Methods of formulation administration to the kidneys

Abstract

The present disclosure provides methods of administering a formulation to the renal pyramid of a subject.

Description

METHODS OF FORMULATION ADMINISTRATION TO THE KIDNEYS1. FIELD

[0001] The present disclosure generally relates to methods of administering a formulation to the kidneys.2. CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from U.S. Provisional Application No. 63 / 653,614 fded on May 30, 2024, the disclosure of which is incorporated by reference in its entirety.3. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (TORQ 010 01WO SeqList ST26.xml; Size: 4,591 bytes; and Date of Creation: May 29, 2025) are herein incorporated by reference in its entirety.4. BACKGROUND

[0004] Limited drug accessibility to nephrons, the functional units of the kidneys, hinders the effective and precise drug administration needed for optimal therapeutic outcomes of kidney diseases. On one hand, the complex structure and intricate filtration mechanisms of the kidneys create barriers for effective drug administration to the kidneys. Additionally, kidney disorders often involve cystic formations, altered blood flow, and fibrotic changes within the renal tissue, posing additional challenges in effective drug administration to the kidneys. On the other hand, conventional drug administration to the kidneys often involves systemic administration such as oral administration and intravenous infusion, which can cause off-target effects and systemic toxicity. Overcoming these obstacles requires innovative methods of kidneyspecific formulation administration, designed to enhance formulation localization within the nephrons and to minimize off-target effects.5. SUMMARY

[0005] The present disclosure provides a method of administering a formulation to the renal pyramid of a subject, comprising: administering the formulation through the retro- ureteral route into one or more nephrons in the renal pyramid.

[0006] In some embodiments, the administration is non-surgical. In some embodiments, the formulation is administered through a catheter.

[0007] In some embodiments, the ureter is occluded during the administration to prevent the formulation from draining from the renal pelvis, and the ureter is occluded at a location lower than the location of the end of the catheter. In some embodiments, the ureter is occluded at the upper ureter near the ureteropelvic junction or at the ureteropelvic junction. In some embodiments, the ureter is occluded by an inflatable balloon.

[0008] In some embodiments, the method does not comprise isolating the kidney from systemic circulation. In some embodiments, the renal artery is not blocked or occluded during the administration. In some embodiments, the renal vein is not blocked or occluded during the administration.

[0009] In some embodiments, the formulation is administered at a constant rate. In some embodiments, the formulation is administered at a constant rate of about 0.1 mL / min to about 20 mL / min. In some embodiments, the formulation is administered at a constant rate of about 0.1 mL / min to about 3 mL / min. In some embodiments, the formulation is administered at a constant rate of about 0.5 mL / min. In some embodiments, the formulation is administered at a constant rate of 1 mL / min. In some embodiments, the formulation is administered at a constant rate of 2 mL / min.

[0010] In some embodiments, the administered volume of the formulation is about 5 mL to about 40 mL per kidney. In some embodiments, the administered volume of the formulation is about 15 mL or about 30 mL per kidney. In some embodiments, the administered volume of the formulation is about 20 mL to about 25 mL per kidney. In some embodiments, the administered volume of the formulation is about 25 mL per kidney. In some embodiments, the administered volume of the formulation is 25 mL per kidney. In some embodiments, the administered volume of the formulation is a fixed volume. In some embodiments, the administered volume is not dependent on the subject’s body weight. In some embodiments, the administered volume is not dependent on the size of the renal pelvis.

[0011] In some embodiments, the administered volume of the formulation is based on the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 0.1 mb to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 10 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is 10 mL larger than the volume of the renal pelvis of each kidney of the subject.

[0012] In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration. In some embodiments the intrarenal pressure is above about 40 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the intrarenal pressure is above baseline pressure during the administration. In some embodiments, the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

[0013] In some embodiments, the maximum intrarenal pressure during the administration is less than about 175 mm Hg, less than about 150 mm Hg, less than about 125 mm Hg, or less than about 100 mm Hg. In some embodiments, the maximum intrarenal pressure during the administration is less than about 125 mm Hg. In some embodiments, the maximum intrarenal pressure during the administration is less than about 100 mm Hg.

[0014] In some embodiments, the ureter remains occluded for about 5 minutes to about 60 minutes after the administration. In some embodiments, the ureter remains occluded for about 10 minutes or about 30 minutes after the administration.

[0015] In some embodiments, the intrarenal pressure is below 200 mm Hg after the administration. In some embodiments, the intrarenal pressure is below 100 mm Hg after the administration. In some embodiments, the intrarenal pressure is above baseline pressure after the administration. In some embodiments, the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

[0016] In some embodiments, the formulation comprises a pharmaceutically acceptable carrier or excipient.

[0017] In some embodiments, the formulation is a therapeutic formulation. In some embodiments, the therapeutic formulation is for treating a kidney disease. In some embodiments, the therapeutic formulation is a gene therapy. In some embodiments, thetherapeutic formulation comprises a therapeutically effective amount of recombinant adeno-associated virus (rAAV). In some embodiments, the therapeutically effective amount of rAAV is between about 108viral genomes (vg) and about 1015vg per kidney. In some embodiments, the therapeutically effective amount of rAAV is between about 1012vg and about 1015vg per kidney. In some embodiments, the therapeutically effective amount of rAAV is between about 1014vg and about 1015vg per kidney. In some embodiments, the therapeutically effective amount of rAAV is between about 108vg / mL and about 1015vg / mL. In some embodiments, the therapeutically effective amount of rAAV is between about IO10vg / mL and about 1014vg / mL. In some embodiments, the therapeutically effective amount of rAAV is between about 1012vg / mL and about 1013vg / mL.

[0018] In some embodiments, the rAAV comprises a vector comprising a transgene. In some embodiments, the rAAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

[0019] In some embodiments, the rAAV comprises a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, AAV.kl3, AAV.k20, or a variant thereof.

[0020] In some embodiments, the capsid protein is a variant of AAV9. In some embodiments, the AAV capsid protein transduces kidney cells.

[0021] In some embodiments, the capsid protein is an AAV.kl3 capsid protein. In some embodiments, the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.kl3 capsid increases transduction of kidney cells by at least about 5%, at least about 10%,at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AAV9 capsid.

[0022] In some embodiments, the capsid protein is an AAV.k20 capsid protein. In some embodiments, the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV.k20 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AAV9 capsid.

[0023] In some embodiments, the method increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to transduction in kidney cells using intravenous administration. In some embodiments, the kidney cells are proximal tubule cells, distal tubule cells, or collecting duct cells.

[0024] In some embodiments, the method results in the transduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, or more of the nephrons in the kidney.

[0025] In some embodiments, the present disclosure provides a method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering a therapeutic formulation comprising an rAAV to the subject by performing the method described herein.

[0026] In some embodiments, the subject is a human. In some embodiments, the subject is seropositive for the rAAV prior to administration of the therapeutic formulation comprising the rAAV. In some embodiments, the subject is seronegative for the rAAV prior to administration of the therapeutic formulation comprising the rAAV.

[0027] In some embodiments, the subject has autosomal dominant polycystic kidney disease (ADPKD). In some embodiments, the subject has ADPKD1. In some embodiments, the subject has ADPKD2 In some embodiments, the method inhibits or ameliorates renal cyst development.

[0028] In some embodiments, the transgene encodes a therapeutic RNA or a therapeutic protein In some embodiments, the therapeutic RNA is a circular RNA. In some embodiments, the therapeutic RNA is a miRNA inhibitor such as a miRNA sponge or tough decoy. In some embodiments, the therapeutic protein is polycystin-1 or a fragment thereof. In some embodiments, the therapeutic protein is polycystin-2 or a fragment thereof.

[0029] In some embodiments, the formulation is a diagnostic formulation. In some embodiments, diagnostic formulation is for kidney scan.6. BRIEF DESCRIPTION OF THE FIGURES

[0030] Fig. 1 shows the manual administration of contrast dye in pigs at an approximate rate of 3-20 mL / min. (*Represents damage to the renal calyx or cortex).

[0031] Fig. 2 is an exemplary retroureteral administration (RUA) schematic in pigs utilizing a syringe and syringe infusion pump. The schematic shows the syringe and syringe infusion pump that is filled with the formulation, the pressure transducer that measures pressure throughout the procedure, the balloon catheter placed through the urethra, bladder, and ureter, and (4) the catheter balloon placed in the upper ureter near the ureteropelvic junction.

[0032] Figs. 3A and 3B show pressure changes associated with the loss of balloon occlusion in RUA. Fig. 3A shows the pressure changes associated with a total dose of 20 mL administered at a constant rate of 2 mL / min and Fig. 3B shows the pressure changes associated with a total dose of 15 mL administered at a constant rate of 1 mL / min. In both Fig. 3 A and 3B, pressure increases following adjustment of the balloon for complete occlusion of the ureter.

[0033] Figs. 4A-4C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 20 mL administered at a constant rate of 5 mL / min. Fig. 4A shows the intrarenal pressure monitored via a pressure transducer located at the base of the catheter. Fig. 4B shows a fluoroscopy image taken early during the dosing period and Fig. 4C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0034] Figs. 5A-5C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 20 mL administered at a constant rate of 1 mL / min. Fig. 5A shows the intrarenal pressure monitored via a pressure transducer located at the base of the catheter. Fig. 5B shows a fluoroscopy image taken early during the dosing period and Fig. 5C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0035] Figs. 6A-6C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 15 mL administered at a constant rate of 2 mL / min. Fig. 6A shows the intrarenal pressure monitored via a pressure transducer located at the base of the catheter. Fig. 6B shows a fluoroscopy image taken early during the dosing period and Fig. 6C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0036] Figs. 7A-7C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 15 mL administered at a constant rate of 1 mL / min. Fig. 7A shows the intrarenal pressure monitored via a pressure transducer located at the base of the catheter. Fig. 7B shows a fluoroscopy image taken early during the dosing period and Fig. 7C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0037] Figs. 8A-8C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 12 mL administered at a constant rate of 1 mL / min. Fig. 8A shows the intrarenal pressure monitored via a pressure transducerlocated at the base of the catheter. Fig. 8B shows a fluoroscopy image taken early during the dosing period and Fig. 8C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0038] Figs. 9A-9C shows the pressure changes and results of the contrast dye administration in pigs at a total volume of 12 mL administered at a constant rate of 1 mL / min. Fig. 9A shows the intrarenal pressure monitored via a pressure transducer located at the base of the catheter. Fig. 9B shows a fluoroscopy image taken early during the dosing period and Fig. 9C shows a fluoroscopy image taken at the end of the contrast dye solution administration.

[0039] Fig. 10 shows the intrarenal pressure following the administration of 15 mL or 20 mL of contrast dye solution at a constant rate of 1 mL / min, which was then held in the kidney for either a 15 or 30 min dwell period.

[0040] Figs. 11A-11D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 2, animal 1 (15 mL volume, 15 min dwell time). Fig. 11A shows the right kidney 2 min into the dosing period, Fig 11B shows the right kidney at the end of the dosing period, Fig. 11C shows the right kidney at the end of the dwell period and Fig. 11D shows the left kidney 2 min into dosing.

[0041] Figs. 12A-12D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 2, animal 2 (15 mL volume, 15 min dwell time). Fig. 12A shows the right kidney 2 min into the dosing period, Fig 12B shows the right kidney at the end of the dosing period, Fig. 12C shows the right kidney at the end of the dwell period and Fig. 12D shows the left kidney 2 min into dosing.

[0042] Figs. 13A-13D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 2, animal 3 (15 mL volume, 15 min dwell time). Fig. 13A shows the right kidney 2 min into the dosing period, Fig 13B shows the right kidney at the end of the dosing period, Fig. 13C shows the right kidney at the end of the dwell period and Fig. 13D shows the left kidney 2 min into dosing.

[0043] Figs. 14A-14D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 3, animal 1 (15 mL volume, 30 min dwell time). Fig. 14Ashows the right kidney 2 min into the dosing period, Fig 14B shows the right kidney at the end of the dosing period, Fig. 14C shows the right kidney at the end of the dwell period and Fig. 14D shows the left kidney 2 min into dosing.

[0044] Figs. 15A-15D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 3, animal 2 (15 mL volume, 30 min dwell time). Fig. 15A shows the right kidney 2 min into the dosing period, Fig 15B shows the right kidney at the end of the dosing period, Fig. 15C shows the right kidney at the end of the dwell period and Fig. 15D shows the left kidney 2 min into dosing.

[0045] Figs. 16A-16D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 3, animal 3 (15 mL volume, 30 min dwell time). Fig. 16A shows the right kidney 2 min into the dosing period, Fig 16B shows the right kidney at the end of the dosing period, Fig. 16C shows the right kidney at the end of the dwell period and Fig. 16D shows the left kidney 2 min into dosing.

[0046] Figs. 17A-17D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 4, animal 1 (20 mL volume, 15 min dwell time). Fig. 17A shows the right kidney 2 min into the dosing period, Fig 17B shows the right kidney at the end of the dosing period, Fig. 17C shows the right kidney at the end of the dwell period and Fig. 17D shows the left kidney 2 min into dosing.

[0047] Figs. 18A-18D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 4, animal 2 (20 mL volume, 15 min dwell time). Fig. 18A shows the right kidney 2 min into the dosing period, Fig 18B shows the right kidney at the end of the dosing period, Fig. 18C shows the right kidney at the end of the dwell period and Fig. 18D shows the left kidney 2 min into dosing.

[0048] Figs. 19A-19D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 4, animal 3 (20 mL volume, 15 min dwell time). Fig. 19A shows the right kidney 2 min into the dosing period, Fig 19B shows the right kidney at the end of the dosing period, Fig. 19C shows the right kidney at the end of the dwell period and Fig. 19D shows the left kidney 2 min into dosing.

[0049] Figs. 20A-20D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 5, animal 1 (20 mL volume, 30 min dwell time). Fig. 20A shows the right kidney 2 min into the dosing period, Fig 20B shows the right kidney at the end of the dosing period, Fig. 20C shows the right kidney at the end of the dwell period and Fig. 20D shows the left kidney 2 min into dosing.

[0050] Figs. 21A-21D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 5, animal 2 (20 mL volume, 30 min dwell time). Fig. 21A shows the right kidney 2 min into the dosing period, Fig 2 IB shows the right kidney at the end of the dosing period, Fig. 21C shows the right kidney at the end of the dwell period and Fig. 21D shows the left kidney 2 min into dosing.

[0051] Figs. 22A-22D show fluoroscopy images of the kidneys at various stages of solution administration (contrast dye into the right kidney and formulation buffer into the left kidney) for Group 5, animal 3 (20 mL volume, 30 min dwell time). Fig. 22A shows the right kidney 2 min into the dosing period, Fig 22B shows the right kidney at the end of the dosing period, Fig. 22C shows the right kidney at the end of the dwell period and Fig. 22D shows the left kidney 2 min into dosing.

[0052] Fig. 23 shows the average pig renal pelvis volume among the animals that received contrast dye solution (9 kidneys).

[0053] Figs. 24A-24H shows hematology measurements for each group of animals assessed. Blood was collected at the noted days prior to and after dosing and the following hematology parameters were measured: Fig. 24A Red blood cell count (le6 / pL), Fig.24B White blood cell count le3 / pL), Fig. 24C Neutrophils (le3 / pL), Fig. 24D Lymphocytes (le3 / pL), Fig. 24E Monocytes (le3 / pL), Fig. 24F Basophils (le3 / pL), Fig. 24G Eosinophils (le3 / pL), and Fig. 24H Platelet count (le3 / pL).

[0054] Figs. 25A-25C shows coagulation measurements for each group of animals assessed. Blood was collected at the noted days prior to and after dosing and the following coagulations assays were performed: Fig. 25A Prothrombin time (sec), Fig. 25B Fibrinogen (mg / dL), and Fig. 25C Activated partial thromboplastin time (sec).

[0055] Figs. 26A and 26B show liver enzyme measurements for each group of animals assessed. Blood was collected at the noted days prior to and after dosing and thefollowing liver enzyme activities were measured: Fig. 26A Alanine aminotransferase (U / L) and Fig. 26B Aspartate aminotransferase (U / L).

[0056] Figs. 27A-27R shows clinical chemistry measurements for each group of animals assessed. Blood was collected at the noted days prior to and after dosing and the following clinical chemistry markers were measured: Fig. 27A Alkaline phosphatase (U / L), Fig. 27B Total bilirubin (mg / dL), Fig. 27C Sorbitol dehydrogenase (U / L), Fig. 27D Gamma glutamyl transferase (U / L), Fig. 27E Albumin (g / dL), Fig. 27F Albumin / globulin ratio (ratio), Fig. 27G Globulin (g / dL), Fig 27H Total protein (g / dL), Fig. 271 Urea (mg / dL), Fig. 27J Cholesterol (mg / dL), Fig. 27K Creatinine (mg / dL), Fig. 27L Glucose (mg / dL), Fig. 27M Triglycerides (mg / dL), Fig. 27N Phosphorous (mg / dL), Fig. 270 Calcium (mg / dL), Fig. 27P Potassium (mEq / L), Fig. 27Q Chloride (mEq / L), and Fig. 27R Sodium (mEq / L).

[0057] Figs. 28A-28C shows urine analysis measurements for each group of animals assessed. Urine was collected at the noted days prior to and after dosing and the following parameters were measured: Fig. 28A Urine volume (mL), Fig. 28B Specific gravity (urine specific gravity), and Fig. 28C pH (urine pH)

[0058] Fig. 29A shows intrarenal pressure in pigs during RUA of 80 mL of contrast dye at a constant rate of 1 mL / min, 2 mL / min, or 5 mL / min. Each data point represents the mean intrarenal pressure and bidirectional bars represent the standard error of the mean. Fig. 29B shows the intrarenal pressure overtime in pigs during the dwell period following RUA with 80 mL of contrast dye at a constant rate of 1 mL / min, 2 mL / min, or 5 mL / min. Each data point represents the mean intrarenal pressure and bidirectional bars represent the standard error of the mean. Fig. 29C shows the maximum intrarenal pressure for each pig during RUA of contrast dye at a constant rate of 1 mL / min, 2 mL / min, or 5 mL / min. The average intrarenal pressure is shown above each graphed group. Fig. 29D shows the volume at which the maximum intrarenal pressure is achieved for each pig administered contrast dye at a constant rate of 1 mL / min, 2 mL / min, or 5 mL / min. Maximum intrarenal pressure was reached after 23 mL of contrast dye on average across the three different flow rates.

