from baseline was also observed at 12 weeks with 1 mg/kg RG-NG-1015; however, the change was not statistically significant (see Figures 10A and 10B for statistical analysis). The polycystic protein pattern was consistent with the tissue PK profile in the preclinical studies. The study also showed an increase in the absolute changes in PC1 and PC2 levels with treatment with 2 mg/kg RG-NG-1015 compared to 1 mg/kg RG-NG-1015. See Figures 8A-C, 9A-9C, and 10A-10B. In Figure 10A, for example, with 2 mg/kg RG-NG-1015, a statistically significant increase in urinary PC1 from baseline was observed on days 57, 86, 99, and 113 of administration. In Figure 10B, for 2 mg/kg RG-NG-1015, a statistically significant increase in urinary PC2 from baseline was observed on day 57.

Studies also showed that, under treatment with 1 mg/kg and 2 mg/kg RG-NG-1015 (RGLS8429) for 3 months (Q2W), the mean polycystic ovary syndrome (PCOS) levels increased, as shown in the absolute changes (Figures 12A-B) and the percentage changes (Figures 13A-B) of PCOS and PCOS. Compared with 1 mg/kg RG-NG-1015, 2 mg/kg RG-NG-1015 showed a greater increase in mean PCOS levels.

Compared with placebo, urine measurements of PC1 and PC2 showed greater bioactivity of RG-NG-1015 (RGLS8429) at 2 mg/kg, which was most evident after three months of administration. Based on urinary polycystic protein analysis, a mechanical dose-response was observed at the 2 mg/kg dose level.

This study also demonstrates that polycystic protein is an effective pharmacodynamic biomarker (i.e., for dose range determination) because urinary polycystic protein exhibits appropriate PK/PD correlations and can be used as a pharmacodynamic biomarker in ADPKD. See Figures 14A and 14B. As shown in Figures 14A-14B, dose-response responses were observed at 0.3 mg/kg and 1 mg/kg RGLS4326 (NCT04536688) and 1 mg/kg RG-NG-1015 (RGLS8429). When the RGLS4326 and RG-NG-1015 datasets were combined, a positive correlation was observed between PC1 and the two measured PK parameters ( _Cmax and AUC _last ). Furthermore, similar pharmacodynamic responses were observed between RGLS 4326 and RG-NG-1015 at a dose level of 1 mg/kg.

Renal function parameters and renal MRI measurements: Based on published longitudinal studies, ADPKD patients experience approximately 6% renal growth per year, therefore, the expected renal volume increase is approximately 1%-2% within 12 weeks.

The study-end MRI was used to evaluate the impact on novel imaging biomarkers characterizing cystic structures. Exploratory results from the MRI image analysis are shown in Figures 15A-C and 16A-C.

Figures 15A-15B show the changes in high-adjusted total renal volume (htTKV) and total renal cyst volume (TKCV) in individuals receiving 1 mg/kg RG-NG-1015, 2 mg/kg RG-NG-1015, and placebo. Figure 15C shows the correlation between changes in TKCV and htTKV.

Table 24 shows the mean htTKV change (%) in the 2 mg/kg group, 1 mg/kg group, and placebo group: HtTKV% 2mpk 1mpk Pb - All average value -0.84 +1.79 +0.52 SD 3.11 5.90 3.13

Table 25 shows the mean TKCV change (%) in the 2 mg/kg group, 1 mg/kg group, and placebo group: TKCV% 2mpk 1mpk Pb - All average value -0.53 +4.36 +0.47 SD 3.90 8.21 1.94

Figures 16A and 16B show the changes in total liver volume (TLV) and total liver cyst volume (TLCV) in individuals receiving 1 mg/kg RG-NG-1015, 2 mg/kg RG-NG-1015, and placebo. Figure 16C shows the correlation between changes in TLCV and changes in TLV.

Table 26 shows the mean TLV change (%) in the 2 mg/kg group, 1 mg/kg group, and placebo group: TLV% 2mpk 1mpk Pb - All average value -1.03 -2.99 +5.03 SD 7.91 7.65 5.61

Table 27 shows the mean absolute TLCV changes in the 2 mg/kg group and the placebo group: Abs TLCV (mL) 2mpk Pb average value -1.72 +0.71 SD 14.95 1.966

Figure 17A shows the exploratory correlation between changes in PC1 and changes in HtTKV. Table 28 shows simple linear regressions for placebo, 1 mg/kg, and 2 mg/kg RG-NG-1015: Simple linear regression R ² p-value placebo 0.0092 0.857 RGLS8429 (1mg/kg) 0.0211 0.709 RGLS8429 (2 mg/kg) 0.0719 0.425

Figure 17B shows the exploratory correlation between changes in PC1 and changes in eGFR. Table 29 shows simple linear regressions for placebo, 1 mg/kg, and 2 mg/kg RG-NG-1015: Simple linear regression R ² p-value placebo 0.0021 0.930 RGLS8429 (1mg/kg) 0.2418 0.179 RGLS8429 (2 mg/kg) 0.4027 0.036

Figure 17C shows the exploratory correlation between changes in PC2 and HtTKV. Table 30 shows simple linear regressions for placebo, 1 mg/kg, and 2 mg/kg RG-NG-1015: Simple linear regression R ² p-value placebo 0.0728 0.605 RGLS8429 (1mg/kg) 0.0639 0.570 RGLS8429 (2 mg/kg) 0.0373 0.512

Figure 17D shows the exploratory correlation between changes in PC2 and eGFR. Table 31 shows simple linear regressions for placebo, 1 mg/kg, and 2 mg/kg RG-NG-1015: Simple linear regression R ² p-value placebo 0.0766 0.596 RGLS8429 (1mg/kg) 0.5902 0.197 RGLS8429 (2 mg/kg) 0.2249 0.006

For Cohort 1, renal function parameters did not show significant changes over the short-term 12-week dosing period. Baseline measurements were consistent with the ADPKD Mayo imaging classification stage. No significant changes were observed in renal function measures (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM 1) over the 12 weeks.

For Cohort 2, exploratory results from MRI imaging analysis are as follows: • The mean change in htTKV was -0.84% in the 2 mg/kg group and +0.52% in the placebo group; • Four of the 11 individuals receiving 2 mg/kg showed a >2% decrease in htTKV, a decrease in TKCV, and an increase in both urinary PC1 and PC2; • Changes in TKCV correlated with changes in htTKV; • Decreased liver volume and liver cyst volume were observed in some patients treated with RG-NG-1015; • No significant changes in renal function measures (i.e., eGFR, UACR, SCr, BUN, U-NGAL, U-KIM 1) were observed over 12 weeks, as expected based on short-term treatment and a small number of individuals.

Some case highlights are described below: • Individual 1: Highest increase in PC1 and PC2* o A 47-year-old male diagnosed in 2006 o Baseline eGFR 66 mL/min and htTKV 941 mL/mo D113 MRI: htTKV decreased by 4.96%; TKCV decreased by 4.34%; • Individual 2: Second highest increase in PC1* o A 44-year-old female diagnosed in 2019 o Baseline eGFR 65 mL/min and htTKV 1253 mL/mo D113 MRI: htTKV decreased by 6.28%; TKCV decreased by 6.93%; • Individual 3: Second highest increase in PC2* o A 29-year-old male diagnosed in 2020 o Baseline eGFR 88 mL/min and htTKV 1162 mL/mo D113 MRI: htTKV decreased by 4.22%; TKCV decreased by 2.73%. • In cohort 2, among the four active individuals with htTKV decrease >2%, PC1 and PC2 were increased in all four individuals. *Mean percentage change in polycystic ovary syndrome (PC) between day 85 and day 113.

The results of cohort 2 showed that, compared with placebo, ADPKD patients had numerical improvements in total volume and cyst volume in the kidneys and liver.

Table 32 provides baseline characteristics for queue 3 and the updated placebo group, which includes additional individuals, relative to the placebo group described in Table 22: Baseline features RG-NG-1015 (3 mg/kg) N=12 Placebo (queues 1 and 2) N=10 Average age (SD) 48 (11) 45 (12) female n (%) 4 (33%) 4 (40%) White people n (%) 11 (92%) 7 (70%) Average BMI (SD) 27 (4) 27 (4) Tolvaptan usage in the first 3 months (n (%)) 2 (17%) 1 (10%) Average eGFR (mL/min/1.73m2) (SD) 61 (16) 55 (17) htTKV (mL/m) average value (SD) 1393 (459) 1763 (859) Mayo Imaging Classification 1C/1D/1E (%) 58%/25%/17% 20%/60%/20% Gene mutation PKD1/PKD2/ neither (%) 75%/8%/17% 80%/0%/20%

As described in this paper for columns 1 and 2, the individuals in column 3 were analyzed. Figures 19 and 20 show the data for the placebo group and columns 1, 2, and 3. The data for columns 1 and 2 are the same as those presented above. The placebo group has additional individuals compared to the data presented above.

Figures 19A and 19B show individual subplots of urinary PC1/CD133 and urinary PC2/CD133 ratios for all cohorts in Part A at baseline and at the end of the study. Increases in urinary PC1/CD133 and PC2/CD133 were observed after treatment with 3 mg/kg, and these increases were more consistent with those observed after treatment with 1 mg/kg and 2 mg/kg.

The absolute changes in urinary PC1/CD133 and PC2/CD133 ratios during treatment were also measured in three individuals in the cohort. As shown in Figures 20A and 20B, the mean absolute increases were generally similar in the 2 mg/kg and 3 mg/kg groups up to day 99. Furthermore, the 3 mg/kg group showed more consistent response and statistical significance at all post-baseline follow-ups.

Analysis of the percentage change in urinary PC1/CD133 and PC2/CD133 ratios from baseline to the end of the study revealed a dose-responsive increase in these ratios, with the largest increase observed in the 3 mg/kg cohort. See Figures 21A and 21B.

Figures 22A and 22B show the changes in high-adjusted total renal volume (htTKV) and total renal cyst volume (TKCV) in individuals receiving 1 mg/kg RG-NG-1015, 2 mg/kg RG-NG-1015, 3 mg/kg, and placebo. Data from the 1 mg/kg and 2 mg/kg groups are also shown in Figures 15A-15C. Figure 22C shows the correlation between changes in TKCV and changes in htTKV.