[0059] Fig. 30 shows fluoroscopy images of pig kidneys during RUA of contrast dye at a constant rate of 1 min / mL, 2 min / mL, or 5 mL / min. The images represent the first signs of damage visible by fluoroscopy for the indicated animal at the indicated flow rate. A black box indicates that no damage was observed at any time point for thatanimal. Arrows point to signs of damage. The numerical values shown on the images represent the volume (mL) at which the first signs of damage are observed.

[0060] Fig. 31 shows fluoroscopy images of the contralateral untreated pig kidneys following RUA of contrast dye at a constant rate of 1 min / mL, 2 min / mL, or 5 mL / min. Systemic leakage was detected in the majority of pigs administered contrast dye at a constant rate of 5 mL / min by RUA. Dashed boxes outline the presence of contrast dye in the renal pelvis of the contralateral kidney. A black box indicates that no contrast dye was observed following the RUA procedure for that animal.

[0061] Fig. 32 shows quantitative analysis of fluoroscopy images of pig kidneys following the RUA procedure. Five pigs were administered contrast dye at a constant rate of 1 mL / min (Group 1) or 2 mL / min (Group 2) using the RUA procedure. The volume (mL) represents the total amount of contrast agent administered by RUA to each kidney, whereas the fold change represents the signal intensity in the renal cortex at each administered dosing volume normalized to the baseline image (fluoroscopic image captured immediately before dosing). The mean normalized signal (fold change) from contrast agent throughout the kidney is represented as an individual data point with bidirectional error bars representing the standard error of the mean. The dashed vertical lines indicate the volume range at which the signal (fold change) begins to plateau (20-30 mL).

[0062] Fig. 33A shows a schematic of the study design evaluating unilateral or bilateral RUA of AAV.kl3 and AAV.k20 in pigs. A self-complementary AAV genome containing a CBA promoter driving expression of the mCherry transgene with an SV40 polyadenylation signal was packaged into AAV.k20 and AAV.kl3. The AAV formulation was injected into the pig kidney via RUA at various doses and tissues were harvested 28 days later for analysis. Fig. 33B shows a schematic of the kidney biopsy strategy for treated (ipsilateral) kidneys with 23 total biopsies collected from cortex, outer medulla, and inner medulla regions.

[0063] Figs. 34A and 34B show AAV biodistribution (Fig. 34A) and RNA (Fig. 34B) expression in whole kidney and kidney regions (cortex, outer medulla and inner medulla) of pigs following unilateral or bilateral RUA of formulation buffer, AAV.kl3, or AAV.k20. Data shown are the means of three animals with error bars depicting standard error of the mean. The dashed vertical line in each graph represents the lower limit of quantification.

[0064] Fig. 35 shows AAV biodistribution in whole liver and individual liver lobes (right medial, right lateral, left medial, and left lateral) following unilateral or bilateral RUA of formulation buffer, AAV.kl3, or AAV.k20. Data shown are the means of three animals with error bars depicting standard error of the mean. The dashed vertical line represents the lower limit of quantitation.

[0065] Figs. 36A and 36B show AAV biodistribution (Fig. 36A) and mCherry RNA (Fig. 36B) expression in the contralateral untreated whole kidney and kidney regions (cortex, outer medulla and inner medulla) following RUA of formulation buffer, AAV.kl3, or AAV.k20. The dashed vertical line in each graph represents the lower limit of quantification.

[0066] Fig. 37 shows a schematic of the study design evaluating RUA of AAV.k20 with a 10 minute or 30 minute dwell period in seronegative or seropositive pigs. A single-stranded AAV genome containing a CBA promoter driving expression of the mCherry transgene with an SV40 polyadenylation signal was packaged into AAV.k20. The AAV formulation was injected into the pig kidney via RUA at various doses and tissues were harvested 28 days later for analysis

[0067] Figs. 38A and 38B show AAV biodistribution (Fig. 38A) and mCherry RNA (Fig. 38B) expression in whole kidney and kidney regions (cortex, outer medulla and inner medulla) of seronegative or seropositive pigs following RUA of formulation buffer or AAV.k20 with a 10 minute or 30 minute dwell period. Data shown are the means of three animals with error bars depicting standard error of the mean. The dashed vertical line in each graph represents the lower limit of quantification.

[0068] Figs. 39A and 39B show AAV biodistribution in contralateral untreated kidney (Fig. 39A) and liver (Fig. 39B) from seronegative or seropositive pigs following RUA of formulation buffer or AAV.k20 with a 10 minute or 30 minute dwell period. Data shown are the means of three animals with error bars depicting standard error of the mean. The dashed vertical line in each graph represents the lower limit of quantification.

[0069] Fig. 40 shows mCherry protein expression by ELISA in whole kidney and kidney regions (cortex, outer medulla and inner medulla) of seronegative pigs following RUA of formulation buffer or AAV.k20 with a 30 minute dwell period. mCherry protein was normalized to the total amount of protein in the kidney sample. The dashed vertical line represents the lower limit of quantification.

[0070] Fig. 41 shows mCherry protein expression by immunohistochemistry (IHC) in ipsilateral treated kidneys of seronegative or seropositive pigs following RUA of formulation buffer or AAV.k20 with a 10 minute or 30 minute dwell period. The top panels are shown at 0.5X magnification and the bottom panels are shown at 4X magnification. Cell types positive for mCherry expression were predominantly cortical and medullary collecting duct tubules and distal tubules. No mCherry protein expression was detected in pig kidneys administered formulation buffer.

[0071] Fig. 42 shows a schematic of the study design evaluating RUA of various AAV.k20 expression cassettes. A single-stranded genome containing either a CBA or PGK promoter that drives expression of a codon optimized and CpG-depleted hPKD2 transgene with an HA tag and bGH polyadenylation signal was packaged into AAV.k20. The AAV formulation was injected into the pig kidney via RUA at various doses, flow rates, and both with and without renal artery (RA) occlusion, and kidneys were harvested 28 days later for analysis.

[0072] Fig. 43 shows intrarenal pressure over time in pigs during RUA of formulation buffer or AAV.k20. Each point represents the mean intrarenal pressure at that interval The bidirectional bars represent the standard error of the mean.

[0073] Figs. 44A and 44B show AAV biodistribution (Fig. 44A) and hPKD2 RNA (Fig. 44B) expression in ipsilateral treated kidneys following RUA of formulation buffer or AAV.k20 in pigs. Data shown are the means of three animals with error bars depicting standard error of the mean. The dashed vertical line in each graph represents the lower limit of quantification.

[0074] Figs. 45 shows AAV biodistribution in contralateral untreated kidneys following RUA of formulation buffer or AAV.k20 in pigs. The dashed vertical line in each graph represents the lower limit of quantification. Each bar represents the mean of three individual pigs and error bars represent the standard error of the mean.

[0075] Figs. 46A and 46B show AAV biodistribution (Fig. 46A) and hPKD2 RNA (Fig. 46B) expression in liver following RUA of formulation buffer or AAV.k20 in pigs. The dashed vertical line in each graph represents the lower limit of quantification. Each bar represents the mean of three individual pigs and error bars represent the standard error of the mean.

[0076] Fig. 47 shows polycystin-2 (PC2) protein expression by CE-SDS in ipsilateral treated kidneys following RUA of formulation buffer or various doses of AAV.k20 ata constant rate of 1 mL / min or 2 mL / min. Protein expression levels were normalized to the protein expression levels observed in animals treated with formulation buffer. Data shown are the means of three animals with error bars depicting standard error of the mean.

[0077] Fig. 48A shows human polycystin-2-HA (hPC2-HA) protein expression in ipsilateral treated kidney sections following RUA of formulation buffer or AAV.k20 by IHC. Brown staining indicates positivity for hPC2-HA. Cell types positive for polycystin-2 expression were predominantly cortical and medullary collecting duct tubules, distal tubules, and proximal tubules. Each image is shown at 0.5X magnification. Fig. 48B shows quantitative image analysis of the area of each kidney section staining positive for hPC2-HA in treated ipsilateral kidneys following RUA of formulation buffer or AAV.k20 in pigs.

[0078] Fig. 49 shows body weight before and after unilateral RUA administration of formulation buffer or AAV.k20 in pigs.

[0079] Figs. 50A-50H show clinical chemistry measurements before and after RUA administration of formulation buffer or AAV k20 in pigs. Blood was collected at the noted days and the following clinical chemistry markers were measured: alanine transaminase (ALT) (Fig. 50A), aspartate transaminase (AST) (Fig. 50B), alkaline phosphatase (ALP) (Fig. 50C), gamma-glutamyl transferase (GGT) (Fig. 50D), succinate dehydrogenase (SDH) (Fig. 50E), total protein (TPRO) (Fig. 50F), blood urea nitrogen (BUN) (Fig. 50G), and creatinine (Fig. 50H). Group 1 is formulation buffer, 1 mL / min; Group 2 is CBA, 1E14 vg, 1 mL / min; Group 3 is CBA, 3E14 vg, 1 mL / min; Group 4 is PGK, 3E14 vg, 1 mL / min; Group 5 is CBA, 3E14 vg, 2 mL / min; Group 6 is CBA, 3E14 vg, 0.5 mL / min; and Group 7 is CBA, 1E14 vg, 2 mL / min, RA occlusion.

[0080] Figs. 51A-51D show hematology tests in pigs following RUA of AAV.k20, including assessment of platelets (Fig. 51A), white blood cells (WBC) (Fig. 51B), lymphocytes (Fig. 51C), and neutrophils (Fig. 51D). Group 1 is formulation buffer, 1 mL / min; Group 2 is CBA, 1E14 vg, 1 mL / min; Group 3 is CBA, 3E14 vg, 1 mL / min; Group 4 is PGK, 3E14 vg, 1 mL / min; Group 5 is CBA, 3E14 vg, 2 mL / min; Group 6 is CBA, 3E14 vg, 0.5 mL / min; and Group 7 is CBA, 1E14 vg, 2 mL / min, RA occlusion.7. DETAILED DESCRIPTION

[0081] The present disclosure provides non-surgical methods of administering a formulation to the renal pyramid (e.g., nephron) of a subject via the retro-ureteral route.

[0082] The methods described herein are advantageous for being able to administer formulations to nephrons in a non-surgical and non-systemic manner. This localized administration increases the potency and safety of kidney-directed therapeutics and diagnostics.

[0083] Existing methods for administering formulations to the kidneys have several limitations. Some methods are not aimed for administration beyond the renal pelvis, thus not suitable for administration into the nephrons (e.g., the retrograde pyelogram method; Wang et al., J Endourol, 27(3):288-93 (2013) and Donin et al., Urology, 99:270-77 (2017)).

[0084] Some methods involve surgery. Specifically, a retrograde injection (e.g., Chung et al., Nephron Extra., l(l):217-23 (2011)) requires an incision to the abdomen to access the kidney and ureter. The ureters and renal artery are then clamped to prevent systemic leaking following dose administration, and after administration of the product the hole in the abdomen is then sutured. Similarly, a retrograde renal vein injection (e g., Rocca et al., Gene Ther., 21(6):618-28 (2014)) involves an incision in the abdomen to access the kidney. After administration a clamp on the vein is held in place for a set time and then the hole in the abdomen is sutured. This method only has indirect access to tubule cells and cannot easily reach the collecting duct. Another method is subcapsular injection (e.g., Rubin et al., Hum Gene Ther., (12): 1559-71 (2019)), where the injection route focuses the administration of the drug around the area of the kidney that is directly injected and, therefore, may not allow for widespread administration across the kidney without multiple injection points. This route could also damage the renal parenchyma and blood vessels within the kidney. These surgical methods can be highly invasive and carry a risk of infection, tissue damage, surgical error, complications, etc. W02024 / 042022 disclosed a loco-perfusion system, where a drug can be directly administered to the kidney with a perfusion method through the renal artery and vein. However, given this system only delivers through the blood vessels, it has indirect access to tubule cells and cannot easily reach the collecting duct. In addition, this method may disrupt the blood flow to the kidney, which greatly increases the risk of the procedure to damage the kidney.7.1. Definitions

[0085] Unless otherwise defined herein, technical and scientific terms used in the present description have meanings that are commonly understood by those of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa unless the content clearly dictates otherwise. In the event that any description of a term set forth conflicts with any document incorporated herein by reference, the description of the term set forth below shall control.

[0086] The terms “a”, “an”, and “the”, as used herein, include plural references unless the context clearly dictates otherwise. As such, the terms “a,” “an,” “one or more,” and “at least one” are used interchangeably herein. In addition, reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.

[0087] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device or the method being employed to determine the value, or the variation that exists among the samples being measured Unless otherwise stated or otherwise evident from the context, the term “about” means within 10% above or below the reported numerical value (except where such number would exceed 100% of a possible value or go below 0%). When used in conjunction with a range or series of values, the term “about” applies to the endpoints of the range or each of the values enumerated in the series, unless otherwise indicated. As used in this application, the terms “about” and “approximately” are used as equivalents.

[0088] The term “and / or”, as used herein, refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0089] The term “between”, as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

[0090] The terms “comprise” and its grammatical equivalents, as used herein, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0091] The terms “including”, “includes”, “included”, and other forms, as used herein, are not limiting.

[0092] The term “administer”, “administration”, or “administering”, as used herein refers to the act of injecting or otherwise physically administering a substance (e.g., a pharmaceutical composition provided herein) to a subject (e.g., human), such as by oral, mucosal, topical, intradermal, parenteral, intravenous, intravitreal, intraarticular, subretinal, intramuscular, intrathecal administration and / or any other method of physical administration described herein or known in the art. The administration can be systemic or to a specific tissue.

[0093] The term “coding sequence” or a polynucleotide which “encodes” a polypeptide, as used herein, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence may be located 3’ to the coding sequence. A Kozak sequence may be located 5’ to the coding sequence.

[0094] As used herein, an “effective amount” or “therapeutically effective amount” is an amount or dose of a composition (e g. a therapeutic composition, compound, or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition. The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic composition (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type, disease stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, namely by monitoring a subject’ s response to administration of a composition and adjusting the dosage accordingly (see e.g. Remington: The Science and Practice of Pharmacy (Gennaro A, ed., Mack Publishing Co., Easton, PA, U.S., 19th ed., 1995)).

[0095] The term “formulation”, as used herein, refers to a solution capable of being administered to a subject for the treatment or diagnostic of a particular disease or disorder.

[0096] The term “non-surgical”, as used herein, refers to a medical procedure used to diagnose, measure, monitor, or treat a disease or disorder that does not involve incision into the skin.

[0097] The term “recombinant” means a genetic entity distinct from that generally found in nature. As applied to a polynucleotide or gene, this means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures that result in the production of a construct that is distinct from a polynucleotide found in nature.

[0098] The term “recombinant AAV vector (rAAV vector)”, as used herein, refers to a polynucleotide vector comprising a nucleic acid sequence from an AAV and one or more heterologous sequences (i.e., nucleic acid sequence not of AAV origin). In some embodiments, the one or more heterologous sequences are flanked by at least one, in some embodiments two, AAV inverted terminal repeat sequences (ITRs). In some embodiments, such rAAV vectors can be replicated and packaged into infectious viral capsid particles, e.g., when present in a host cell that has been infected with a suitable helper virus (or that is expressing suitable helper functions) and that is expressing AAV rep and cap gene products (i.e., AAV Rep and Cap proteins). An rAAV vector may be incorporated into a larger polynucleotide (e.g., in a chromosome or in another vector such as a plasmid used for cloning or transfection), and can be “rescued” by replication and encapsidation in the presence of AAV packaging functions and suitable helper functions. An rAAV vector can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed with lipids, encapsulated within liposomes, and encapsidated in a viral capsid particle, particularly an AAV particle. An rAAV vector can be packaged into an AAV capsid to generate a “recombinant adeno-associated virus (rAAV).”

[0099] As used herein, the term “subject” refers to any subject, e.g., a human or a nonhuman mammal, for whom diagnosis, prognosis, or therapy is desired. The term “subject” may mean a human or non-human mammal affected, likely to be affected, or suspected to be affected with a disease. The terms “subject” and “patient” are used interchangeably herein. In some embodiments, a subject is a mammal. A mammalincludes primates, such as humans, monkeys, chimpanzee, and apes, and non-primates such as domestic animals, including laboratory animals (such as rabbits and rodents, e.g., guinea pig, rat, or mouse) and household pets and farm animals (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals, such as wildlife, birds, reptile, fish, or the like. As used herein, the term “a subject in need thereof’ includes subjects that could or would benefit from the methods described herein. Subj ects in need of treatment include, without limitation, those already with the disease, disorder, or condition, those prone to having the disease, disorder, or condition, those in which the disease, disorder, or condition is suspected, as well as those in which the disease, disorder, or condition is to be prevented, ameliorated, or reversed. In some embodiments, the subject is seropositive for an rAAV prior to administration of the formulation comprising the rAAV. In some embodiments, the subject is seronegative for an rAAV prior to administration of the formulation comprising the rAAV.

[0100] The term “transduced”, as used herein, refers to a process by which a transgene is introduced into a host cell from a virus particle.

[0101] The terms “treatment” and “treating”, as used herein, refer to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and / or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying, or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0102] The term “vector”, as used herein, refers to a molecule or moiety which transports, transduces, or transfects a nucleic acid molecule of interest into a host cell or tissue. Vectors applicable for use include, for example, plasmids, phage vectors, viralvectors, episomes, and artificial chromosomes. A vector may include sequences that direct autonomous replication in a cell, or may include sequences sufficient to allow integration into host cell DNA. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0103] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed (Sambrook et al., HaRBor Laboratory Press 2001 ); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0104] In some embodiments, the intrarenal pressure is maintained above baseline during or after administration of the formulation via RUA. The term “baseline” as used herein refers to an intrarenal pressure between about 0 mm Hg and about 20 mm Hg. In some embodiments, the term “baseline” refers to an intrarenal pressure of about 1 mm Hg, about 2 mm Hg, about 3 mm Hg, about 4 mm Hg, about 5 mm Hg, about 6 mm Hg, about 7 mm Hg, about 8 mm Hg, about 9 mm Hg, about 10 mm Hg, about 11 mm Hg, about 12 mm Hg, about 13 mm Hg, about 14 mm Hg, about 15 mm Hg, about16 mm Hg, about 17 mm Hg, about 18 mm Hg, about 19 mm Hg, or about 20 mm Hg. In some embodiments, the intrarenal pressure is measured using a pressure transducer. In some embodiments, the intrarenal pressure is measured by a pressure wire.7.2. Methods of Formulation Administration to the Kidneys

[0105] Provided herein are methods of administering a formulation to the renal pyramid of a subject. In some embodiments, the formulation is administered through the retro-ureteral route. In some embodiments, the formulation is not directly administered through a renal artery or a renal vein. In some embodiments, the formulation is administered to one or more nephrons in the renal pyramid. In some embodiments, the method comprises administering the formulation through the retro- ureteral route into one or more nephrons in the renal pyramid. In some embodiments, the administration is non-surgical. In some embodiments, the formulation is administered through a catheter.