Figure 23A illustrates the correlation between PC1 changes and htTKV changes. Table 33 presents a simple linear regression of the updated placebo group and all RGLS8429 A-columns for the data in Figure 23A: Simple linear regression R ² p-value placebo 0.0207 0.6920 RGLS8429 (1mg/kg) 0.0211 0.7092 RGLS8429 (2 mg/kg) 0.0720 0.4250 RGLS8429 (3 mg/kg) 0.1683 0.2390

Figure 23B illustrates the correlation between the compared PC2 and htTKV changes. Table 34 presents a simple linear regression for the updated placebo group and all RGLS8429 A-columns using the data in Figure 23B: Simple linear regression R ² p-value placebo 0.0820 0.4226 RGLS8429 (1mg/kg) 0.0639 0.5117 RGLS8429 (2 mg/kg) 0.0373 0.5694 RGLS8429 (3 mg/kg) 0.2581 0.1399

Figure 24 illustrates the correlation between the compared PC1 and eGFR changes. Table 35 presents a simple linear regression of the updated placebo group and all RGLS8429 A-columns for the data in Figure 24: Simple linear regression R ² p-value placebo 0.0110 0.7734 RGLS8429 (1mg/kg) 0.2418 0.1787 RGLS8429 (2 mg/kg) 0.4027 0.0360 RGLS8429 (3 mg/kg) 0.0984 0.3476

Compared to the lower-dose cohort, RGLS8429 at a dose of 3 mg/kg demonstrated a more consistent increase in urinary PC1 and PC2 in patients. From baseline to the end of the study, urinary PC1 and PC2 changed in a dose-responsive manner, with statistical significance observed at the 3 mg/kg dose (compared to placebo). Furthermore, exploratory MRI imaging analysis showed that three months of administration of RGLS8429 at 3 mg/kg reduced htTKV.

Similar to the 1 mg/kg and 2 mg/kg doses, the 3 mg/kg dose demonstrated a favorable safety and tolerability profile.

Part I. Results B : Analysis of results for 14 out of 30 individuals in queue 4. Table 36 provides baseline characteristics for queue 4: Baseline characteristics of 4 individuals in a row (N=14) Average age (in years) 47 Average eGFR (mL/min/1.73m2) 59 htTKV (mL/m) average value 1479 Mayo Category 1C/1D/1E (%) 7 (50%) / 4 (30%) / 3 (20%) Gene mutation PKD1/PKD2/neither (%) 9 (64%) / 1 (7%) / 4 (29%)

The primary endpoint of the study was to evaluate the effects of RG-NG-1015 (RGLS8429) on ADPKD biomarkers in 14 individuals, including changes in urinary PC1 and PC2 from baseline to the end of the study (day 113). Geometric least squares and statistical analyses were performed on log-transformed data for percentage changes. CD133 was used as a normalization control. Placebo results for individuals receiving placebo in Summary A are presented. Table 37 shows the geometric least squares and statistical analyses of the percentage changes in the ratios of PC1 and CD133 relative to baseline at the end of the study. PC1/CD133 ratio Geometric mean Log SE upper limit lower limit Upper limit standard error Lower limit standard error 300 mg (N = 14) 36.85 0.0733 47.26 27.18 10.41 9.67 Placebo (aggregate, N = 10) 5.09 0.0851 14.42 -3.48 9.33 8.57

The data in Table 37, as well as the corresponding data for the 1 mg/kg, 2 mg/kg, and 3 mg/kg doses, are also shown in Figure 25A. The geometric least squares mean of the percentage change is presented. Error bars represent standard errors. ANCOVA analysis was performed using logarithmic scaling to account for non-normal distributions. Data for one individual in each of the 3 mg/kg and 300 mg/kg fixed-dose groups were unavailable.

Table 38 presents the geometric least squares mean and statistical analysis of the percentage change in the ratio of PC2 to CD133 relative to the baseline at the end of the study: PC2/CD133 ratio Geometric mean Log SE upper limit lower limit Upper limit standard error Lower limit standard error 300 mg (N = 14) 31.11 0.0900 43.46 19.83 12.35 11.28 Placebo (aggregate, N = 10) -9.9 0.1030 -0.12 -18.72 9.78 8.82

The data in Table 38, as well as the corresponding data for the 1 mg/kg, 2 mg/kg, and 3 mg/kg doses, are also shown in Figure 25B. The geometric least squares mean of the percentage change is presented. Error bars represent standard errors. ANCOVA analysis was performed using logarithmic scaling to account for non-normal distributions. Data for one individual in each of the 3 mg/kg and 300 mg/kg fixed-dose groups were unavailable.

A secondary objective of this study was to evaluate the effect of RG-NG-1015 (RGLS8429) on height-adjusted total renal volume (htTKV) for each of the 14 individuals from baseline to the end of the study (day 113). Geometric least squares mean and statistical analysis were performed on the percentage change of logarithmic transformation data. Placebo outcomes for individuals receiving placebo in Summary A are presented. Table 39 shows the geometric least squares mean and statistical analysis of the percentage change of htTKV relative to baseline at the end of the study. htTKV change% Geometric mean Log SE upper limit lower limit Upper limit standard error Lower limit standard error 300 mg (N = 14) 0.38 0.01 1.65 -0.87 1.27 1.25 Placebo (aggregate, N = 10) 2.56 0.01 4.04 1.10 1.48 1.46

Data from Table 39, as well as the corresponding data for the 1 mg/kg, 2 mg/kg, and 3 mg/kg cohorts, are shown in Figure 26. Geometric least squares mean data of percentage change are presented. Error bars represent standard errors. ANCOVA analysis was performed using logarithmic scaling to account for non-normal distributions. Data were unavailable for two individuals in the 3 mg/kg group and one individual in the 300 mg fixed group. One individual in the 300 mg/kg fixed group experienced renal cyst rupture, and therefore only the contralateral kidney result for this individual is included. Variation between dosing groups is expected due to the small size of each cohort, the length of dosing intervals, and the inherent variability of eGFR.