[0106] In some embodiments, the retro-ureteral method comprises: (a) inserting a cystoscope into the urethra of a subject and advancing to the bladder; (b) identifying a ureteral orifice in the bladder of the subject using the cystoscope; (c) advancing a guidewire from the cystoscope through the bladder into the ureteral orifice to the upper ureter, ureteropelvic junction, or renal pelvis of the subject; (d) removing the cystoscope, (e) advancing a balloon catheter into the upper ureter, ureteropelvic junction, or renal pelvis and inflating the balloon to occlude the ureter; (f) priming the dosing system with formulation; (g) injecting the formulation through the catheter into the renal pelvis and renal parenchyma of the subject at a controlled rate using a syringe pump; and (h) allowing the balloon to remain inflated after the administration for a period of time (i.e. a dwell period). In some embodiments, a pressure transducer was added to the end of the catheter to measure intrarenal pressure during and after the administration. In some embodiments, the intrarenal pressure is maintained above baseline during and after the administration. In some embodiments, the renal artery or renal vein is not occluded or blocked during or after the administration. In some embodiments, the administered volume of the formulation is a fixed volume that is not dependent on the subject’s body weight or size of the renal pelvis. In some embodiments, the formulation is an AAV formulation. In some embodiments, the ureter is occluded during the administration to prevent the formulation from draining from the renal pelvis. This allows the formulation to stay within the renal pelvis for a longerperiod of time and to maintain an increased pressure within the renal pelvis, both of which are helpful for drug administration to the renal pyramid (e.g., nephron).

[0107] In some embodiments, the method comprises priming the dosing system before RUA of the formulation to the subject. As used herein, “priming the dosing system” refers to filling the dosing system with the formulation prior to inj ecting the formulation through the catheter into the renal pelvis and renal parenchyma of the subject. In some embodiments, the dosing system is primed with about 1 mL to about 10 mL of the formulation. In some embodiments, the dosing system is primed with about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL of the formulation. In some embodiments, the dosing system is primed with about 1 mL to about 2 mL of the formulation. In some embodiments, the dosing system is primed with about 1.0 mL, about 1.25 mL, about 1.5 mL, about 1.75 mL, or about 2.0 mL of the formulation. In some embodiments, the dosing system is primed with about 1.75 mL of the formulation. In some embodiments, the dosing system is a catheter, an extension set, a syringe and any other additional components through which the formulation makes contact with.

[0108] In some embodiments, the ureter is occluded at a location lower than the location of the end of the catheter. A skilled artisan would understand that the upper end of the ureter is connected with the renal pelvis and the lower end of the ureter is connected with the bladder. Therefore, the location of the end of the catheter is closer to the renal pelvis than the location of the occlusion.

[0109] In some embodiments, the ureter is occluded at the upper ureter near the ureteropelvic junction. In some embodiments, the ureter is occluded at the ureteropelvic junction. In some embodiments, the ureter is occluded at the upper ureter near the ureteropelvic junction due to the difficulty in reaching the ureteropelvic junction. In some embodiments, the difficulty is caused by more curvature in the ureters of the subject.

[0110] In some embodiments, the ureter is occluded by an inflatable balloon. In some embodiments, the inflatable balloon is connected with the catheter (i.e., a balloon catheter).

[0111] In some embodiments, the method further comprises occluding a renal blood vessel of the kidney selected from the group consisting of: a renal artery, a renal vein, and a combination thereof, prior to administering the formulation. In someembodiments, the renal blood vessel is occluded with a clamp or a balloon catheter. In some embodiments, occluding the renal blood vessel blocks the kidney from systemic circulation.

[0112] As used herein, the term “systemic circulation” refers to a flow of blood through a subject’s vascular system from the heart to organs and tissues throughout the body (including kidneys) and back to the heart. The vascular system in the kidney comprises a renal artery, which supplies oxygenated blood to the kidney, and a renal vein which carries deoxygenated blood away from the kidney.

[0113] In some embodiments, the method described herein does not isolate the kidney from systemic circulation during the performance of said method. In some embodiments, the method described herein does not occlude or block the renal artery, renal vein, or a combination thereof. In some embodiments, the method described herein does not occlude or block the renal artery. In some embodiments, the method described herein does not occlude or block the renal vein. In some embodiments, the method described herein does not occlude or block the renal vein or the renal artery.

[0114] In some embodiments, the formulation is manually delivered into the ureter using a plunger associated with a catheter or cannula. In some embodiments, the formulation is manually delivered into the ureter using an automatic injection, e.g., a pump (a syringe pump, peristaltic pump, etc ).

[0115] In some embodiments, the formulation is injected over a time period of about 1 second to about 3 minutes, about 1 second to about 2 minutes, about 1 second to about 1 minute, about 30 seconds to about 1 minute, about 1 minute to about 2 minutes, about 30 seconds to about 2 minutes. In some embodiments, the formulation is injected over a time period of about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 11 seconds, about 12 seconds, about 13 seconds, about 14 seconds, about 15 seconds, about 16 seconds, about 17 seconds, about 18 seconds, about 19 seconds, about 20 seconds, about 21 seconds, about 22 seconds, about 23 seconds, about 24 seconds, about 25 seconds, about 26 seconds, about 27 seconds, about 28 seconds, about 29 seconds, about 30 seconds, about 31 seconds, about 32 seconds, about 33 seconds, about 34 seconds, about 35 seconds, about 36 seconds, about 37 seconds, about 38 seconds, about 39 seconds, about 40 seconds, about 41 seconds, about 42 seconds, about 43 seconds, about 44 seconds, about 45 seconds,about 46 seconds, about 47 seconds, about 48 seconds, about 49 seconds, about 50 seconds, about 51 seconds, about 52 seconds, about 53 seconds, about 54 seconds, about 55 seconds, about 56 seconds, about 57 seconds, about 58 seconds, about 59 seconds, about 1 minute, about 1.25 minutes, about 1.5 minutes, about 1.75 minutes, about 2 minutes, about 2.25 minutes, about 2.5 minutes, about 2.75 minutes, about 3 minutes, about 3.25 minutes, about 3.5 minutes, about 3.75 minutes, about 4 minutes, about 4.25 minutes, about 4.5 minutes, about 4.75 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes about 10 minutes, about 11 minutes, about 12 minutes about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, or more.

[0116] In some embodiments, the formulation is administered at different rates during the administration process. In some embodiments, the formulation is administered at a constant rate. In some embodiments, the formulation is administered at the rate of about 0.1 mL / min to about 20 mL / min. In some embodiments, the formulation is administered at the rate of at least about 0.1 mL / min. In some embodiments, the formulation is administered at the rate of at most about 20 mL / min. In some embodiments, the formulation is administered at the rate of about 0.1 mL / min to about 0.5 mL / min, about 0.1 mL / min to about 1 mL / min, about 0.1 mL / min to about 2 mL / min, about 0.1 mL / min to about 3 mL / min, about 0.1 mL / min to about 4 mL / min, about 0.1 mL / min to about 5 mL / min, about 0.1 mL / min to about 6 mL / min, about 0.1 mL / min to about 7 mL / min, about 0.1 mL / min to about 8 mL / min, about 0.1 mL / min to about 10 mL / min, about 0.1 mL / min to about 20 mL / min, about 0.5 mL / min to about 1 mL / min, about 0.5 mL / min to about 2 mL / min, about 0.5 mL / min to about 3 mL / min, about 0.5 mL / min to about 4 mL / min, about 0.5 mL / min to about 5 mL / min, about 0.5 mL / min to about 6 mL / min, about 0.5 mL / min to about 7 mL / min, about 0.5 mL / min to about 8 mL / min, about 0.5 mL / min to about 10 mL / min, about 0.5 mL / min to about 20 mL / min, about 1 mL / min to about 2 mL / min, about 1 mL / min to about 3 mL / min, about 1 mL / min to about 4 mL / min, about 1 mL / min to about 5 mL / min, about 1 mL / min to about 6 mL / min, about 1 mL / min to about 7 mL / min, about 1 mL / min to about 8 mL / min, about 1 mL / min to about 10 mL / min, about 1 mL / min to about 20 mL / min, about 2 mL / minto about 3 mL / min, about 2 mL / min to about 4 mL / min, about 2 mL / min to about 5 mL / min, about 2 mL / min to about 6 mL / min, about 2 mL / min to about 7 mL / min, about2 mL / min to about 8 mL / min, about 2 mL / min to about 10 mL / min, about 2 mL / min to about 20 mL / min, about 3 mL / min to about 4 mL / min, about 3 mL / min to about 5 mL / min, about 3 mL / min to about 6 mL / min, about 3 mL / min to about 7 mL / min, about3 mL / min to about 8 mL / min, about 3 mL / min to about 10 mL / min, about 3 mL / min to about 20 mL / min, about 4 mL / min to about 5 mL / min, about 4 mL / min to about 6 mL / min, about 4 mL / min to about 7 mL / min, about 4 mL / min to about 8 mL / min, about4 mL / min to about 10 mL / min, about 4 mL / min to about 20 mL / min, about 5 mL / min to about 6 mL / min, about 5 mL / min to about 7 mL / min, about 5 mL / min to about 8 mL / min, about 5 mL / min to about 10 mL / min, about 5 mL / min to about 20 mL / min, about 6 mL / min to about 7 mL / min, about 6 mL / min to about 8 mL / min, about 6 mL / min to about 10 mL / min, about 6 mL / min to about 20 mL / min, about 7 mL / min to about 8 mL / min, about 7 mL / min to about 10 mL / min, about 7 mL / min to about 20 mL / min, about 8 mL / min to about 10 mL / min, about 8 mL / min to about 20 mL / min, or about 10 mL / min to about 20 mL / min. In some embodiments, the formulation is administered at the rate of about 0.1 mL / min, about 0.5 mL / min, about 1 mL / min, about 2 mL / min, about 3 mL / min, about 4 mL / min, about 5 mL / min, about 6 mL / min, about 7 mL / min, about 8 mL / min, about 10 mL / min, or about 20 mL / min.

[0117] In some embodiments, the administered volume of the formulation is about 5 mL to about 40 mL. In some embodiments, the administered volume of the formulation is at least about 5 mL. In some embodiments, the administered volume of the formulation is at most about 40 mL. In some embodiments, the administered volume of the formulation is about 5 mL to about 10 mL, about 5 mL to about 15 mL, about 5 mL to about 20 mL, about 5 mL to about 25 mL, about 5 mL to about 30 mL, about 5 mL to about 35 mL, about 5 mL to about 40 mL, about 10 mL to about 15 mL, about 10 mL to about 20 mL, about 10 mL to about 25 mL, about 10 mL to about 30 mL, about 10 mL to about 35 mL, about 10 mL to about 40 mL, about 15 mL to about 20 mL, about 15 mL to about 25 mL, about 15 mL to about 30 mL, about 15 mL to about 35 mL, about 15 mL to about 40 mL, about 20 mL to about 25 mL, about 20 mL to about 30 mL, about 20 mL to about 35 mL, about 20 mL to about 40 mL, about 25 mL to about 30 mL, about 25 mL to about 35 mL, about 25 mL to about 40 mL, about 30 mL to about 35 mL, about 30 mL to about 40 mL, about 35 mL to about 40 mL, about 40mL to about 45 mL, about 45 mL to about 50 mL, about 50 mL to about 55 m , about 55 mL to about 60 mL, about 60 mL to about 65 mL, about 65 mL to about 70 mL, about 70 mL to about 75 mL, about 75 mL to about 80 mL, about 80 mL to about 85 mL, about 85 mL to about 90 mL, about 90 mL to about 95 mL, or about 95 mL to about 100 mL. In some embodiments, the administered volume of the formulation is about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, about 40 mL, about 41 mL, about 42 mL, about 43 mL, about 44 mL, about 45 mL, about 46 mL, about 47 mL, about 48 mL, about 49 mL, about 50 mL, about 51 mL, about 52 mL, about 53 mL, about 54 mL, about 55 mL, about 56 mL, about 57 mL, about 58 mL, about 59 mL, about 60 mL, about 61 mL, about 62 mL, about 63 mL, about 64 mL, about 65 mL, about 66 mL, about 67 mL, about 68 mL, about 69 mL, about 70 mL, about 71 mL, about 72 mL, about 73 mL, about 74 mL, about 75 mL, about 76 mL, about 77 mL, about 78 mL, about 79 mL, about 80 mL, about 81 mL, about 82 mL, about 83 mL, about 84 mL, about 85 mL, about 86 mL, about 87 mL, about 88 mL, about 89 mL, about 90 mL, about 91 mL, about 92 mL, about 93 mL, about 94 mL, about 95 mL, about 96 mL, about 97 mL, about 98 mL, about 99 mL, about 100 mL, or more. In some embodiments, the administered volume of the formulation is 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, 50 mL, 51 mL, 52 mL, 53 mL, 54 mL, 55 mL, 56 mL, 57 mL, 58 mL, 59 mL, 60 mL, 61 mL, 62 mL, 63 mL, 64 mL, 65 mL, 66 mL, 67 mL, 68 mL, 69 mL, 70 mL, 71 mL, 72 mL, 73 mL, 74 mL, 75 mL, 76 mL, 77 mL, 78 mL, 79 mL, 80 mL, 81 mL, 82 mL, 83 mL, 84 mL, 85 mL, 86 mL, 87 mL, 88 mL, 89 mL, 90 mL, 91 mL, 92 mL, 93 mL, 94 mL, 95 mL, 96 mL, 97 mL, 98 mL, 99 mL, or 100 mL. In some embodiments, the administered volume of the formulation provided herein is for each kidney. In some embodiments, the administered volume of the formulation is a fixed volume. In some embodiments, theadministered volume is not dependent on the subject’s body weight. In some embodiments, the administered volume is not dependent on the size of the renal pelvis.

[0118] In some embodiments, the administered volume of the formulation is based on the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 0.1 mL to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 0.5 mL to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 1 mL to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 2 mL to about 25 mL larger than the volume of each kidney of the renal pelvis of the subject. In some embodiments, the administered volume of the formulation is about 5 mL to about 20 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, or about 30 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is about 10 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is 10 mL larger than the volume of the renal pelvis of each kidney of the subject. In some embodiments, the administered volume of the formulation is a fixed volume. In some embodiments, the administered volume is not dependent on the size of the renal pelvis.

[0119] In some embodiments, the volume of the renal pelvis is estimated morphologically using images obtained through computed tomography (CT), magnetic resonance imaging (MRI), or sonography using ultrasound (US). In some embodiments, the volume of the renal pelvis is determined directly by injecting a fluorescent or contrast agent into the renal pelvic space at the time of the procedure. A skilled artisan would understand how to measure the volume of the renal pelvis of the subject.

[0120] In some embodiments, the administered volume of the formulation is about 0.1 mL to about 0.3 mL / kg, about 0.1 mL to about 0.4 mL / kg, about 0.1 mL to about 0.5 mL / kg, about 0.2 mL to about 0.4 mL / kg, about 0.2 mL to about 0.5 mL / kg, about 0.2 mL to about 0.6 mL / kg, about 0.3 mL to about 0.5 mL / kg, about 0.3 mL to about 0.6 mL / kg, about 0.3 mL to about 0.7 mL / kg, about 0.4 mL to about 0.6 mL / kg, about 0.4 mL to about 0.7 mL / kg, about 0.4 mL to about 0.8 mL / kg, about 0.5 mL to about 0.7 mL / kg, about 0.5 mL to about 0.8 mL / kg, about 0.5 mL to about 0.9 mL / kg, about 0.6 mL to about 0.8 mL / kg, about 0.6 mL to about 0.9 mL / kg, about 0.6 to about 1 mL / kg, about 0.7 mL to about 0.9 mL / kg, about 0.7 mL to about 1 mL / kg, or about 0.8 to about 1 mL / kg. In some embodiments, the administered volume of the formulation is about 0.25 mL / kg. In some embodiments, the administered volume of the formulation is about 0.3 mL / kg. In some embodiments, the administered volume of the formulation is about 0.35 mL / kg In some embodiments, the administered volume of the formulation is a fixed volume. In some embodiments, the administered volume is not dependent on the subject’s body weight.

[0121] In some embodiments, the method comprises administering a formulation through a catheter into the renal pelvis and renal parenchyma of a subject. In some embodiments, the method further comprises administering a volume of fluid through the catheter to flush the catheter line of the remaining formulation. In some embodiments, the volume of fluid is about 1 mL, about 2 mL, about 3 mL, about 4 mL, about 5 mL, about 6 mL, about 7 mL, about 8 mL, about 9 mL, or about 10 mL. In some embodiments, the volume of fluid is about 3 mL. In some embodiments, the fluid is a buffer, such as normal saline (i.e., sodium chloride in water). In some embodiments, the fluid is a buffer and the buffer is phosphate buffered saline. In some embodiments, the buffer comprises a detergent (e.g. , Pluronic F-68®), a cryoprotectant (e.g. , trehalose or mannitol), or a stabilizer (e.g., histidine). In some embodiments, the fluid is a formulation buffer that can vary in composition depending on the manufacturer.

[0122] In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below 190 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 170 mm Hg during theadministration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 160 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 150 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 140 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 130 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 120 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 110 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 100 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 90 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 80 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 70 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 60 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 50 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 40 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 30 mm Hg during the administration. In some embodiments, the intrarenal pressure is below a level that can be harmful to the kidney. A skilled artisan would understand and determine the pressure level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is above baseline during the administration. In some embodiments, the intrarenal pressure is measured by placing a pressure monitor on the end of the catheter. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer. In some embodiments, the formulation is administered at a constant rate of 0.5 mL / min, 1 mL / min, or 2 mL / min.

[0123] In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 200 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below 190 mm Hg during theadministration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 170 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 160 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 150 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 140 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 130 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 120 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 110 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 100 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 90 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 80 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 70 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 60 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about 50 mm Hg during the administration. In some embodiments, the intrarenal pressure is above about 40 mm Hg and below about a level that can be harmful to the kidney. A skilled artisan would understand and determine the pressure level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is above baseline during the administration. In some embodiments, the intrarenal pressure is measured by placing a pressure monitor on the end of the catheter. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer. In some embodiments, the formulation is administered at a constant rate of 0.5 mL / min, 1 mL / min, or 2 mL / min.

[0124] In some embodiments, the maximum intrarenal pressure during the administration is less than about 200 mmHg, less than about 190 mmHg, less than about 180 mmHg, less than about 170 mmHg, less than about 160 mmHg, less than about 150mmHg, less than about 140 mmHg, less than about 130 mmHg, less than about 120 mmHg, less than about 110 mmHg, or less than about 100 mmHg during the administration of the formulation. In some embodiments, the maximum intrarenal pressure is 200 mm Hg, 190 mm Hg, 180 mm Hg, 170 mm Hg, 160 mm Hg, 150 mm Hg, 140 mm Hg, 130 mm Hg, 120 mm Hg, 110 mm Hg, 100 mm Hg, 90 mm Hg, 80 mm Hg, 70 mm Hg, 60 mm Hg, 50 mm Hg, or any value therebetween, during the administration of the formulation. In some embodiments, the formulation is administered at a constant rate of 0.5 mL / min, 1 mL / min, or 2 mL / min. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0125] In some embodiments, maximum intrarenal pressure is achieved after an administered volume of about 1 mL, 5 mL, about 6 mL, about 7 mL, about 8 mb, about 9 mL, about 10 mL, about 11 mL, about 12 mL, about 13 mL, about 14 mL, about 15 mL, about 16 mL, about 17 mL, about 18 mL, about 19 mL, about 20 mL, about 21 mL, about 22 mL, about 23 mL, about 24 mL, about 25 mL, about 26 mL, about 27 mL, about 28 mL, about 29 mL, about 30 mL, about 31 mL, about 32 mL, about 33 mL, about 34 mL, about 35 mL, about 36 mL, about 37 mL, about 38 mL, about 39 mL, or about 40 mL. In some embodiments, the maximum intrarenal pressure is 200 mm Hg, 190 mm Hg, 180 mm Hg, 170 mm Hg, 160 mm Hg, 150 mm Hg, 140 mm Hg, 130 mm Hg, 120 mm Hg, 110 mm Hg, 100 mm Hg, or lower. In some embodiments, the formulation is administered at a constant rate of 1 mL / min, 2 mL / min, or 5 mL / min. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0126] In some embodiments, the intrarenal pressure is measured during the administration. In some embodiments, the intrarenal pressure is measured every 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, or 100 minutes during the administration of the formulation. In some embodiments, the formulation is administered at a constant rate of 0.5 mL / min, 1 mL / min, or 2 mL / min. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0127] In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 160 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 140 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 120 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 100 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 80 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 60 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 1 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 40 mm Hg during the administration. In some embodiments, the intrarenal pressure is below a level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is above baseline during the administration. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0128] In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2mL / min and the intrarenal pressure is above about 20 mm Hg and below about 160 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 140 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 120 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 100 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 80 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 60 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 2 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 40 mm Hg during the administration. In some embodiments, the intrarenal pressure is below a level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is above baseline during the administration. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0129] In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 180 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 160 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 140 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 120mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 100 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 80 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 60 mm Hg during the administration. In some embodiments, the formulation is administered at a constant rate of at least about 0.5 mL / min and the intrarenal pressure is above about 20 mm Hg and below about 40 mm Hg during the administration. In some embodiments, the intrarenal pressure is below a level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is maintained above baseline during the administration In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0130] In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 190 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 180 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 170 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 160 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 150 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 140 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 130 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 120 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 110 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 100 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 90 mm Hg during the dwell period.In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 80 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 70 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 60 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 50 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 40 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is above about 20 mm Hg and below about 30 mm Hg during the dwell period. In some embodiments, the intrarenal pressure is below a level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is maintained above baseline during the dwell period. A skilled artisan would understand and determine the pressure level that can be harmful to the kidney. In some embodiments, the intrarenal pressure is measured by placing a pressure monitor on the end of the catheter. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0131] In some embodiments, the maximum intrarenal pressure is 200 mm Hg, 190 mm Hg, 180 mm Hg, 170 mm Hg, 160 mm Hg, 150 mm Hg, 140 mm Hg, 130 mm Hg, 120 mm Hg, 110 mm Hg, 100 mm Hg, 90 mm Hg, 80 mm Hg, 70 mm Hg, 60 mm Hg, 50 mm Hg, or any value therebetween, during the dwell period. As used herein, the “maximum intrarenal pressure” is the highest pressure measured and / or recorded within the kidney during the RUA procedure. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0132] In some embodiments, the intrarenal pressure is measured during the dwell period. In some embodiments, the intrarenal pressure is measured every 10 seconds, 15 seconds, 30 seconds, 45 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, or 30 minutes during the dwell period. In some embodiments, the intrarenal pressure is measured by a pressure wire. In some embodiments, the intrarenal pressure is measured using a pressure transducer.

[0133] In some embodiments, the ureter remains occluded for about 5 minutes to about 60 minutes after the administration. In some embodiments, this aims to elongate the dwell time of the formulation in the renal pelvis and to maintain an increased pressurewithin the renal pelvis, both of which are helpful for drug administration to the renal pyramid (e g., nephron). In some embodiments, the ureter remains occluded for about 5 minutes to about 55 minutes after the administration. In some embodiments, the ureter remains occluded for about 10 minutes to about 50 minutes after the administration. In some embodiments, the ureter remains occluded for about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, or about 50 minutes after the administration.

[0134] In some embodiments, the formulation is a therapeutic formulation. In some embodiments, the therapeutic formulation is a slow-release formulation.

[0135] In some embodiments, the therapeutic formulation is for treating bladder cancer. In some embodiments, the therapeutic formulation is for treating a kidney disease. In some embodiments, the kidney disease is chronic kidney disease. In some embodiments, the kidney disease is end-stage renal disease. In some embodiments, the kidney disease is a genetic kidney disease. In some embodiments, the kidney disease is autosomal dominant polycystic kidney disease (ADPKD). In some embodiments, the kidney disease is autosomal recessive PKD (ARPKD) In some embodiments, the kidney disease is diabetic nephropathy. In some embodiments, the kidney disease is IgA nephropathy. In some embodiments, the kidney disease is Alport syndrome. In some embodiments, the kidney disease is cystinosis. In some embodiments, the kidney disease is kidney cancer. In some embodiments, the kidney disease is Gitelman syndrome. In some embodiments, the kidney disease is Nephronophthisis (NPHP). In some embodiments, the kidney disease is glomerular disease. In some embodiments, the kidney disease is Fabry disease. In some embodiments, the kidney disease is familial amyloidosis. In some embodiments, the kidney disease is congenital nephrotic syndrome. In some embodiments, the kidney disease is proximal tubule disease. In some embodiments, the kidney disease is autosomal dominant tubulointerstitial kidney disease (ADTKD). In some embodiments, the kidney disease is cystinuria. In some embodiments, the kidney disease is proximal renal tubular acidosis. In some embodiments, the kidney disease is Dent disease. In some embodiments, the kidney disease is thick ascending limb disease. In some embodiments, the kidney disease is distal convoluted tubule disease. In some embodiments, the kidney disease is Bartter syndrome. In some embodiments, the kidney disease is collecting duct disease. In some 1embodiments, the kidney disease is Liddle syndrome. In some embodiments, the kidney disease is distal renal tubular acidosis.

[0136] In some embodiments, the therapeutic formulation is for treating a kidney- associated disorder. In some embodiments, the kidney-associated disorder is selected from the group consisting of: autosomal dominant polycystic kidney disease (ADPKD); ADPKD1, ADPKD2, Alport syndrome; autosomal dominant tubulointerstitial kidney disease (ADTKD); medullary cystic kidney disease; nephronophthisis; Bartter Syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; Thin Basement Membrane Nephropathy; cystinuria; Liddle syndrome; Papillorenal syndrome; and cystinosis. In some embodiments, the kidney disease or kidney-associated disorder is selected from the group consisting of: Apparent mineralocorticoid excess, Autosomal dominant hypocalcemia, Autosomal dominant hypomagnesemia, Bartter type 1, Bartter type 2, Bartter type 3, Bartter type 4a, Bartter type 4b, Bartter type 5, Congenital adrenal hyperplasia type 1, Congenital adrenal hyperplasia type 2, Congenital adrenal hyperplasia type 4, Congenital adrenal hyperplasia type 5, Cystinuria A, Cystinuria B, Dent disease type 1, Dent disease type 2 / Lowe syndrome, Dicarboxylic aminoaciduria, Distal RTA, EAST / SeSAME syndrome, Fanconi Bickel syndrome, Fanconi renotubular syndrome 1, Fanconi renotubular syndrome 2, Fanconi renotubular syndrome 3, Fanconi renotubular syndrome 4, Gitelman syndrome, Glucocorticoid remediable aldosteronism, Hartnup disorder, Hereditary hypophosphatemic rickets with hypercalciuria, HNFIB-related kidney disease, Hyperphenylalaninemia BH4- deficient, Hypomagnesemia type 1 / hypomagnesemia with secondary hypocalcemia, Hypomagnesemia type 2, Hypomagnesemia type 3 / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, Hypomagnesemia type 4, Hypomagnesemia type 5 / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, Hypomagnesemia, seizures, and mental retardation type 1, Hypomagnesemia, seizures, and mental retardation type 2, Iminoglycinuria, Kenny-Caffey syndrome type 2, Liddle syndrome, Lysinuric protein intolerance, Neonatal inflammatory skin and bowel disease type 2, Nephrogenic diabetes insipidus, Nephrogenic syndrome of inappropriate antidiuresis, Pseudohypoaldosteronism type 1, Pseudohypoaldosteronism type 1A, Pseudohypoaldosteronism type 2b, Pseudohypoaldosteronism type 2c,Pseudohypoaldosteronism type 2d, Pseudohypoaldosteronism type 2e, Renal tubular acidosis type 3, and X-linked hypophosphatemic rickets.

[0137] In some embodiments, treating refers to the treatment of a disease in a mammal, e.g., in a human, including (a) inhibiting the disease, i.e., arresting disease development or preventing disease progression; (b) relieving the disease, i.e., causing regression of the disease state or relieving one or more symptoms of the disease; and (c) curing the disease, i.e., remission of one or more disease symptoms. In some embodiments, treatment results in an improvement or remediation of the symptoms of the disease. In some embodiments, treatment may refer to a short-term (e.g., temporary and / or acute) and / or a long-term e.g., sustained) improvement or remediation in one or more disease symptoms. In some embodiments, the improvement is an observable or measurable improvement. In some embodiments, the improvement is an improvement in the general feeling of well-being of the subject. In some embodiments, the method inhibits or ameliorates renal cyst development.

[0138] In some embodiments, the therapeutic formulation comprises one or more pharmaceutical compositions for treating a kidney disease or condition. In some embodiments, the active ingredient of the therapeutic formulation is a small molecule for treating a kidney disease or condition. In some embodiments, the therapeutic formulation comprises a large molecule (e g., an antibody) for treating a kidney disease or condition. In some embodiments, the therapeutic formulation comprises stem cells for treating a kidney disease or condition. In some embodiments, the therapeutic formulation comprises a gene therapy for treating a kidney disease or condition. In some embodiments, the therapeutic formulation comprises a therapeutic RNA. In some embodiments, the therapeutic formulation comprises a therapeutic protein.

[0139] In some embodiments, the active ingredient of the therapeutic formulation is a small molecule for treating a kidney disease or condition. In some embodiments, the active ingredient of the therapeutic formulation is an activator of phosphodiesterase-4 (PDE4). In some embodiments, the activator of PDE4 is LoAc (Mironid). In some embodiments, the active ingredient of the therapeutic formulation is an activator of nuclear factor 2 erythroid 2 (NRF2). In some embodiments, the activator of NRF2 is AL-1311 (AceLink Therapeutics). In some embodiments, the active ingredient of the therapeutic formulation is an activator of AMP-activated protein kinase (AMPK). In some embodiments, the activator of AMPK is PXL-770 (Poxel). In some embodiments,the active ingredient of the therapeutic formulation is an inhibitor of mitochondrial Complex I. In some embodiments, the active ingredient of the therapeutic formulation is an inhibitor of receptor tyrosine kinase (RTK). In some embodiments, the inhibitor of RTK is tesevatinib (Sanofi). In some embodiments, the active ingredient of the therapeutic formulation is an inhibitor of Xanthine Oxidase. In some embodiments, the inhibitor of Xanthine Oxidase is XRx-008 (XORTX Therapeutics). In some embodiments, the active ingredient of the therapeutic formulation is an inhibitor of a vasopressin receptor. In some embodiments, the inhibitor of a vasopressin receptor is tolvaptan. In some embodiments, the active ingredient of the therapeutic formulation is an inhibitor of angiotensin converting enzyme (ACE). In some embodiments, the active ingredient of the therapeutic formulation is an inhibitor of sodium-glucose cotransporter-2 (SGLT2). In some embodiments, the active ingredient of the therapeutic formulation is an agonist of a somatostatin receptor (e g., SST3). In some embodiments, the active ingredient of the therapeutic formulation is an antagonist of V2 receptor. In some embodiments, the antagonist of V2 receptor is Jynarque (Otsuka Holdings). In some embodiments, the active ingredient of the therapeutic formulation is an angiotensin receptor blocker (ARB). In some embodiments, the active ingredient of the therapeutic formulation is a pain reliever. In some embodiments, the pain reliever is acetaminophen. In some embodiments, the active ingredient of the therapeutic formulation is an antibiotic. In some embodiments, the active ingredient of the therapeutic formulation is cysteamine bitartrate. In some embodiments, the active ingredient of the therapeutic formulation is pasireotide. In some embodiments, the active ingredient of the therapeutic formulation is JNJ-0237 (Johnson & Johnson).

[0140] In some embodiments, the therapeutic formulation comprises a gene therapy for treating a kidney disease or condition. In some embodiments, the gene therapy is delivered by lipid nanoparticles. In some embodiments, the gene therapy is delivered by viral vectors. Examples of viral vectors for gene therapy include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors, and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.

[0141] In some embodiments, the vectors disclosed herein comprise a promoter operably linked to a transgene. In some embodiments, the promoter is a ubiquitous promoter such as a cytomegalovirus (CMV) promoter, a CAG promoter, a chicken beta actin (CBA) promoter, an SV40 promoter, a ubiquitin promoter, a GAPDH promoter, a phosphoglycerate kinase (PGK) promoter, or a rous sarcoma virus (RSV) promoter. In some embodiments, the promoter is a kidney-specific or cell type-specific promoter, such as a mouse polycystin 2 (PKD2) promoter, a human PKD2 promoter, a fibrocystin promoter (PKHD1), a podocin (NPHS2) promoter, a nephrin (NPHS1) promoter, a synaptopodin promoter, a WT1 promoter, a podocalyxin prompter, a PAX8 promoter, a gamma-glutamyltransferase (GGT) promoter, a sodium-glucose cotransporter-2 (SGLT2) promoter, a kidney androgen-regulated protein (KAP) promoter, a megalin promoter, a carbonic anhydrase II (CAII) promoter, an organic anion transporter 1 (OAT1, SLC22A6) promoter, a sodium phosphate cotransporter 2a (NPT2a) promoter, an E-cadherin (ECAD) promoter, a glucose-6-phosphatase (G6Pase) promoter, a kidney-specific cadherin (KSP-C) promoter, an elongation factor-1 alpha (EF-la) promoter, a Na-K-Cl cotransporter 2 (NKCC2, SLC12A1) promoter, a WNK1 promoter, a WNK4 promoter, a v-type proton ATPase subunit Bl (ATP6V1B1) promoter, a thiazide-sensitive Na-Cl cotransporter (NCC) promoter, a calcium-sensing receptor (CaSR) promoter, an epithelial sodium channel (ENaC) promoter, a uromodulin promoter, an aquaporin 1 (AQP1) promoter, an aquaporin 2 (AQP2) promoter, a vasopressin receptor 2 (V2R) promoter, a transient receptor potential vanilloid 4 (TRPV4) promoter, a renin promoter, a parathyroid hormone receptor promoter, a smooth muscle alpha-actin (ACTA2) promoter, a COL4A1 promoter, a platelet-derived growth factor receptor beta (PDGFRU) promoter, a smooth muscle protein 22-a (SM22a) promoter, a smooth muscle myosin heavy chain (SM- MHC) promoter, a neuron-glial antigen 2 (NG2, chondroitin sulphate proteoglycan 4 (CSPG4)) promoter, a desmin promoter, a regulator of G-protein signaling-5 (RGS5) promoter, a fibroblast-specific protein 1 (FSP1) promoter, a transcription factor 21 (TCF21) promoter, a COL1A2 promoter, a COL3A1 promoter, a vimentin promoter, a TIE2 promoter, a von Willebrand factor (vWF) promoter, a CD31 promoter, or a VE- cadherin promoter. In some embodiments, the transgene encodes a therapeutic RNA or therapeutic protein. In some embodiments, the vector is a viral vector such as rAAV.

[0142] In some embodiments, the vectors described herein comprise a gene encoding a selectable marker, which includes, but is not limited to, a protein whose expression can be readily detected such as a fluorescent or luminescent protein or an enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance (“detectable markers”). Detectable markers include green fluorescent protein (GFP) blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and variants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also detectable markers. In some embodiments, the vector is a viral vector such as rAAV.

[0143] In some embodiments, the formulation comprises a pharmaceutically acceptable carrier or excipient.

[0144] In some embodiments, the formulation comprises at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is a membrane, a lipid bilayer and / or a polymeric carrier (e g., a lipid nanoparticle).

[0145] In some embodiments, the formulation comprises at least one pharmaceutically acceptable carrier. Illustrative pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions. Examples of suitable aqueous and nonaqueous carriers that may be used in the formulations described herein include water, ethanol, polyols e.g., glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, such as vegetable oils and injectable organic esters.