When analyzing the percentage change in htTKV based on MIC, RG-NG-1015 (RGLS8429) at a 300 mg dose consistently affected the percentage change in htTKV, independent of MIC. Table 40 shows the percentage change in htTKV for individual individuals receiving either a placebo or a 300 mg dose, based on MIC: htTKV change% MIC 1C MIC 1D MIC 1E placebo 300 mg placebo 300 mg placebo 300 mg 3.97 3.45 0.29 -3.26 0.48 -2.92 2.49 -4.1 -4.49 0.97 7.97 2.19 -2.79 3.68 3.98 0.93 10.27 -0.82 -1.79 3.98 2.25 10.06 -2.21

No significant changes were observed in renal function measures (i.e., eGFR, UACR, SCr, cystin-C, BUN, U-KIM 1) over 12 weeks, as expected based on short-term treatment and a small number of individuals.

Table 41 shows the rate of change in eGFR in the 300 mg group compared to the placebo group: Change rate of eGFR (mL/min/ ^m² /year) Annual average change 95% CL lower limit change 95% CL upper limit change 300 mg (N = 14) -0.899 -3.072 1.274 Placebo (N = 10) -2.270 -4.886 0.345

In summary, a fixed dose of 300 mg RG-NG-1015 (RGLS8429) was found to have a favorable safety and tolerability profile. Urinary PC1 and PC2 levels were consistent with optimal miR-17 inhibition. Notably, TKV measurements after three months of treatment with 300 mg RG-NG-1015 (RGLS8429) indicated an effect on disease progression. Similar results are expected for the remaining individuals in cohort 4.

Example 10: Modeling of RG-NG-1015 (RGLS8429) Target Binding Study Data The efficacy of RG-NG-1015 (RGLS8429) was evaluated in a KspCre; Pkd1 F/RC ( Pkd1 -F/RC) mouse model. Pkd1 -F/RC mice were subcutaneously injected with different doses of RG-NG-1015, a control oligonucleotide at 20 mg/kg, or PBS every day at 8, 10, 12, and 15 days of age (N=8-13 mice/group). Another group of Pkd1-F/RC mice were subcutaneously injected with RG-NG-1015 at 20 mg/kg at 8 and 12 days of age. 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, fixed in 10% formalin, dehydrated, and embedded in paraffin using a standard protocol. The samples were sectioned at 5 μm and stained with hematoxylin and eosin.

As shown in Figures 18A-18B, RGLS8429 demonstrates its dose-responsive efficacy in reducing the kidney weight to body weight ratio (KW/BW). (Figure 18B, left: individual KW/BW by dose level and regimen; right: calculated percentage of inhibition (CPI) per KW/BW against individual renal concentrations of RG-NG-1015. The estimated renal concentration corresponding to 50% inhibition per KW/BW is shown, i.e., the IC50 value). Following administration of RGLS8429, both kidney size and the number of cysts decreased in a dose-responsive manner. See Figure 18A.

Wild-type C57BL6 mice were administered single SC doses of RGLS8429 or RGLS4326 at doses of 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 harvested, and target binding (miR-17 shift from high molecular weight polyribosomes) was measured by miPSA analysis (Androsavich, Nucleic Acids Res. 44, e13 (2016)). The calculated percentage inhibition (CPI) of target binding in mouse kidneys was plotted against individual renal concentrations of RGLS8429. The estimated renal concentration corresponding to 80% inhibition of miR-17, i.e., the IC80 value, is shown. See Figure 18C. The predicted AUC was approximately 12,494 h*ug/g, and it was used as a benchmark to drive renal exposure with maximum target binding (miR-17 inhibition of approximately 80%).

Based on recent results from cohorts 1 and 2, RG-NG-1015 at doses of 1 mg/kg (cohort 1) and 2 mg/kg (cohort 2) in ADPKD patients is projected to produce renal exposures of approximately 6,052 h*ug/g and 8,900 h*ug/g, respectively (lower than the projected baseline renal exposure of approximately 12,494 h*ug/g). Based on these results, a dose of 3 mg/kg (cohort 3) is expected to produce a projected human renal exposure of approximately 12,494 h*ug/g.

Plasma-to-tissue modeling showed that 1 mg/kg was less than half the dose-response curve. Data suggested that higher doses might exhibit a greater urinary polycystic protein response.

Based on extensive non-clinical analysis and PK/PD modeling, renal exposure associated with peak miR-17 target binding is expected to be achieved in humans at >2.4 mg/kg.