[0146] In some embodiments, the formulation comprises at least one additional pharmaceutically acceptable excipient. Non-limiting examples of a pharmaceutically acceptable excipient include arginine, arginine sulfate, citric acid, glycerol, hydrochloric acid, mannitol, methionine, polysorbate, sodium chloride, sodium citrate, sodium hydroxide, sorbitol, sucrose, trehalose, and / or water.

[0147] In some embodiments, the formulation comprises a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include both acid and base addition salts. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2- di chloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid,benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, ptoluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2- diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. In some embodiments, organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. Suitable salts are further described in P. Heinrich Stahl, Camille G. Wermuth (Eds.), Handbook of Pharmaceutical Salts Properties, Selection, and Use; 2002.

[0148] In some embodiments, the formulation comprises at least one pharmaceutically acceptable buffer. Non-limiting examples of suitable buffers include acetate, citrate, histidine, phosphate, histidine, Tris, tartrate, glycine, glutamate, and succinate buffers. In some embodiments, the formulation comprises an aqueous carrier comprising a pharmaceutically acceptable buffer. In some embodiments, the formulation comprisesa salt and / or powder, such as, e.g. a freeze-dried, lyophilized, dehydrated, and / or cryodesiccated composition comprising a pharmaceutically acceptable buffer (e.g., sodium citrate).

[0149] In some embodiments, the formulation comprises one or more pharmaceutically acceptable surfactants, emulsifying agents and lubricants, preservative agents, cryoprotective agents, antioxidants, clarifying agents, suspending agents, thickening agents, chelating agents, wetting agents, dispersing agents, stabilizers, isotonic agents, tonicity-adjusting agents, coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives, and antioxidants.

[0150] In some embodiments, the formulations described herein comprise a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0151] In some embodiments, the therapeutic formulation comprises a therapeutically effective amount of recombinant adeno-associated virus (rAAV). In some embodiments, the rAAV comprises a vector comprising a transgene. The rAAV vector can be derived from any AAV serotype or a variant thereof. In some embodiments, the rAAV vector is derived from AAV1, AAV2, AAV2i8, AAV3-B, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

[0152] In some embodiments, the rAAV vector comprises at least one inverted terminal repeat (ITR). In some embodiments, the ITRs provided herein may be derived from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV2i8, AAV3, AAV3- B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV 12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof. In some embodiments, the ITRs used in the vectors described herein have a wild-type nucleic acid sequence. In some embodiments, the ITRs used in thevectors described herein are not wild-type, but instead, comprise, e.g., an insertion, deletion or substitution of one or more nucleotides.

[0153] In some embodiments, the transgene encodes a therapeutic RNA. In some embodiments, the therapeutic RNA is a circular RNA. In some embodiments, the therapeutic RNA is an antisense oligonucleotide. In some embodiments, the therapeutic RNA is a ribozyme. In some embodiments, the therapeutic RNA is siRNA. In some embodiments, the therapeutic RNA is shRNA. In some embodiments, the therapeutic RNA is miRNA. In some embodiments, the therapeutic RNA is self-amplifying RNA. In some embodiments, the therapeutic RNA is a miRNA inhibitor. In some embodiments, the therapeutic RNA is a tough decoy. In some embodiments, the therapeutic RNA is a miRNA sponge. In some embodiments, the therapeutic RNA is an activating RNA. In some embodiments, the therapeutic RNA is a repressive RNA.

[0154] In some embodiments, the transgene encodes a therapeutic protein. In some embodiments, the therapeutic protein is a wildtype or functional protein or a fragment thereof that is absent or non-functional in the subject. In some embodiments, the therapeutic protein is a wildtype or functional protein or a fragment thereof that is expressed at a decreased level in the subject. In some embodiments, the therapeutic protein is wildtype or functional poly cystin- 1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional polycystin-2 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional fibrocystin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional DAZ interacting zinc finger protein 1-like or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional transient receptor potential cation channel subfamily M member 6 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional collagen type IV alpha 3 chain or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional collagen type IV alpha 4 chain or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional collagen type IV alpha 5 chain or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional galactosidase alpha or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional fibrinogen alpha chain or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional apolipoprotein Al or a fragment thereof. In someembodiments, the therapeutic protein is wildtype or functional lysozyme or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional beta-2- microglobulin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional NPHS1 adhesion molecule, nephrin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional NPHS2 stomatin family member, podocin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional glucosidase II alpha subunit or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional PKHD1 ciliary IPT domain containing fibrocystin / polyductin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional mucin 1, cell surface associated or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional uromodulin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional renin or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional HNF1 homeobox B or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional SEC61 translocon subunit alpha lor a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 3 member 1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 7 member 9 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 4 member 4 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional cystinosin, lysosomal cystine transporter or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional chloride voltage-gated channel 5 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional OCRL inositol polyphosphate-5-phosphatase or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 12 member 1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional potassium inwardly rectifying channel subfamily J member 1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional chloride voltage-gated channel Kb or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional barttin CLCNK type accessory subunit beta or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional chloride voltage-gatedchannel Ka or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional MAGE family member D2 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 12 member 3 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional sodium channel epithelial 1 subunit alpha or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional sodium channel epithelial 1 subunit beta or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional sodium channel epithelial 1 subunit gamma or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional ATPase H+ transporting VO subunit a4 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional ATPase H+ transporting VI subunit Bl or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional forkhead box II or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional solute carrier family 4 member 1 or a fragment thereof. In some embodiments, the therapeutic protein is wildtype or functional WD repeat domain 72 or a fragment thereof.

[0155] In some embodiments, the transgene encodes a CRISPR / Cas system (e.g., a CRISPR / Cas9 system). In some embodiments, the transgene encodes an aptamer. In some embodiments, the transgene encodes a miRNA inhibitor. In some embodiments, the transgene encodes an RNA editor. In some embodiments, the transgene encodes an integrase. In some embodiments, the miRNA inhibitor is an anti-miR-17 oligonucleotide. In some embodiments, the anti-miR-17 oligonucleotide is RGLS4326 (Regulus Therapeutics). In some embodiments, the anti-miR-17 oligonucleotide is RGLS8429 (Regulus Therapeutics).

[0156] In some embodiments, the rAAV comprises a vector comprising a transgene and an AAV capsid. In general, an AAV capsid comprises three proteins, VP1, VP2 and VP3, each of which is encoded by splice variants of the AAV cap gene. VP2 and VP3 are truncated versions of VP1, and thus have sequences that are also comprised by VP1. Generally, the amino acid sequence of VP1 defines the serotype of the capsid. In some embodiments, the capsid is a capsid of AAV1, AAV2, AAV2i8, AAV3-B, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof. In some embodiments, the capsid is an AAV9 capsidvariant, such as AAV.kl3 or AAV.k20, as described in PCT Publication No. WO / 2024 / 206226, which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the AAV capsid is a variant of a parental wild-type AAV capsid, wherein the AAV capsid improves transfer and / or expression of the transgene described herein in one or more regions or parts of the kidney compared to the parental wild-type AAV capsid. In some embodiments, the AAV capsid comprises one or more mutations, e.g., one or more amino acid substitutions, amino acid deletions, or heterologous peptide insertions, compared to a parental wild-type AAV capsid, such as a naturally-occurring capsid protein from which it was derived. In some embodiments, the amino acid sequence of the AAV capsid (e.g., VP1, VP2, and / or VP3) is identical to the amino acid sequence of the parental wild-type AAV capsid except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acid residues, e.g., except for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions. In some embodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid. Exemplary AAV capsids with improved transfer and / or expression in kidney cells and kidney-related cells, as well as methods of using AAV particles comprising such AAV capsids to efficiently deliver a cargo to one or more regions or parts of kidney is disclosed in PCT Publication No. WO / 2024 / 206226

[0158] In some embodiments, the rAAV comprises a vector comprising a transgene and an AAV capsid. In some embodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid comprises an amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV capsid comprises the amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV capsid protein consists of the amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0159] In some embodiments, the rAAV comprises a vector comprising a transgene and an AAV capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid. In some embodiments, the AAV capsid comprises an amino acid sequence of SEQ ID NO:3 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV capsid comprises the amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1. In some embodiments, the AAV capsid protein consists of the amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0160] Exemplary amino acid sequences for AAV9 capsid variants are provided below. For wild-type AAV9, bolded residues indicate position 452-458 in the AAV9 VP1 subunit.Table 1. Exemplary AAV Capsids

[0161] In some embodiments, the AAV capsid transduces one or more cell types in a kidney. In some embodiments, the AAV capsid transduces one or more regions or parts of a kidney. In some embodiments, the AAV capsid has tropism for one or more kidney cell types. In some embodiments, the kidney cell is a mesangial cell. In some embodiments, the kidney cell is a podocyte. In some embodiments, the kidney cell is a glomerular endothelial cell. In some embodiments, the kidney cell is a parietal cell. In some embodiments, the kidney cell is a proximal tubule cell. In some embodiments, the kidney cell is a descending limb cell. In some embodiments, the kidney cell is a thin ascending limb cell and / or a thick ascending limb cell. In some embodiments, the kidney cell is a distal convoluted tubule cell. In some embodiments, the kidney cell is a connecting tubule cell. In some embodiments, the kidney cell is a principal cell. In some embodiments, the kidney cell is a intercalated cell In some embodiments, the kidney cell is a vascular smooth muscle cell. In some embodiments, the kidney cell is an endothelial cell. In some embodiments, the kidney cell is a fibroblast. In some embodiments, the kidney cell is an immune cell. In some embodiments, the immune cell is a natural killer cell, a T cell, a B cell, a macrophage, and / or a monocyte. In some embodiments, the AAV capsid has tropism for one or more regions or parts of a kidney. In some embodiments, the region or part of the kidney is the cortex In some embodiments, the region or part of the kidney is the medulla. In some embodiments, the region or part of kidney is the renal column. In some embodiments, the region or part of the kidney is the renal pyramid. In some embodiments, the region or the part of the kidney is the distal tubule e.g., a distal convoluted tubule). In some embodiments, the region or the part of the kidney is the proximal tubule (e.g., a proximal convoluted tubule). In some embodiments, the region or the part of the kidney is the collecting tubule. In some embodiments, the region or the part of the kidney is the Loop of Henle. In some embodiments, the region or the part of the kidney is the glomerulus. In some embodiments, the region or part of the kidney is the renal pelvis. In some embodiments, the region or part of the kidney is the major calyx. In some embodiments, the region or part of kidney is the minor calyx. In some embodiments, the region or part of the kidney is the papillae. In some embodiments, the region or part of the kidney is the ureter. In some embodiments, the region or part of kidney is the proximal tubule . In someembodiments, the AAV capsid is an AAV.kl3 capsid. In some embodiments, the AAV capsid is an AAV.k20 capsid.

[0162] In some embodiments, the AAV capsid variant transduces one or more kidney cells, kidney-derived cell types, and / or kidney-related cell types more efficiently than that of the wild-type AAV capsid. In some embodiments, the AAV capsid variant increases or improves transduction efficiency in the kidney by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more compared to the wild-type AAV capsid.

[0163] In some embodiments, the AAV.kl3 capsid transduces one or more kidney cells, kidney-derived cell types, and / or kidney-related cell types more efficiently than that of the wild-type AAV9 capsid. In some embodiments, the AAV.kl3 capsid increases or improves transduction efficiency in the kidney by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to the wild-type AAV9 capsid.

[0164] In some embodiments, the AAV.k20 capsid transduces one or more kidney cells, kidney-derived cell types, and / or kidney-related cell types more efficiently than that of the wild-type AAV9 capsid. In some embodiments, the AAV.k20 capsid increases or improves transduction efficiency in the kidney by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to the wild-type AAV9 capsid.

[0165] The rAAV particles described herein may be produced using any suitable method known in the art. For example, a host cell (e g., a mammalian cell) may be engineered to stably express the necessary components for AAV particle production. This can be achieved by integrating a plasmid (or multiple plasmids) comprising AAVrep and cap genes, and a selectable marker, such as an antibiotic (e.g., neomycin or ampicillin) resistance gene into the genome of the cell. The cell can be, e g., an insect or mammalian cell which can then be co-infected with a helper virus (e.g., adenovirus or baculovirus providing the helper functions) and the rAAV vector comprising the 5’ and 3’ AAV ITR. The use of a selectable marker allows for large-scale production of the rAAV. As another non-limiting example, adenovirus or baculovirus rather than plasmids can be used to introduce rep and cap genes into packaging cells. As yet another non-limiting example, both the viral vector containing the 5’ and 3’ AAV ITRs and the rep and cap genes can be stably integrated into the DNA of producer cells, and the helper functions can be provided by a wild-type adenovirus to produce the rAAV.

[0166] In some embodiments, host cells containing the rAAV vectors described herein is rendered capable of providing AAV helper functions to replicate and encapsulate the recombinant nucleic acid molecule provided herein flanked by the AAV ITRs to produce rAAV particles. AAV helper functions are generally AAV-derived coding sequences which can be expressed to provide AAV gene products that, in turn, function in trans for productive AAV replication. AAV helper functions are used herein to complement necessary AAV functions that are missing from the rAAV vectors. In some embodiments, AAV helper functions include one, or both of the major AAV ORFs, namely the rep and cap coding regions, or functional homologues thereof.

[0167] AAV helper functions can be introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of the rAAV vector. For example, AAV helper constructs can be used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions that are necessary for productive AAV infection. Typically, AAV helper constructs lack AAV ITRs and can neither replicate nor package themselves. The AAV helper constructs can be in the form of, e.g., a plasmid, phage, transposon, cosmid, virus, or virion.

[0168] A helper virus for AAV refers to a virus that allows AAV to be replicated and packaged by a host cell. A helper virus provides helper functions that allow for the replication of AAV. A number of such helper viruses have been identified, including adenoviruses, herpesvimses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerous adenoviruses of human, non-humanmammalian and avian origin are known and are available from depositories such as the ATCC. Viruses of the herpes family, which are also available from depositories such as ATCC, include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses (EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV). Examples of adenovirus helper functions for the replication of AAV include E1A functions, E1B functions, E2A functions, VA functions and E4orf6 functions.

[0169] A preparation of AAV is said to be substantially free of helper virus if the ratio of infectious AAV particles to infectious helper virus particles is at least about 102: 1; at least about 104:1, at least about 106:1; or at least about 108: 1. Preparations can also be free of equivalent amounts of helper virus proteins (i .e., proteins as would be present as a result of such a level of helper virus if the helper virus particle impurities noted above were present in disrupted form). Viral and / or cellular protein contamination can generally be observed as the presence of Coomassie staining bands on SDS gels (e g., the appearance of bands other than those corresponding to the AAV capsid proteins VP1, VP2 and VP3).

[0170] In some embodiments, the host cell is also capable of providing or is provided with non AAV-derived functions or “accessory functions” to produce rAAV particles. Accessory functions are non AAV-derived viral and / or cellular functions upon which AAV is dependent for its replication, such as non AAV proteins and RNAs that are required in AAV replication, including those involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of Cap expression products and AAV capsid assembly. In some embodiments, viral-based accessory functions can be derived from a known helper virus.

[0171] A variety of host cells can be used to produce rAAV particles described herein. Suitable host cells for producing AAV particles from the nucleic acid molecules and AAV vectors provided herein include microorganisms, yeast cells, insect cells, and mammalian cells. Typically, such cells can be, or have been, used as recipients of a heterologous nucleic acid molecule and can grow in, e g., suspension culture and a bioreactor.

[0172] In some embodiments, the cell is a mammalian host cell, for example, a HEK293, HEK293-T, A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC 1, BSC 40, BMT 10, VERO, W138, HeLa, 293, Jurkat, 2V6.11, Saos, C2C12, L, HT1080, HepG2, primary fibroblast, hepatocyte, and myoblast cells.

[0173] In some embodiments, the cell is an insect cell, for example an Sf9, SF21, SF900+, or a drosophila cell lines, mosquito cell lines, e.g., Aedes albopictus derived cell lines, domestic silkworm cell lines, e.g. Bombyxmori cell lines, Trichoplusiani cell lines such as High Five cells or Lepidoptera cell lines such as Ascalapha odorata cell lines. In some embodiments, insect cells are cells from the insect species which are susceptible to baculovirus infection, including High Five, Sf9, Se301, SeIZD2109, SeUCRl, Sf900+, Sf21, BTI-TN-5BI-4, MG-1, Tn368, HzAml, BM-N, Ha2302, Hz2E5 and Ao38. For example, large scale production of recombinant AAV in cells, including Sf9 insect cells, has been described by Kotin RM. Hum Mol Genet. 20 (Rl) : R2-R6 (2011) doi: 10.1093 / hmg / ddrl41. Methodology for molecular engineering and expression of polypeptides in insect cells is described, for example, in Summers and Smith. A Manual of Methods for Baculovirus Vectors and Insect Culture Procedures, Texas Agricultural Experimental Station Bull. No. 7555, College Station, Tex (1986); King, L A. and R.D. Possee, The baculovirus expression system. Chapman and Hall, United Kingdom (1992); O’Reilly, D R , L.K. Miller, V.A. Luckow. Baculovirus Expression Vectors: A Laboratory Manual, New York (1992), W.H. Freeman and Richardson, C D , Baculovirus Expression Protocols, Methods in Molecular Biology, volume 39 (1995).

[0174] In some embodiments, as a result of the infection of the host cell with a helper virus and / or an accessory function vector, a recombinant AAV particle is produced, and the produced rAAV particle is infectious, replication-defective virus, and includes an AAV protein shell that encapsulates a heterologous nucleotide sequence of interest flanked on both sides by AAV ITRs.

[0175] rAAV particles can be purified from the host cell using a purification method known in the art, such as chromatography, CsCl gradients, and other methods as described, for example, in U.S. Pat. Nos. 6,989,264 and 8,137,948 and WO 2010 / 148143. In some embodiments, residual helper virus can be inactivated using known methods, e.g., by heating.

[0176] The effective amount of the therapeutic formulation administered to a subject will depend on a variety of factors, several of which will differ from patient to patient including the disorder being treated and the severity of the disorder; activity of the specific agent(s) employed; the age, body weight, general health, sex and diet of the patient; the timing of administration, the duration of the treatment; drugs used incombination; the judgment of the prescribing physician; and like factors known in the medical arts.

[0177] In some embodiments, the therapeutic formulation comprises a viral vector or viral particle. In some embodiments, the viral vector or viral particle is administered at a concentration of vector genomes per milliliter (vg / mL) in the range of about 105vg / mL to about 1018vg / mL. In some embodiments, the concentration is in the range of about 105vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of about 105vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of about 105vg / mL to about IO10vg / mL. In some embodiments, the concentration is in the range of about 105vg / mL to about 108vg / mL. In some embodiments, the concentration is in the range of about 107vg / mL to about 1018vg / mL. In some embodiments, the concentration is in the range of about 107vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of about 107vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of about 107vg / mL to about IO10vg / mL. In some embodiments, the concentration is in the range of about 109vg / mL to about 1018vg / mL. In some embodiments, the concentration is in the range of about 109vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of about 109vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of about 1011vg / mL to about 1018vg / mL. In some embodiments, the concentration is in the range of about 1011vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of about 1011vg / mL to about 1013vg / mL. In some embodiments, the concentration is in the range of about 1011vg / mL to about 1012vg / mL. In some embodiments, the concentration is in the range of about 1012vg / mL to about 1013vg / mL. In some embodiments, the concentration is in the range of about 1013vg / mL to about 1014vg / mL. In some embodiments, the concentration is in the range of about 1014vg / mL to about 1015vg / mL. In some embodiments, the concentration is in the range of about 1015vg / mL to about 1016vg / mL. In some embodiments, the concentration is in the range of about 1016vg / mL to about 1017vg / mL. In some embodiments, the concentration is in the range of about 1017vg / mL to about 1018vg / mL. In some embodiments, the concentration is about 105vg / mL. In some embodiments, the concentration is about 106vg / mL. In some embodiments, the concentration is about 107vg / mL. In some embodiments, the concentration is about 108vg / mL. In some embodiments, theconcentration is about 109vg / mL. In some embodiments, the concentration is about IO10vg / mL. In some embodiments, the concentration is about 1011vg / mL. In some embodiments, the concentration is about 1012vg / mL. In some embodiments, the concentration is about 1013vg / mL. In some embodiments, the concentration is about 1014vg / mL. In some embodiments, the concentration is about 1015vg / mL. In some embodiments, the concentration is about 1016vg / mL. In some embodiments, the concentration is about 1017vg / mL. In some embodiments, the concentration is about 1018vg / mL. In some embodiments, the concentration is 4.5xlOnvg / mL. In some embodiments, the concentration is S.OxlO11vg / mL. In some embodiments, the concentration is 1.5xl012vg / mL. In some embodiments, the concentration is 5.0xl012vg / mL. In some embodiments, the concentration is l.OxlO13vg / mL. In some embodiments, the concentration is 1.5xl013vg / mL. In some embodiments, the viral vector or viral particle is an rAAV vector or rAAV particle In some embodiments, the formulation comprises the viral vector or viral particle at a concentration that is effective to treat a kidney disease or condition.

[0178] In some embodiments, the viral vector or viral particle is administered to a subject at a dose of lxl05to IxlO20vector genomes (vg). In some embodiments, the dose is about IxlO5to IxlO18vg. In some embodiments, the dose is about lxl05to IxlO16vg. In some embodiments, the dose is about IxlO5to IxlO14vg. In some embodiments, the dose is about IxlO5to IxlO12vg. In some embodiments, the dose is about IxlO5to IxlO10vg. In some embodiments, the dose is about IxlO5to IxlO8vg. In some embodiments, the dose is about IxlO7to IxlO18vg. In some embodiments, the dose is about IxlO7to IxlO16vg. In some embodiments, the dose is about IxlO7to IxlO14vg. In some embodiments, the dose is about IxlO7to IxlO12vg. In some embodiments, the dose is about IxlO7to IxlO10vg. In some embodiments, the dose is about IxlO7to IxlO8vg. In some embodiments, the dose is about IxlO9to IxlO18vg. In some embodiments, the dose is about IxlO9to IxlO16vg. In some embodiments, the dose is about IxlO9to IxlO14vg. In some embodiments, the dose is about IxlO9to IxlO12vg. In some embodiments, the dose is about IxlO9to IxlO10vg. In some embodiments, the dose is about IxlO11to IxlO18vg. In some embodiments, the dose is about IxlO11to IxlO16vg. In some embodiments, the dose is about IxlO11to IxlO14vg. In some embodiments, the dose is about IxlO11to IxlO12vg. In some embodiments, the dose is about IxlO13to IxlO18vg. In some embodiments, the dose is about IxlO13toIxlO16vg. In some embodiments, the dose is about IxlO13to IxlO14vg. In some embodiments, the dose is about IxlO15to IxlO18vg. In some embodiments, the dose is about IxlO15to IxlO16vg. In some embodiments, the dose is about lxl05vg. In some embodiments, the dose is about lxl06vg. In some embodiments, the dose is about IxlO7vg. In some embodiments, the dose is about lxlO8vg. In some embodiments, the dose is about IxlO9vg. In some embodiments, the dose is about IxlO10vg. In some embodiments, the dose is about IxlO11vg. In some embodiments, the dose is about lxl012vg. In some embodiments, the dose is about lxl013vg. In some embodiments, the dose is about lxl014vg. In some embodiments, the dose is about IxlO15vg. In some embodiments, the dose is about lxl016vg. In some embodiments, the dose is about lxl017vg. In some embodiments, the dose is about lxl018vg. In some embodiments, the dose is about IxlO19vg. In some embodiments, the dose is about IxlO20vg. In some embodiments, the dose is 9.0xl012vg. In some embodiments, the dose is lxl013vg. In some embodiments, the dose is 3.0xl013vg. In some embodiments, the dose is IxlO14vg. In some embodiments, the dose is 3.0xl014vg. In some embodiments, the dose is 6.0xl014vg. In some embodiments, the dose is 1.0xl015vg In some embodiments, the viral vector or viral particle is an rAAV vector or rAAV particle. In some embodiments, the dose is the total dose. In some embodiments, the dose is for each administration. In some embodiments, the dose of the vector or viral particle provided herein is for each kidney.

[0179] In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1010to about 1 x 1015viral genomes (vg) per kilogram (kg). In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1011vg / kg to about 1 x 1014vg / kg. In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1012vg / kg to about 1 x 1014vg / kg. In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1013vg / kg to about 1 x 1014vg / kg. In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1014vg / kg to about 1 x 1015vg / kg. In some embodiments, the viral vector or viral particle is administered at a dose of at least about 1 x IO10vg / kg, at least about 1 x 1011vg / kg, at least about 1 x 1012vg / kg, at least about 1 x 1013vg / kg, at least about 1 x 1014vg / kg, or at least about 5 x 1015vg / kg. In some embodiments, the viral vector or viral particle is administered at a dose of about 1 x 1012vg / kg.

[0180] In some embodiments, the method described herein treats the kidney in a subject in need thereof. In some embodiments, the method treats one or more nephrons in the renal pyramid. In some embodiments, the method treats the inner medulla of the kidney In some embodiments, the method treats the outer medulla of the kidney. In some embodiments, the method treats the cortex of the kidney. In some embodiments, the method treats the glomerulus of the nephron. In some embodiments, the method treats the juxtaglomerular apparatus of the nephron.

[0181] In some embodiments, the method described herein treats a kidney cell in a subject in need thereof. In some embodiments, the method described herein treats at least one of mesangial cells, endothelial cells, epithelial cells, fibroblasts, glomerular cells (e.g., glomerular endothelial cells), podocytes, glomerular endothelial cells, parietal cells (e.g, parietal epithelial cells), tubular epithelial cells, interstitial cells, tubule cells, proximal tubule cells, distal tubule cells (e.g, distal convoluted tubule cells), descending limb cells, thin ascending limb cells, thick ascending limb cells, principal cells, connecting tubule cells, intercalated cells, Loop of Henle cells, collecting duct cells, pericytes, vascular cells, vascular smooth muscle cells, or immune cells (e.g, natural killer cells, T cells, B cells, macrophages, and / or monocytes).

[0182] In some embodiments, the method described herein transduces kidney tissue with a therapeutic formulation in a subject in need thereof. In some embodiments, the method transduces one or more nephrons in the renal pyramid. In some embodiments, the method transduces the portion of the nephron within the inner medulla. In some embodiments, the method transduces the portion of the nephron within the outer medulla. In some embodiments, the method transduces the portion of the nephron within the cortex. In some embodiments, the method transduces the glomerulus of the nephron. In some embodiments, the method transduces cells in a region or part of the kidney comprising the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

[0183] In some embodiments, the method described herein transduces at least one cell population in the kidney with the therapeutic formulation described herein. In some embodiments, the method described herein transduces at least one of mesangial cells, endothelial cells, epithelial cells, fibroblasts, glomerular cells, podocytes, glomerular endothelial cells, parietal epithelial cells, tubule cells, proximal tubule cells, distaltubule cells, Loop of Henle cells, collecting duct cells, pericytes, or vascular smooth muscle cells.

[0184] The number of administrations of the therapeutic formulation to a subject may vary. In some embodiments, introducing the therapeutic formulation into the subject may be a one-time event. In some embodiments, the one-time treatment is administered to a single kidney. In some embodiments, the one-time treatment is administered to both kidneys. In some embodiments, such treatment may require an on-going series of repeated treatments (e.g., once per month, once per year, or multiple times per month or year). In some embodiments, the repeated treatments are for a single kidney. In some embodiments, the repeated treatments are for both kidneys. In some embodiments, the repeated treatments are for a single kidney, and the one-time treatment is for another single kidney. In some embodiments, multiple administrations of the therapeutic formulation may be required before an effect is observed. The exact protocols depend upon the disease or condition, the stage of the disease and parameters of the individual subject being treated.

[0185] In some embodiments, the efficacy of the method described herein, is determined, for example, by tests of rAAV transduction of the kidney (e g., qPCR, dPCR, ddPCR, sequencing or imaging for rAAV genomes) or expression of an rAAV- delivered reporter gene or transgene (e g., RNA or protein-based assays such as RT- qPCR, RT-dPCR, RT-ddPCR, ELISA, histological staining, or flow cytometry), anti- rAAV serology (e.g., in the urine or blood), or occurrence of symptoms related to a kidney-associated disease or condition for which the rAAV was administered to treat.

[0186] In some embodiments, the method described herein results in the transduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or more of the cells in the kidney with the rAAV.

[0187] In some embodiments, the method described herein results in the transduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about 85%, or more of the nephrons in the kidney with the rAAV.

[0188] In some embodiments, the formulation is a diagnostic formulation. In some embodiments, the diagnostic formulation is for kidney scan. In some embodiments, the diagnostic formulation comprises a radioactive tracer.

[0189] In some embodiments, the subject is human. In some embodiments, the subject is non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, bovines, horses, household cats, tigers and other large cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, and birds e.g., chickens, turkeys, and ducks). In some embodiments, the subject may be a neonate, a juvenile, or an adult.

[0190] In some embodiments, the subject is a human and the human is seropositive for an rAAV prior to administration of the formulation comprising the rAAV. A subject that is seropositive has antibodies in the circulating serum that neutralize the rAAV to be administered. In some embodiments, the circulating serum antibodies do not neutralize the rAAV in the kidney upon administration. In some embodiments, the neutralizing antibodies are not present in the kidney fluids and / or urine, even if neutralizing antibodies are circulating in the serum. In some embodiments, a subsequent administration of an rAAV as described herein is performed without resulting in a substantial inflammatory response in the kidney.

[0191] In some embodiments, the subject is a human and the human is seronegative for an rAAV prior to administration of the formulation comprising the rAAV. A subject that is seronegative does not have antibodies that neutralize the rAAV to be administered.8. EMBODIMENTS

[0192] Embodiment 1. A method of administering a formulation to the renal pyramid of a subject, comprising: administering the formulation through the retro-ureteral route into one or more nephrons in the renal pyramid.

[0193] Embodiment 2. The method of embodiment 1, wherein the administration is non- surgical.

[0194] Embodiment 3. The method of embodiment 1 or 2, wherein the formulation is administered through a catheter.

[0195] Embodiment 4. The method of embodiment 3, wherein the ureter is occluded during the administration to prevent the formulation from draining from the renal pelvis, and wherein the ureter is occluded at a location lower than the location of the end of the catheter.

[0196] Embodiment 5. The method of embodiment 4, wherein the ureter is occluded at the upper ureter near the ureteropelvic junction or at the ureteropelvic junction.

[0197] Embodiment 6. The method of embodiment 4 or 5, wherein the ureter is occluded by an inflatable balloon.

[0198] Embodiment 7. The method of any one of embodiments 1-6, wherein the method does not comprise isolating the kidney from systemic circulation.

[0199] Embodiment 8. The method of any one of embodiments 1-7, wherein the renal artery is not blocked or occluded during the administration.

[0200] Embodiment 9. The method of any one of embodiments 1-8, wherein the renal vein is not blocked or occluded during the administration.

[0201] Embodiment 10. The method of any one of embodiments 1-9, wherein the formulation is administered at a constant rate.

[0202] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the formulation is administered at a constant rate of about 0.1 mL / min to about 20 mL / min.

[0203] Embodiment 12. The method of embodiment 11, wherein the formulation is administered at a constant rate of about 0.1 mL / min to about 3 mL / min.

[0204] Embodiment 13. The method of embodiment 11 or 12, wherein the formulation is administered at a constant rate of about 0.5 mL / min.

[0205] Embodiment 14. The method of embodiment 11 or 12, wherein the formulation is administered at a constant rate of 1 mL / min.

[0206] Embodiment 15. The method of embodiment 11 or 12, wherein the formulation is administered at a constant rate of 2 mL / min.

[0207] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the administered volume of the formulation is about 5 mL to about 40 mL per kidney.

[0208] Embodiment 17. The method of embodiment 16, wherein the administered volume of the formulation is about 15 mL to about 30 mL per kidney.

[0209] Embodiment 18. The method of embodiment 16 or 17, wherein the administered volume of the formulation is about 20 mL to about 25 mL per kidney.

[0210] Embodiment 19. The method of any one of embodiments 16-18, wherein the administered volume of the formulation is about 25 mL per kidney.

[0211] Embodiment 20. The method of any one of embodiments 16-19, wherein the administered volume of the formulation is 25 mL per kidney.

[0212] Embodiment 21. The method of any one of embodiments 1-19, wherein the administered volume of the formulation is a fixed volume.

[0213] Embodiment 22. The method of any one of embodiments 1-20, wherein the administered volume is not dependent on the subject’s body weight.

[0214] Embodiment 23. The method of any one of embodiments 1-21, wherein the administered volume is not dependent on the size of the renal pelvis.

[0215] Embodiment 24. The method of any one of embodiments 1 to 20, wherein the administered volume of the formulation is based on the volume of the renal pelvis of each kidney of the subject.

[0216] Embodiment 25. The method of embodiment 24, wherein the administered volume of the formulation is about 0.1 mL to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject.

[0217] Embodiment 26. The method of embodiment 19 or 20, wherein the administered volume of the formulation is about 10 mL larger than the volume of the renal pelvis of each kidney of the subject.

[0218] Embodiment 27. The method of any one of embodiments 24-26, wherein the administered volume of the formulation is 10 mL larger than the volume of the renal pelvis of each kidney of the subject.

[0219] Embodiment 28. The method of any one of embodiments 1 to 27, wherein the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration.

[0220] Embodiment 29. The method of embodiment 28, wherein the intrarenal pressure is above about 40 mm Hg and below about 180 mm Hg during the administration.

[0221] Embodiment 30. The method of any one of embodiments 1-28, wherein the intrarenal pressure is above baseline pressure during the administration.

[0222] Embodiment 31. The method of embodiment 30, wherein the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

[0223] Embodiment 32. The method of any one of embodiments 1 to 31, wherein the maximum intrarenal pressure during the administration is less than about 175 mm Hg,less than about 150 mm Hg, less than about 125 mm Hg, or less than about 100 mm Hg

[0224] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the maximum intrarenal pressure during the administration is less than about 125 mm Hg.

[0225] Embodiment 34. The method of any one of embodiments 1 to 33, wherein the maximum intrarenal pressure during the administration is less than about 100 mm Hg.

[0226] Embodiment 35. The method of any one of embodiments 1 to 34, wherein the ureter remains occluded for about 5 minutes to about 60 minutes after the administration.

[0227] Embodiment 36. The method of embodiment 35, wherein the ureter remains occluded for about 10 minutes after the administration.

[0228] Embodiment 37. The method of embodiment 35, wherein the ureter remains occluded for about 30 minutes after the administration.

[0229] Embodiment 38. The method of any one of embodiments 1-37, wherein the intrarenal pressure is below 200 mm Hg after the administration.

[0230] Embodiment 39. The method of any one of embodiments 1-38, wherein the intrarenal pressure is maintained below 100 mm Hg after the administration.

[0231] Embodiment 40. The method of any one of embodiments 1-39, wherein the intrarenal pressure is maintained above baseline pressure after the administration

[0232] Embodiment 41. The method of embodiment 40, wherein the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

[0233] Embodiment 42. The method of any one of embodiments 1-41, wherein the formulation comprises a pharmaceutically acceptable carrier or excipient.

[0234] Embodiment 43. The method of any one of embodiments 1 to 42, wherein the formulation is a therapeutic formulation.

[0235] Embodiment 44. The method of embodiment 43, wherein the therapeutic formulation is for treating a kidney disease.

[0236] Embodiment 45. The method of embodiment 43 or 44, wherein the therapeutic formulation is a gene therapy.

[0237] Embodiment 46. The method of any one of embodiments 43-45 , wherein the therapeutic formulation comprises a therapeutically effective amount of recombinant adeno-associated virus (rAAV).

[0238] Embodiment 47. The method of embodiment 46, wherein the therapeutically effective amount of rAAV is between about 108viral genomes (vg) and about 1015vg per kidney.

[0239] Embodiment 48. The method of embodiment 46 or 47, wherein the therapeutically effective amount of rAAV is between about 1012vg and about 1015vg per kidney.

[0240] Embodiment 49. The method of any one of embodiments 46-48, wherein the therapeutically effective amount of rAAV is between about 1014vg and about 1015vg per kidney.

[0241] Embodiment 50. The method of embodiment 46, wherein the therapeutically effective amount of rAAV is between about 108vg / mL and about 1015vg / mL.