Figure 1. Purine nucleobase structure. Figure 2A-2C . Efficacy of RG-NG-1015 in the Pkd1 -F/RC model of PKD. Effects of treatments on (2A) kidney weight to body weight ratio, (2B) blood urea nitrogen (BUN) level, and (2C) blood creatinine level. Figure 3. Maximum tolerated dose (MTD) studies and comparative dose assessments of RG-NG-1001, RGLS4326, and RG-NG-1017. Single intracerebral ventricular (ICV) injections 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; RG-NG-1017) were administered to male C57BL/6J mice at different dose levels in 4 μL volumes, and the mice were monitored for 7 days. The mortality rates of the three different compounds at different doses are indicated. Figures 4A-4F illustrate the evaluation of the activity of RG-NG-1015 and RGLS4326 against miR-17 (4A), miR-20a (4B), miR-93 (4C), and miR106(a) (4D) luciferase sensor activities in HeLa cells in vivo. This study describes the evaluation of the activity of luciferase sensors containing the full-length 3' untranslated regions (UTRs) of the miR-17 direct target genes PKD1 (4E) and PKD2 (4F). Figures 5A-5D show the pharmacokinetics and target binding of RGLS4326 and RG-NG-1015 after a single subcutaneous administration in C57BL6 mice (measured by miPSA). Plasma concentration (5A), tissue concentration (5B), renal target binding (5C), and hepatic target binding (5D) are presented. Figures 6A-6E show the effects of RG-NG-1015 at different doses and regimens, and in combination with tolvaptan, on the Pcy /DBA mouse PKD model. The administration schedule is shown in Figure 6A, and the legends for the graphs in Figures 6C-6E are shown in Figure 6B. Kidney weight/body weight (6C), cyst area (%) (6D), and urinary Ngal/Cr (6E) are displayed. Error bars represent standard deviations. Compared with the Pcy mediator treatment group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.001, (ns)p >0.05; one-way ANOVA Bonferroni's multiple comparison test. Compared with the tolvaptan-only treatment group, #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.001, (ns)p >0.05; one-way ANOVA with Sadik's multiple comparison test. Compared with the dose-matched RG-NG-1015-only treatment group, $p < 0.05, $$p < 0.01, $$$p < 0.001, $$$$p < 0.001, (ns)p >0.05; one-way ANOVA with Sadik's multiple comparison test. Figures 7A-7D . Measurement of urinary polycystic protein-1 (PC1; Figure 7A) and polycystic protein-2 (PC2; Figure 7C) levels in healthy patients and patients with chronic kidney disease and ADPKD (shown as PC1/CD133 ratio or PC2/CD133 ratio). HV: healthy volunteers; CKD: chronic kidney disease, including samples from patients with CKD stages 2-4, including T1D (type 1 diabetes), T2D (type 2 diabetes), AKF (acute renal failure), HT (hypertension), and COPD (chronic obstructive pulmonary disease); ADPKD: autosomal dominant polycystic kidney disease, where the Mayo Clinic classification is based on htTKV and age; CD133: Prominin-1 has been shown to co-localize with PC1 and PC2 on urocytosomnoid vesicles in ADPKD patient samples (Hogan et al., J Am Soc Nephrol. 2009 Feb; 20(2):278-288). *p-value, one-way ANOVA and Dunnett's correction compared to HV. Urinary PC1 (Fig. 7B) and PC2 (Fig. 7D) levels (shown as PC1/CD133 ratio or PC2/CD133 ratio) and mean _D85-D113 values were measured at placebo and baselines of 1 mg/kg and 2 mg/kg RG-NG-1015 (RGLS8429). Figs. 8A-8C . Absolute changes in urinary PC1/CD133 ratio from individual baselines for 2 mg/kg RG-NG-1015 (RGLS8429) (Fig. 8A), 1 mg/kg RG-NG-1015 (Fig. 8B), and placebo (Fig. 8C). Figures 9A - 9C . Absolute changes in urinary PC2/CD133 ratios from individual baselines for 2 mg/kg RG-NG-1015 (Figure 9A), 1 mg/kg RG-NG-1015 (Figure 9B), and placebo (Figure 9C). Figures 10A-10B . Exploratory regression analysis of the absolute changes in urinary PC1/CD133 (Figure 10A) and PC2/CD133 (Figure 10B) ratios from baseline for 2 mg/kg RG-NG-1015, 1 mg/kg RG-NG-1015, and placebo. Solid circles represent RG-NG-1015 treatment (1 mg/kg Q2W ×7), and solid squares represent RG-NG-1015 treatment (2 mg/kg Q2W ×7). Hollow circles represent placebo (pair 1+2). For RG-NG-1015 (1 mg/kg): #, PC1 change from baseline; statistical significance of PC1/CD133 ratio by Wilcoxon matched-pairs signed-rank test at days 85 and 86. #, PC2 change from baseline; statistical significance of PC2/CD133 ratio by Wilcoxon matched-pairs signed-rank test at day 113. For RG-NG-1015 (2 mg/kg): #, PC1 change from baseline; statistical significance of PC1/CD133 ratio by Wilcoxon matched-pairs signed-rank test at days 57, 86, 99, and 113. # Changes in PC2 from baseline; statistical significance of PC2/CD133 ratios based on Wilcarson-matched sign-rank test by day 57. Exploratory regression analysis was performed using nonlinear regression (second-order polynomial). For curve fitting purposes, individuals with absolute increases in PC1/CD133=3.85 and PC2/CD133=0.033 at day 113 were excluded. Figures 11A-11B . Changes in urinary PC1/CD133 (Figure 11A) and PC2/CD133 (Figure 11B) ratios from baseline were measured for 2 mg/kg RG-NG-1015, 1 mg/kg RG-NG-1015, and placebo, and are shown in the group mean plot. Urinary polycystic protein levels were measured before, during, and 28 days after the last dose (and the 7th dose) of RG-NG-1015 administered every two weeks for 113 days. Urinary PC1 levels are shown in Figure 11A, and urinary PC2 levels are shown in Figure 11B. Solid circles represent RG-NG-1015 treatment (1 mg/kg Q2W ×7) (total N=9 individuals, with Mayo imaging classes 1C/1D/1E=5/3/1). Hollow circles represent placebo cohort 1+2 (Q2W ×7) (total N=6 individuals, with Mayo imaging classes 1C/1D/1E=1/4/1). Solid squares represent RG-NG-1015 treatment (2 mg/kg Q2W ×7) (total N=11 individuals, with Mayo Cognitive Imaging Classes 1C/1D/1E=5/4/1). Figures 12A-12B show the mean change in polycystic protein levels (mean of all available measurements between day 85 and day 116) from baseline after 3 months of Q2W administration for 2 mg/kg RG-NG-1015, 1 mg/kg