[0242] Embodiment 51. The method of embodiment 46 or 50, wherein the therapeutically effective amount of rAAV is between about 1010vg / mL and about 1014vg / mL.

[0243] Embodiment 52. The method of embodiment 46, 50, or 51, wherein the therapeutically effective amount of rAAV is between about 1012vg / mL and about 1013vg / mL.

[0244] Embodiment 53. The method of any one of embodiments 46-52, wherein the rAAV comprises a vector comprising a transgene.

[0245] Embodiment 54. The method of embodiment 53, wherein the transgene encodes a therapeutic RNA or a therapeutic protein.

[0246] Embodiment 55. The method of embodiment 54, wherein the therapeutic RNA is a circular RNA.

[0247] Embodiment 56. The method of embodiment 54 or 55, wherein the therapeutic RNA is a miRNA inhibitor.

[0248] Embodiment 57. The method of embodiment 56, wherein the miRNA inhibitor is a miRNA sponge or tough decoy.

[0249] Embodiment 58. The method of embodiment 54, wherein the therapeutic protein is polycystin-1 or a fragment thereof.

[0250] Embodiment 59. The method of embodiment 45, wherein the therapeutic protein is polycystin-2 or a fragment thereof.

[0251] Embodiment 60. The method of any one of embodiments 46-59, wherein the rAAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4,AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LKO3, AAVrh74, AAV44-9, or a variant thereof.

[0252] Embodiment 61. The method of any one of embodiments 46-60, wherein the rAAV comprises a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, AAV.kl3, AAV.k20, or a variant thereof.

[0253] Embodiment 62. The method of embodiment 61, wherein the capsid protein is a variant of AAV9.

[0254] Embodiment 63. The method of embodiment 61 or 62, wherein the AAV capsid protein transduces kidney cells.

[0255] Embodiment 64. The method of any one of embodiments 61-63, wherein the capsid protein is an AAV.kl3 capsid protein.

[0256] Embodiment 65. The method of embodiment 64, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0257] Embodiment 66. The method of embodiment 64 or 65, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0258] Embodiment 67. The method of any one of embodiments 64-66, wherein the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0259] Embodiment 68. The method of any one of embodiments 64-67, wherein the AAV.kl3 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AAV9 capsid.

[0260] Embodiment 69. The method of any one of embodiments 61-63, wherein the capsid protein is an AAV.k20 capsid protein.

[0261] Embodiment 70. The method of embodiment 69, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0262] Embodiment 71. The method of embodiment 69 or 70, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 at positions 452- 458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0263] Embodiment 72. The method of any one of embodiments 69-71, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

[0264] Embodiment 73. The method of any one of embodiments 69-72, wherein the AAV.k20 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wild-type AAV9 capsid.

[0265] Embodiment 74. The method of any one of embodiments 46-73, wherein the method increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to transduction in kidney cells using intravenous administration.

[0266] Embodiment 75. The method of any one of embodiments 63, 68, 73, or 74, wherein the kidney cells are proximal tubule cells, distal tubule cells, or collecting duct cells.

[0267] Embodiment 76. The method of any one of embodiments 46-73, wherein the method results in the transduction of at least about 5%, at least about 10%, at least about15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or more of the nephrons in the kidney.

[0268] Embodiment 77. A method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering a therapeutic formulation comprising an rAAV to the subject by performing the method of any one of embodiments 46-76.

[0269] Embodiment 78. The method of any one of embodiments 1-77, wherein the subject is a human.

[0270] Embodiment 79. The method of any one of embodiments 46-78, wherein the subject is seropositive for the rAAV prior to administration of the therapeutic formulation comprising the rAAV.

[0271] Embodiment 80. The method of any one of embodiments 46-78, wherein the subject is seronegative for the rAAV prior to administration of the therapeutic formulation comprising the rAAV.

[0272] Embodiment 81. The method of any one of embodiments 1-80, wherein the subject has autosomal dominant polycystic kidney disease (ADPKD).

[0273] Embodiment 82. The method of embodiment 81, wherein the subject has ADPKD 1.

[0274] Embodiment 83. The method of embodiment 81, wherein the subject has ADPKD2

[0275] Embodiment 84. The method of any one of embodiments 1-83, wherein the method inhibits or ameliorates renal cyst development.

[0276] Embodiment 85. The method of any one of embodiments 1 to 42, wherein the formulation is a diagnostic formulation.

[0277] Embodiment 86. The method of embodiment 85, wherein the diagnostic formulation is for a kidney scan.9. EXAMPLES

[0278] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, andare not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.9.1. EXAMPLE 1 — Retroureteral administration (RUA) can be used to administer solutions to the nephrons of the renal pyramids

[0279] Initial studies were performed to assess whether a non-surgical RUA procedure could be used to administer a solution to kidney nephrons.

[0280] For these initial studies, female Yorkshire pigs were anesthetized before administration of contrast dye solution via RUA. A rigid cystoscope was used to insert a guidewire through the pigs’ urethra, bladder, and ureter. Subsequently, a catheter with an inflatable balloon was placed into the upper ureter near the ureteropelvic junction The approximate location of the balloon was then verified via fluoroscopy by filling the balloon with contrast dye and infusing a small amount of contrast dye solution into the renal pelvis. The balloon was then fully inflated to occlude the ureter. Next, 20 mL of contrast dye solution was manually injected into the left kidney and then subsequently the right kidney through a Cook occlusion balloon catheter over periods of approximately 1 -6 minutes. After administration of the dose solution was completed, 3 mL of vehicle was administered to flush the catheter line of remaining dose solution. The balloon remained inflated after dosing to allow for a 30-minute dwell time. Dose administration was visualized using contrast media and x-ray fluoroscopy. A pressure transducer (Centurion) was added to the end of the catheter to periodically measure pressure.

[0281] Results are shown in Fig. 1. As shown, the administration of between 10 and 20 mL of a contrast dye solution resulted in observable contrast dye solution in the renal pelvis and occasionally in the nephrons of the renal pyramids. However, manual administration of contrast dye led to varying average rates of administration (3-20 mL / min) that did not correlate with the pressure measurements taken at the end of dose. While the average rates of administration across the entire dosing period are shown inFig. 1, the actual rate of administration during dosing likely fluctuated above and / or below the reported values given the variance observed with manual administration. This variance likely resulted in the renal fomiceal or renal cortex ruptures that were observed in two of the kidneys from treated animals. For this reason, all subsequent experiments utilized a syringe pump to allow for controlled dosing of contrast dye at a constant rate.9.2. EXAMPLE 2 — RUA of 12-20 mL solution at a controlled rate of 1 or 2 mL / min results in consistent administration of solution to the nephrons while preventing forniceal ruptures

[0282] Additional studies were then conducted to optimize the RUA procedure to more consistently administer solution to the nephrons without causing damage to the kidneys. A similar method was used as disclosed in Example 1 above. A schematic of the RUA procedure in pigs is shown in Figure 2. Pigs are anesthetized and place in supine position under normothermic conditions to maintain body temperature. A cystoscope was used to insert a guidewire from the bladder into the ureter. The guidewire was advanced to the upper ureter with fluoroscopy imaging and then a balloon occlusion catheter was inserted and balloon inflated to occlude the upper ureter near the ureteropelvic junction. The catheter line was primed with dose solution, connected to a pressure monitor, a three-way stop cock, an extension line, a syringe, and a syringe infusion pump. The syringe was fdled with dose solution and the pressure monitor measured intrarenal pressure throughout the procedure.

[0283] First, pressure changes resulting from loss of balloon occlusion during the RUA procedure were assessed. Results are in Figs. 3 A and 3B. As shown, following an initial increase in pressure upon contrast dye administration initiation, a decrease in pressure was observed in pigs receiving either a total dose volume of 20 mL at a constant rate of 2 mL / min (Fig. 3 A) or a total dose of 15 mL at a constant rate of 1 mL / min (Fig. 3B). The drop in pressure showed that the filled catheter balloon was not completely occluding the kidney ureter and the placement of the balloon catheters were subsequently adjusted to ensure occlusion. A concomitant rise and stabilization in pressure was observed following catheter balloon adjustment under both conditions. These data highlight the importance of complete ureter occlusion with the catheter balloon to maintain dose solution pressure throughout the RUA procedure.

[0284] Results are shown in Figs. 4A-4C, 5A-5C, 6A-6C, 7A-7C, 8A-8C and 9A-9C. As shown, the administration of a total contrast dye solution volume between 12 and20 mL resulted in observable contrast dye solution in the nephrons of the renal pyramids. Administration at a controlled rate of 5 mL / min using a syringe pump resulted in a larger increase in pressure during dosing than at 1 or 2 mL / min, while administration at a controlled rate of 1 or 2 mL / min using a syringe pump did not result in any observable fornix ruptures. Moreover, catheter balloon occlusion allowed for stabilized increased pressure conditions during the RUA procedure that were alleviated following balloon deflation.

[0285] In sum, this study demonstrated more consistent administration of solution to the nephrons with no damage to the kidneys and more stabilized pressure conditions.9.3. EXAMPLE 3 — Dwell time does not affect the RUA administration of 15-20 mL solution to the nephrons

[0286] Additional studies were conducted to further optimize RUA procedure parameters including dose volume and dwell time. A similar method was used as disclosed in Example 1. In this instance, the contrast dye solution was injected into the right kidney at a constant rate of 1 mL / min employing a syringe pump. The final dose volume and dwell time for each group are outlined in Table 2. After the dwell time, the catheter balloon was deflated, and the contrast dye solution was allowed to drain. The procedure was then repeated on the left kidney, using formulation buffer instead of contrast dye solution.Table 2. Contrast dye study design assessing total dose volume and dwell time parameters* Right kidney dosed with contrast dye solution; left kidney dosed with formulation buffer only.

[0287] Intrarenal pressure following the administration of the contrast dye solution was measured every minute throughout the procedure. Results are shown in Fig. 10. A slow, consistent rise in intrarenal pressure was observed during the dosing period and peaked around 50-70 mm Hg after 15 mL of the 15 mL dose solution had been administered or plateaued between 80-90 mm Hg after 15 mL of the 20 mL solution had been administered. Pressure was maintained above baseline kidney pressure (Group 1; no dose solution and kidneys are occluded via a balloon catheter) during the dwell period and returned to baseline kidney pressure following deflation of the catheter balloon.

[0288] Fluoroscopy images were taken of the contrast dye solution in the right kidney 2 min into the dosing period (A), at the end of the dosing period (B), at the end of the dwell period (C), and the left kidney 2 min into dosing (D). Results are shown in Figs. 11A-11D, 12A-12D, 13A-13D, 14A-14D, 15A-15D, 16A-16D, 7A-17D, 18A-18D, 19A-19D, 20A-20D, 21A-21D, and 22A-22D. As shown, the total volume doses of 15 or 20 mL resulted in visible contrast dye solution in the kidney nephrons, and contrast remained in nephrons for the dwell time periods of 15 or 30 minutes.9.4. EXAMPLE 4 — Average pig renal volume is 10 mL

[0289] The average pig renal pelvis volume was then determined. The volume needed to fill the renal pelvis was noted for each animal that received administration of contrast dye solution. The average renal pelvis volume was then calculated (n=9 kidneys) Results are in Fig. 23. As shown, an average renal pelvis volume of about 10 mL was observed.9.5. EXAMPLE 5 — All RUA dosing parameters are well-tolerated

[0290] In the study outlined in Table 2, serum, plasma, whole blood, and urine were collected on the noted days prior to and after dosing to obtain a variety of measurements. Results are shown in Figs. 24A-24H, 25A-25C, 26A-26B, 27A-27R, and 28A-28C. A variety of measures were assessed including hematology measurements (Fig. 24A-24H), coagulation measurements (Fig. 25A-25C), liver enzymes (Fig. 26A-26B), clinical chemistry measurements (Fig. 27A-Fig. 27R), and urinalysis measurements (Fig. 28A-28C). As shown, all of the RUA dosing parameters assessed in these studies were well-tolerated. Overall, there were no signs of adverse events from RUA by the assays reported.9.6. EXAMPLE 6 — The effect of RUA flow rate on intrarenal pressure

[0291] A study was conducted to assess the effects of different RUA flow rates and dosing volumes on intrarenal pressure.

[0292] RUA in female Yorkshire pigs was conducted as described in Example 1. Briefly, pigs were administered 80 mL of contrast dye at a flow rate of 1 mL / min, 2 mL / min, or 5 mL / min and intrarenal pressure measurements were recorded every minute throughout the RUA procedure using a pressure transducer. Intrarenal pressure was also measured every 5 minutes throughout the 30-minute dwell period following the RUA procedure (Fig. 29A). Five pigs were dosed in each experimental group.

[0293] The maximum intrarenal pressure was reached by 20 mL to 30 mL of contrast dye during the RUA procedure (Fig. 29A), while intrarenal pressure remained constant during the 30 minute dwell period (Fig. 29B). A higher flow rate during RUA resulted in an increase in maximum intrarenal pressure, with flow rates of 1 mL / min, 2 mL / min, and 5 mL / min having an intrarenal pressure of 100 mm Hg, 127 mm Hg, and 175 mm Hg, respectively (Fig. 29C). Notably, maximum intrarenal pressures were reached after administration of 23 mL of contrast dye on average across the different flow rates (Fig 29D).

[0294] Exemplary fluoroscopy images of kidney damage during RUA are shown in Fig. 30 and gross macroscopic observations immediately after RUA of the entire 80 mL dose solution are detailed in Table 3 below. No signs of kidney damage were observed in animals administered contrast dye at flow rates of 1 mL / min or 2 mL / min at volumes below 40 mL, while a flow rate of 5 mL / min resulted in kidney damage in multiple animals at total volumes of 20 mL and 25 mL (Fig. 30). Systemic leakage was also observed in the majority of pigs treated with contrast dye at 5 mL / min by RUA (Fig. 31).Table 3. Macroscopic ObservationsDilatation (cyst), cranial end was also observed for the contralateral kidneyNVL: No visible lesion

[0295] The fluorescent signal from the contrast dye plateaued in the cortex of the pig kidney after administration of 20 mL to 30 mL of contrast dye at a flow rate of 1 mL / min or 2 mL / min, which suggested that the maximum filling of pig kidneys is within this volume range (Fig. 32).

[0296] Fluoroscopy images were acquired every 1 minute during RUA with contrast dye in the ipsilateral kidney and quantitative analysis of the fluoroscopy images for each ipsilateral kidney was performed by calculating the mean normalized contrast dye signal (fold change) throughout the kidney. As shown in Fig. 32, the volume measures represented the total amount of contrast dye administered by RUA to each kidney, whereas the fold change represented the signal intensity in the renal cortex at each dosing volume normalized to the baseline image (i.e., fluoroscopic image captured immediately before dosing). The volume at which the signal (i.e., fold change) began to plateau was then determined, which indicated the optimal volume for filling the entire kidney with dose solution. Fluoroscopy images were also acquired following the RUA procedure for the contralateral kidney.

[0297] Overall, these findings indicated that RUA of about 20 to 30 mL of solution at a flow rate of 1 mL / min or 2 mL / min resulted in maximum filling of the kidneys with little to no kidney damage. These results support effective dosing at high volumes without safety risk.9.7. EXAMPLE 7— RUA of AAV.kl3 or AAV.k20 in pigs

[0298] A study was conducted to assess AAV.kl3 and AAV.k20 transduction in pig kidneys using RUA.

[0299] A self-complementary AAV genome containing the chicken P-actin (CBA) promoter that drives expression of an mCherry transgene with a SV40 polyadenylation signal was packaged into AAV.k20 and AAV.kl3 capsids (Fig. 33A). Pigs were administered the glucocorticoid, methylprednisolone, orally at 1 mg / kg daily beginning 5 days prior to treatment as a prophylactic immunosuppressant. The AAV formulation was unilaterally injected via RUA into pig kidneys as described in Example 2. The administration flow rate was held constant at 1 mL / min, and the administration volume was calculated by measuring the renal pelvis volume and adding 10 mL. For bilaterial RUA administration, a second RUA procedure was performed after the first RUA procedure for the other kidney. Each experimental group underwent a 30-minute dwell period following the RUA procedure. Tissues were harvested from animals 28 days after RUA. Fig. 33A provides a schematic of the study design and Table 4 shows the various parameters tested in each experimental group. Group 1 was administered formulation buffer alone and served as a negative control throughout the study.Table 4. Experimental Design'Dose level for a kidney with a 10 mL renal pelvis volume and total administered volume of 20 mL.

[0300] Following RUA delivery of the AAV.kl3 and AAV.k20 capsids, AAV biodistribution and mCherry RNA expression were evaluated in the treated (ipsilateral) and untreated (contralateral) kidneys of each animal in the experimental group. For the ipsilateral kidney, five biopsies from the inner medulla, eight biopsies from the outer medulla, and ten biopsies from the cortex were analyzed for each individual animal as depicted in Fig. 33B. The average of all biopsies (N=23) was then used to determine whole kidney AAV biodistribution in the treated kidney for an individual animal. For the contralateral kidney, two biopsies from the inner medulla, three to four biopsies from the outer medulla, and three to five biopsies from the cortex were harvested from each individual animal. The average of all biopsies (N=8-ll) was then used to determine whole kidney AAV biodistribution in the contralateral kidney for an individual animal. AAV genome copy numbers per pg gDNA were calculated by normalizing bGH copy numbers to total pg gDNA for dPCR

[0301] Genomic DNA was extracted from biopsies collected from the various kidney regions and AAV biodistribution was measured by digital PCR (dPCR). For the ipsilateral kidney, RNA was extracted from all kidney biopsies that had high AAV biodistribution as well as additional biopsies with low AAV vector genome copies from each kidney region to determine mCherry RNA expression throughout the kidney (N=3-16 biopsies per kidney). For the contralateral kidney, RNA was extracted from kidney biopsies from each region to determine mCherry RNA expression throughout the kidney (N=2-7 biopsies per kidney). mCherry RNA expression was then quantified by reverse transcription-dPCR (RT-dPCR).

[0302] AAV biodistribution was also determined in the liver of pigs treated with AAV.k20 and AAV.kl3 by RUA. AAV biodistribution was determined by extracting genomic DNA from liver biopsies collected from individual liver lobes (right medial, right lateral, left medial, and left lateral). The average of all lobes (N=4) was used to determine whole liver copy numbers for an individual animal. AAV biodistribution was then measured by dPCR.