RG-NG-1015, and placebo. Figure 12A shows the absolute change in the urinary PC1/CD133 ratio, and Figure 12B shows the absolute change in the urinary PC2/CD133 ratio. Figures 13A-13B show the mean change in polycystic ovary syndrome (PCOS) levels compared to baseline after 3 months of Q2W administration for 2 mg/kg RG-NG-1015, 1 mg/kg RG-NG-1015, and placebo. Figure 13A shows the percentage change in the urinary PC1/CD133 ratio, and Figure 13B shows the percentage change in the urinary PC2/CD133 ratio. Figures 14A-14B show the correlation between PCOS levels and pharmacokinetic (PK) parameters. The correlation between urinary PC1 and single-dose _Cmax is shown in Figure 14A. The correlation _between urinary PC1 and single-dose AUC is shown in Figure 14B. Figures 15A-15C . Changes in high-adjusted total renal volume (htTKV) (Figure 15A) and total renal cyst volume (TKCV) (Figure 15B) at the end of the study compared to baseline 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). Figures 16A-16C. Changes in total liver volume (TLV) (Figure 16A) and total liver cyst volume (TLCV) (Figure 16B) at the end of the study compared to baseline 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). Figures 17A-17D show the exploratory correlation between the change in PC1 (shown as the absolute change in PC1/CD133, average _D85-D113 from the baseline) and the change in htTKV (Figure 17A), and the exploratory correlation between the change in PC1 (shown as the absolute change in PC1/CD133, average _D85-D113 values) and the change in eGFR (Figure 17B). Exploratory correlations were measured between PC2 changes (shown as absolute changes in PC1/CD133, mean _D85–D113 from baseline) and htTKV changes (Fig. 17C), and between PC2 changes (shown as absolute changes in PC1/CD133, mean _D85–D113 values) and eGFR changes (Fig. 17D). Figures 18A–18C . RG-NG-1015 inhibits miR-17 and confers efficacy in a dose-responsive manner. Figure 18A shows cross-sections of the kidneys of Pkd1 ^F/RC mice administered various doses of RG-NG-1015 (RGLS8429), PBS, or 20 mg/kg control oligonucleotides. *A cross-section of the kidneys of age-matched wild-type C57BL6 mice is shown (Lakhia et al. 2022 Nat Commun. 15 Aug 2022; 13(1):4765), for reference only. Pkd1 ^F/RC mice in this treatment group were administered the drug only on days (P) 8 and 12 postnatally. All other Pkd1 F/RC mice in the study were administered the drug at P8, 10, 12, and 15. Figure 18B shows the calculated inhibition of kidney weight/body weight (KW/BW) of various Pkd1 ^{F/RC mouse} renal concentrations of RG-NG-1015 (RGLS8429). Figure 18C shows the calculated % inhibition of miR-17 in the renal concentrations of RGLS-4326 and RG-NG-1015 (RGLS8429) in various WT-C57BL6 mice. Figures 19A-19B are individual subplots of urinary PC1/CD133 ratios (Figure 19A) and PC2/CD133 ratios (Figure 19B) for all cohorts in Part A at baseline and at the end of the study. Baseline values are calculated as the average of values before screening and administration. End of study (EOS) is the average of values from day 85 to day 113 (D85-113). Figures 20A-20B show the absolute changes in urinary PC1/CD133 (Figure 20A) and PC2/CD133 (Figure 20B) during treatment. Absolute changes are based on the original data (unaltered). # Indicates statistical significance at the 0.05 significance level, assessed by Wilcarson's matched-pair (one-tailed) signed-rank test compared to baseline within the treatment group. "Scr1" and "Scr2" indicate data from the first and second screening visits to individuals. In Figures 20A-20B, the four bars for each group, from left to right, represent: placebo, 1 mg/kg RGLS8429, 2 mg/kg RGLS8429, and 3 mg/kg RGLS8429. Figures 21A-21B. Percentage changes in urinary PC1/CD133 (Figure 21A) and urinary PC2/CD133 (Figure 21B) from baseline to the end of the study. Log-scaling transformation was used for percentage changes, and mixed-model randomized coefficient regression analysis was employed to explain non-normal distributions. Figures 22A-22C. Individual subplots showing the changes in height-adjusted total renal volume (htTKV) (Figure 22A) and total renal cystic volume (TKCV) (Figure 22B) for all cohorts in Part A. Individual subplots showing the correlation between the percentage change in TKCV and the percentage change in htTKV for all cohorts in Part A (Figure 22C). Figures 23A-23B. Correlation between the absolute changes in the urinary PC1/CD133 ratio (mean _{D85 -D113} from baseline) (Figure 23A) and the percentage change in htTKV (from baseline to EOS). Absolute variation (PC) and percentage variation (htTKV) data and statistical analysis were performed on the raw data without logarithmic scaling. Figure 24. Correlation between the absolute variation of urinary PC1/CD133 (mean _D85-D113 from baseline) and the absolute variation of eGFR (from baseline to EOS). Absolute variation (PC and eGFR) and statistical analysis were performed on the raw data without logarithmic scaling. Figures 25A-25B. Percentage variation of urinary PC1/CD133 (mean _D85-D113 from baseline) (Figure 25A) and PC2/CD133 (mean _D85-D113 from baseline) (Figure 25B) for all columns in parts A and B. Geometric least squares mean data of percentage variation are shown. Error bars represent standard errors. ANCOVA analysis was performed using log-scaling to account for non-normal distributions. Data for one individual in each of the 3 mg/kg and 300 mg/kg fixed groups were unavailable. Figure 26. Percentage change in htTKV for each treatment group in parts A and B. Geometric least squares mean of percentage change data is shown. Error bars represent standard errors. ANCOVA analysis was performed using log-scaling to account for non-normal distributions. Data for two individuals in the 3 mg/kg group and one individual in the 300 mg fixed group were unavailable. One individual in the 300 mg/kg fixed group experienced renal cyst rupture; therefore, only contralateral kidney results were included in the analysis.

TW202539703A_114108718_SEQL.xml

Claims (77)

A method for treating polycystic kidney disease includes administering a modified oligonucleotide or a pharmaceutically acceptable salt thereof to an individual in need at a fixed dose between about 150 mg and about ₃₅₀ mg, wherein the modified _{oligonucleotide has the structure 5'-ASGSCMAFFCFUFUMUUSAS - 3} ', wherein the nucleoside followed by the _subscript "M" is a 2'- _{O - methyl} nucleoside; the nucleoside followed by the subscript "F" is a 2' _- fluoro nucleoside; and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, and wherein each cytosine is an unmethylated cytosine. The method of claim 1, wherein the modified oligonucleotide or its pharmaceutically acceptable salt is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg. As in the method of claim 1 or 2, the pharmaceutically acceptable salt is a sodium salt. A method for treating polycystic kidney disease includes administering a modified oligonucleotide to an individual in need at a fixed dose between about 150 mg and about 350 mg, wherein the modified oligonucleotide has the following structure: Or a medically acceptable salt. The method of claim 4, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg. If the method is requested in item 4 or 5, the pharmaceutically acceptable salt is sodium salt. The method of any of claims 1 to 6, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent. The method of claim 7, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution. The method described in claim 8, wherein the sterile aqueous solution is a saline solution. A method for treating polycystic kidney disease includes administering a modified oligonucleotide to an individual in need at a fixed dose between about 150 mg and about 350 mg, wherein the modified oligonucleotide has the following structure: . The method of claim 10, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg. The method of claim 10 or 11, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a pharmaceutically acceptable diluent. The method of claim 12, wherein the pharmaceutically acceptable diluent is a sterile aqueous solution. The method of claim 13, wherein the sterile aqueous solution is a saline solution. The method described in any of claims 1 to 14, wherein the individual suffers from polycystic kidney disease. If any of the methods described in claims 1 to 15 have been used to diagnose the individual with polycystic kidney disease using clinical, histopathological, and/or genetic criteria. The method of any one of claims 1 to 16, wherein the polycystic kidney disease is somatic dominant polycystic kidney disease (ADPKD). The method of claim 17, wherein the individual has ADPKD of Mayo Imaging Classification 1C, 1D or 1E. The method of any of claims 1 to 18, wherein the individual has an estimated glomerular filtration rate (eGFR) between 30 and 90 mL/min/1.73 ^m² prior to administration of the modified oligonucleotide. The method of any of claims 1 to 19, wherein prior to administration of the modified oligonucleotide, the individual is determined to have reduced levels of polycystin-1 (PC1) and/or polycystin-2 (PC2) in the individual's kidneys, urine, or blood. The method of any of claims 1 to 20, wherein the individual has a mutation selected from either the PKD1 gene or the PKD2 gene. The method of any of claims 1 to 21, wherein the individual has an increased total renal volume. The method of any of claims 1 to 22, wherein the individual suffers from hypertension. The method of any of claims 1 to 23, wherein the individual has impaired renal function. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 150 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 160 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 170 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 180 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 190 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 200 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 210 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 220 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 230 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 240 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 250 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 260 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 270 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 280 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 290 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 300 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 310 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 320 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 330 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 340 mg. The method of any one of claims 1 to 24, wherein the method comprises administering the modified oligonucleotide at a fixed dose of 350 mg. The method of any of claims 1 to 45, wherein the method includes administering the modified oligonucleotide once every 2 weeks. The method of any one of claims 1 to 46, wherein the method includes administering the modified oligonucleotide at least 7 times. The method of any of claims 1 to 47, wherein the modified oligonucleotide is administered subcutaneously. The method of any of claims 1 to 48, wherein the treatment reduces the total renal volume in the individual. The method of any of claims 1 to 49, wherein the treatment slows the rate of increase of total renal volume in the individual. As in the method of request item 49 or 50, where the total renal volume is the height-adjusted total renal volume (htTKV). The method of any of claims 1 to 51, wherein the treatment slows the rate of decline in the glomerular filtration rate in the individual. The method of any of claims 1 to 52, wherein the treatment increases the glomerular filtration rate in the individual. The method of claim 52 or 53, wherein the glomerular filtration rate is an estimated glomerular filtration rate. The method of any of claims 1 to 54, wherein the treatment inhibits or slows the increase in cyst growth in the individual's kidneys and/or liver. The method of claim 55, wherein the treatment inhibits or slows the increase in total cyst volume, number and/or size distribution. The method described in any of claims 1 to 56, wherein the treatment: a) improves or slows the rate of decrease in creatinine clearance in the individual; b) reduces or slows the rate of increase in the albumin:creatinine ratio in the individual; c) reduces or slows the rate of increase in blood urea nitrogen (BUN) levels in the individual; d) reduces or slows the rate of increase in serum creatinine (SCr) levels in the individual; e) increases polycystic protein-1 (PC1) in the individual's urine; f) increases polycystic protein-2 (PC2) in the individual's urine; g) reduces or slows the rate of increase in neutrophil gelatinase-associated lipocalin (NGAL) protein in the individual's urine; and/or h) Reduce or slow the rate of increase of kidney damage molecule-1 (KIM-1) protein in the urine of the individual. The method of any of claims 1 to 57, wherein the administration: a) reduces or slows the rate of increase of mononuclear globulin-1 (MCP-1) in the urine of the individual; b) reduces or slows the rate of increase of β-2-microglobulin (B2M) in the urine of the individual; c) reduces or slows the rate of increase of complement cleavage products C3a and/or Bb in the plasma of the individual; d) reduces or slows the rate of increase of serum insulin-like growth factor-binding protein acid unstable subunits (IGFALS) in the individual; e) reduces or slows the rate of increase of serum pro-AVP in the individual; f) reduces or slows the rate of increase of serum N-acetylglucosamine in the individual; and/or g) Reduce or slow the rate of increase of acute-phase proteins in the individual. The method of any of claims 1 to 58, wherein the treatment causes negligible CNS damage in the individual. The method described in claim 59, wherein the treatment causes virtually no change in the individual’s dysphagia assessment and rating scale (SARA) test scores. The method described in any one of claims 1 to 60 includes: a) measuring the height-adjusted total renal volume (HtTKV) in the individual; b) measuring polycystic protein-1 (PC1) in the individual's urine; c) measuring polycystic protein-2 (PC2) in the individual's urine; d) measuring the blood urea nitrogen (BUN) level in the individual; e) measuring the serum creatinine (SCr) level in the individual; f) measuring the creatinine clearance rate in the individual; g) measuring the urinary albumin:creatinine ratio (UACR) in the individual; h) measuring the estimated glomerular filtration rate (eGFR) in the individual; i) measuring the neutrophil gelatinase-associated lipocalin (NGAL) protein in the individual's urine; j) k) Measure the urinary kinase-1 (KIM-1) protein in the individual; l) Measure the urinary mononuclear globulin-1 (MCP-1) in the individual; m) Measure the urinary β-2 microglobulin (B2M) in the individual; n) Measure the serum insulin-like growth factor-binding protein acid-instable subunit (IGFALS) in the individual; n) Measure the serum CT-proAVP in the individual; o) Measure the serum N-acetylglucosamine in the individual; p) Measure the complement cleavage products C3a and/or Bb in the individual's plasma; and/or q) Measure the total cyst volume, number, and/or size distribution in the individual; and/or r) Measure the individual's SARA test score. The method of any of the requests 1 to 61, wherein the individual is a human individual. The method described in any of claims 1 to 62 has an acceptable safety and tolerability profile. A modified oligonucleotide or a pharmaceutically acceptable salt _thereof for the treatment of polycystic kidney disease, wherein the _{modified oligonucleotide has the structure 5'-ASGSCMAFFCFUFUMUUSAS - 3 '} , wherein the _nucleoside followed by the subscript "M" is a 2'- _O - _{methyl nucleoside} ; the nucleoside followed by the subscript "F" is a 2'-fluoro nucleoside; and the nucleoside followed by the subscript "S" is an S-cEt nucleoside, wherein each cytosine is an unmethylated cytosine; and wherein the modified oligonucleotide is administered at a fixed dose between about 150 mg and about 350 mg. The modified oligonucleotide used in claim 64, wherein the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg or 350 mg. The modified oligonucleotide used in claims 64 or 65, wherein the pharmaceutically acceptable salt is a sodium salt. The modified oligonucleotide used in any of claims 64 to 66, wherein the modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution. The modified oligonucleotide used in any of claims 64 to 67, wherein the polycystic kidney disease is somatic dominant polycystic kidney disease (ADPKD). The modified oligonucleotide used in any of claims 64 to 68, wherein the modified oligonucleotide is administered once every two weeks. The modified oligonucleotide used in any of claims 64 to 69, wherein the modified oligonucleotide is administered at least seven times. The use of a modified oligonucleotide or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of polycystic kidney disease, wherein the _{modified oligonucleotide has the structure 5'-ASGSCMAFFCFUFUMUUSAS - 3 ' ,} wherein the _nucleoside following _{the subscript} "M" is a 2'-O-methyl nucleoside; the nucleoside following the subscript "F" is a 2'- _fluoro nucleoside; and the nucleoside following the subscript "S" is an S-cEt nucleoside, and wherein each cytosine is an unmethylated cytosine; wherein the modified oligonucleotide is formulated for administration at a fixed dose between about 150 mg and about 350 mg. As claimed in claim 71, the modified oligonucleotide is administered at a fixed dose of 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, or 350 mg. For the purposes described in claims 71 or 72, the pharmaceutically acceptable salt is a sodium salt. The modified oligonucleotide is present in a pharmaceutical composition comprising a sterile saline solution, as claimed in any of claims 71 to 73. For the purposes of any of claims 71 to 74, wherein the polycystic kidney disease is somatic dominant polycystic kidney disease (ADPKD). For the purposes of any of claims 71 to 75, the modified oligonucleotide is administered at least once every two weeks. For any of the uses in claims 71 to 76, the modified oligonucleotide is administered at least seven times.