[0303] Animals unilaterally or bilaterally administered AAV.kl3 or AAV.k20 exhibited AAV biodistribution and mCherry RNA expression throughout various regions of the kidney (Figs. 34A and Fig. 34B). Animals unilaterally dosed with AAV.k20 via RUA exhibited the lowest levels of AAV biodistribution in the liver (Fig. 35). AAV biodistribution and RNA expression were undetectable or low incontralateral kidneys of animals unilaterally dosed with either AAV.k20 or AAV.kl3 via RUA (Figs. 36A and 36B).

[0304] To assess safety and tolerability of AAV.k20 and AAV.kl3 delivered by RUA, standard toxicology assessments were performed. There were no treatment-related findings for mortality, clinical signs, body weights, body weight gains, clinical pathology parameters of urinalysis, urine chemistry, or urine biomarker, and no changes to macroscopic pathology or organ weights. Minor variations in hematology and clinical chemistry parameters were observed but were within the normal physiological range and histopathology observations were consistent with common incidental findings in porcine kidney.

[0305] Overall, this study demonstrated successful transduction of kidney cells following RUA of AAV.kl3 and AAV.k20 capsids. Bilateral kidney administration was also effective and well tolerated.9.8. EXAMPLE 8 - RUA of AAV.k20 in seropositive or seronegative Pigs

[0306] The aim of this study was to examine the effect of various dwell periods on kidney transduction in seropositive or seronegative animals.

[0307] A single-stranded AAV genome containing a CBA promoter that drives expression of the mCherry transgene with an SV40 polyadenylation signal was packaged into AAV.k20 (Fig. 37). The AAV.k20 formulation was injected via RUA into pig kidneys as described in Example 2 and kidneys were harvested 28 days later (Fig. 37). Pigs were administered the glucocorticoid, methylprednisolone, orally at 1 mg / kg daily beginning 5 days prior to treatment as a prophylactic immunosuppressant. Table 5 shows the various parameters tested in each experimental group. Group 1 was administered formulation buffer alone and served as a negative control throughout the study.Table 5. Experimental Design'Dose level for a kidney with a 10 mL renal pelvis volume and total administered volume of 20 mL.

[0308] AAV biodistribution and mCherry RNA expression levels were evaluated in kidney and liver following administration of formulation buffer or AAV.k20 as described in Example 7 above. However, for the untreated (contralateral) kidney, three biopsies from the inner medulla, four biopsies from the outer medulla, and five biopsies from the cortex were harvested from each individual animal. The average of all biopsies (N=12) was then used to determine whole kidney AAV biodistribution in the untreated kidney for an individual animal.

[0309] mCherry protein expression was also assessed in the kidneys of seronegative animals administered formulation buffer or AAV.k20 with a 30-minute dwell period using ELISA and immunohistochemistry (IHC). Protein was extracted from biopsies collected from treated (ipsilateral) kidney regions and quantified by bicinchoninic acid (BCA) assay. mCherry protein expression was then measured using a commercially available ELISA kit with mCherry protein levels normalized to the amount of protein in a given sample. For IHC, ipsilateral kidney sections were fixed in 10% neutral -buffer formalin for 24 hours and different kidney regions were sectioned from coronal or sagittal planes. mCherry protein expression was detected using anti-RFP rabbit polyclonal antibody (Rockland: 600-401-379).

[0310] As shown in Figs. 38A and Fig. 38B, a longer dwell period led to overall increased AAV biodistribution and mCherry RNA expression in some kidney regions. Moreover, AAV biodistribution and mCherry RNA expression was also observed in the kidneys of seropositive pigs following RUA delivery of AAV.k20 (Fig. 38A and Fig. 38B). Furthermore, AAV biodistribution was low in the contralateral kidney (Fig. 39A) and liver (Fig. 39B) regardless of dwell time or pig serostatus.

[0311] mCherry protein was also expressed in various kidney regions of animals dosed with AAV.20 via RUA (Fig. 40). mCherry expression was predominantly detected incortical and medullary collecting duct tubules and distal tubules of the ipsilateral kidney (Fig. 41). No mCherry protein expression was detected in the ipsilateral kidneys of animals administered formulation buffer (Fig. 40 and Fig. 41).

[0312] In sum, this study the mean AAV kidney biodistribution and RNA expression with a 30-minute dwell period was higher compared to a 10-minute dwell period, but inter-animal variability was high within each group. Additionally, AAV.k20 successfully transduced kidney cells in seropositive subjects.9.9. EXAMPLE 9 — RUA of AAV.k20 comprising human PKD2 in pigs

[0313] The aim of this study was to evaluate transduction efficiency of various AAV.k20 expression cassettes comprising a human PKD2 (hPKD2)' transgene in pig kidneys by RUA.

[0314] A single-stranded genome containing either a CBA or PGK promoter that drives expression of a hPKD2 transgene with an HA tag and bGH polyadenylation signal was packaged into AAV.k20 (Fig. 42). The AAV.k20 formulation was injected via RUA into pig kidneys as described in Example 2. Group 1 was administered formulation buffer alone and served as a negative control throughout the study. Group 7 occluded the renal artery using an arterial balloon catheter during the RUA procedure.

[0315] Table 6 below shows the study groups and parameters evaluated.Table 6. Experimental Design'Dose level for a kidney with a 10 mL renal pelvis volume and total administered volume of 20 mL.

[0316] Intrarenal pressure measurements were measured every minute during RUA using a pressure transducer in-line with the dosing catheter. Pigs were also assessed for various clinical factors following RUA dosing. Body weights were measured prior to dosing and at least once per week after dosing to assess body weight gain over the 28- day study duration.

[0317] Tissues were harvested 28 days following RUA administration (Fig. 42). Left and right kidneys were weighed at necropsy and the individual kidney weights relative to body weights were determined.

[0318] AAV biodistribution and hPKD2 RNA expression levels were evaluated in kidney and liver following administration of formulation buffer or AAV.k20 as described in Example 7 above. For hPKD2 RNA expression in the liver, RNA copy numbers per 100 ng cDNA were calculated by normalizing bGH copy numbers to total ng cDNA for RT-dPCR.

[0319] PC2 (the hPKD2 gene protein product) expression in ipsilateral pig kidneys following RUA was assessed. Protein lysates were prepared from kidney tissue biopsies and total PC2 protein was measured using the Jess Automated Western Blot System (Capillary Electrophoresis - Sodium Dodecyl Sulfate). An anti-PC2 antibody (Atlas, HPA015794) that detects pig and human PC2 was used to measure total PC2 protein levels. Protein expression levels were normalized to the formulation buffer (FB) vehicle control animals. Three biopsies from the inner medulla, four biopsies from the outer medulla, and one to five biopsies from the cortex were used to determine average PC2 expression in the specified kidney region for each individual animal. The average of all biopsies (N=8-12) was used to determine whole kidney PC2 expression for an individual animal.

[0320] PC2 protein expression was also evaluated in treated ipsilateral kidneys using IHC. Briefly, ipsilateral kidneys were fixed in 10% neutral -buffered formalin for 24 hours and different kidney regions were sectioned from coronal or sagittal planes. PC2 protein expression was detected using an anti-HA rabbit monoclonal antibody (ThermoFisher, MA5-27915). IHC scoring was performed by a board-certified veterinary pathologist using nine sections from different regions of the ipsilateral pig kidney. Staining was graded on a three-point scale of the average strength of signalwithin positive cells (1 = weak staining; 2 = moderate staining; 3= strong staining). Image analysis via ImageJ was performed using the same image sets and the percent area within a kidney section that was positive for 3,3 '-diaminobenzidine (DAB) staining was determined.

[0321] Furthermore, clinical chemistry and hematology tests were conducted in pigs following RUA delivery of formulation buffer or AAV.k20. Blood was collected at five different timepoints for each study group prior to and post-treatment as follows: Day 0 samples were collected 5 to 9 days prior to treatment; Day 7 samples were collected 7 to 8 days post-treatment; Day 15 samples were collected 15 days post-treatment; and Day 28 samples were collected 27 to 29 days post-treatment. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gammaglutamyl transferase (GGT), Sorbitol dehydrogenase (SDH), total protein (TPRO), blood urea nitrogen (BUN), and creatinine were analyzed at each timepoint to assess liver and kidney function. Platelet counts, white blood cell (WBC) counts, lymphocyte counts, and neutrophil counts were measured at each timepoint for hematology assessment.

[0322] The results of the study are shown in Figs. 43-5 ID.

[0323] Intrarenal pressure during RUA administration of formulation buffer or AAV.k20 varied depending on the flow rate with intrarenal pressure not exceeding an average of 120 mm Hg for any experimental group (Fig. 43).

[0324] AAV biodistribution (Fig. 44A) and hPKD2 RNA (Fig. 44B) expression were detected in different regions of the ipsilateral kidneys in pigs administered AAV.k20 via RUA. Notably, average AAV biodistribution in the ipsilateral kidney was comparable when the renal artery was or was not occluded during the RUA procedure (Fig. 44A) indicating that renal artery occlusion is not required and does not significantly improve biodistribution.

[0325] AAV genome expression was lower in the contralateral kidneys compared to the ipsilateral kidneys in pigs administered AAV.k20 via RUA (Fig. 45). AAV genome and hPKD2 RNA expression in pig liver following RUA is shown in Fig. 46A and Fig. 46B, respectively. PC2 protein expression was detected in various regions of the kidney (cortex, outer medulla, and inner medulla) in pigs administered AAV.k20 via RUA (Fig. 47).

[0326] Ipsilateral kidney tissue showed widespread hPC2-HA protein expression in pigs administered AAV.k20 by RUA (Fig. 48). Expression was present in both the cortex and medulla of the ipsilateral kidneys with highest expression in the cranial and caudal poles. Cell types positive for PC2 protein expression included cortical and medullary collecting duct tubules, distal tubules, and proximal tubules. No expression was detected in the kidneys of animals treated with formulation buffer. IHC immunopositivity scoring by a histopathologist is detailed below in Table 7. Image analysis of the same histological sections is shown in Fig. 48B, which revealed that the highest PC2 expression levels were observed in animals administered the AAVk.20 vector comprising the CBA promoter at both 1 mL / min and 2 mL / min flow rates.Table 7. IHC Staining Scores

[0327] There was no impact of AAV.k20 treatment on total body weight (Fig. 49) or total kidney weight (Table 8). Results of clinical chemistry kidney and liver function tests are shown in Figs. 50A-50H, and results of hematology tests are shown in Figs.51A-51D. As shown, all of the RUA dosing parameters assessed in these studies were well-tolerated. There were no signs of adverse events from RUA by the assays reported.Table 8. Summary of Organ WeightsG] - Anova & Dunnett, 2-sided: No significance at p < 0.5[Gl] - Kruskal- Wallis & Dunn, 2-sided: No significance at p < 0.5

[0328] Overall, the average dose solution of 19 mL supported robust expression of the AAVk.20 expression cassette comprising hPKD2. Safety and kidney transduction results were not significantly different between RUA flow rates between 0.5 mL / min to 2 mL / min. Occlusion of the renal artery during the RUA procedure did not result in notable differences in kidney biodistribution or transduction. Administration of nominal doses 1E14 vg / kidney and 3E14 vg / kidney resulted in broad AAV.k20 biodistribution and hPC2-HA expression with generally increasing hPKD2 RNA expression and hPC2-HA levels with increasing dose.9.10. EXAMPLE 10 — RUA of rAAV to the Kidneys of Humans to Treat Kidney Disease

[0329] A study is conducted to assess the administration of a therapeutic formulation to the kidneys of humans for the treatment of a kidney disease, such as ADPKD, using RUA.

[0330] A vector as described herein, encoding a transgene, is packaged into rAAV such as AAV.kl3, AAV.k20, or AAV9. The transgene can be under the control of aconstitutive or kidney specific promoter, non-limiting examples of which are provided herein (e.g., CBA, PGK, etc.). The transgene can encode a therapeutic protein, such as PCI or PC2, or a therapeutic RNA, such as a miRNA inhibitor. Male and / or female subjects are either pre-screened or not pre-screened for neutralizing antibodies against the selected rAAV (e.g., AAV.k20, AAV.kl3, AAV9).

[0331] The rAAV is administered to humans (e.g., 20-90 kg) via RUA. The details of the administration are determined using the results of testing in mammals, such as pigs and / or non-human primates. For example, the rAAV is administered in a total volume that can range from about 2.5 mL to about 30 mL in a subject. In some embodiments, the rAAV is administered in a total volume of about 20 mL to about 30 mL in a subject.

[0332] The rAAV is administered by guiding a catheter through the urethra, bladder, and ureter into the renal pelvis of the kidney as described in Example 2. In some embodiments, the renal artery or renal vein is not blocked or occluded during the RUA procedure. In some embodiments, the renal artery and / or renal vein is occluded (e.g., starting immediately before rAAV administration and ending about 10 minutes to 60 minutes after the rAAV administration) using a balloon catheter, e g., inserted through the femoral artery, femoral vein, carotid artery, internal jugular vein, and the like, as determined by a medical professional.

[0333] The AAV formulation is administered at a constant rate between 0.1 mL / min and 10 mL / min. In some embodiments, the AAV formulation is administered at a constant rate of 1 mL / min or 2 mL / min. The intrarenal pressure during RUA is at or below 200 mmHg, 175 mm Hg, 150 mmHg, 125 mmHg, or lower. The dwell period following RUA administration is between about 5 minutes and about 60 minutes. In some embodiments, the dwell period is between about 10 minutes and about 30 minutes. In some embodiments, the dwell period is 30 minutes. In some embodiments, one kidney is treated with the AAV formulation. In other embodiments, both kidneys are treated with the AAV formulation.

[0334] After a sufficient amount of time (e.g., 1 day, 1 week, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, or more), the human subjects are evaluated for kidney health, which can be specific to the kidney-associated disease, administered rAAV, and / or transgene encoding the therapeutic protein or RNA. The rAAV-administered subjects are compared to a negative control (e g., no rAAV) and / or to a comparative control.9.11. EXAMPLE 11 — Administration of Nucleic Acid Therapeutics by RUA

[0335] A study is conducted to assess administration of nucleic acid therapeutics by RUA. This study includes delivering nucleic acid therapeutics, including in vitro transcribed mRNA, with and without lipid nanoparticles. For each study group one kidney per animal is treated. RUA is performed as described in Example 10.9.12. EXAMPLE 12 — Administration of Small Molecules by RUA

[0336] A study is conducted to assess administration of small molecules by RUA. This study includes delivering therapeutic formulations comprising one or more small molecules as the active ingredient. For each study group one kidney per animal is treated. RUA is performed as described in Example 10.9.13. EXAMPLE 13 — Administration of Antibodies by RUA

[0337] A study is conducted to assess administration of antibodies by RUA This study includes delivering therapeutic formulations comprising one or more antibodies as the active ingredient. For each study group one kidney per animal is treated. RUA is performed as described in Example 10.9.14. EXAMPLE 14 — Administration of Ribonucleoprotein Complexes by RUA

[0338] A study is conducted to assess administration of ribonucleoprotein complex therapeutics by RUA. This study includes delivering ribonucleoprotein complex therapeutics with and without lipid nanoparticles. For each study group one kidney per animal is treated. RUA is performed as described in Example 10.

[0339] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the present description.

[0340] Various publications, articles and patents are cited or described in the background and throughout the specification; each of these references is herein incorporated by reference in its entirety. Discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is for the purpose of providing context for the invention. Such discussion is not an admissionthat any or all of these matters form part of the prior art with respect to any inventions disclosed or claimed.

Claims

WHAT IS CLAIMED:

1. A method of administering a formulation to the renal pyramid of a subject, comprising: administering the formulation through the retro-ureteral route into one or more nephrons in the renal pyramid.

2. The method of claim 1, wherein the administration is non-surgical.

3. The method of claim 1 or 2, wherein the formulation is administered through a catheter.

4. The method of claim 3, wherein the ureter is occluded during the administration to prevent the formulation from draining from the renal pelvis, and wherein the ureter is occluded at a location lower than the location of the end of the catheter.

5. The method of claim 4, wherein the ureter is occluded at the upper ureter near the ureteropelvic junction or at the ureteropelvic junction.

6. The method of claim 4 or 5, wherein the ureter is occluded by an inflatable balloon.

7. The method of any one of claims 1-6, wherein the method does not comprise isolating the kidney from systemic circulation.

8. The method of any one of claims 1-7, wherein the renal artery is not blocked or occluded during the administration.

9. The method of any one of claims 1-8, wherein the renal vein is not blocked or occluded during the administration.

10. The method of any one of claims 1-9, wherein the formulation is administered at a constant rate.

11. The method of any one of claims 1 to 10, wherein the formulation is administered at a constant rate of about 0.1 mL / min to about 20 mL / min.

12. The method of claim 11, wherein the formulation is administered at a constant rate of about 0.1 mL / min to about 3 mL / min.

13. The method of claim 11 or 12, wherein the formulation is administered at a constant rate of about 0.5 mL / min.

14. The method of claim 11 or 12, wherein the formulation is administered at a constant rate of 1 mL / min.

15. The method of claim 11 or 12, wherein the formulation is administered at a constant rate of 2 mL / min.

16. The method of any one of claims 1 to 15, wherein the administered volume of the formulation is about 5 mL to about 40 mL per kidney.

17. The method of claim 16, wherein the administered volume of the formulation is about 15 mL to about 30 mL per kidney.

18. The method of claim 16 or 17, wherein the administered volume of the formulation is about 20 mL to about 25 mL per kidney.

19. The method of any one of claims 16-18, wherein the administered volume of the formulation is about 25 mL per kidney.

20. The method of any one of claims 16-19, wherein the administered volume of the formulation is 25 mL per kidney.

21. The method of any one of claims 1-19, wherein the administered volume of the formulation is a fixed volume.

22. The method of any one of claims 1-20, wherein the administered volume is not dependent on the subject’s body weight.

23. The method of any one of claims 1-21, wherein the administered volume is not dependent on the size of the renal pelvis.

24. The method of any one of claims 1 to 20, wherein the administered volume of the formulation is based on the volume of the renal pelvis of each kidney of the subject.

25. The method of claim 24, wherein the administered volume of the formulation is about 0.1 mL to about 30 mL larger than the volume of the renal pelvis of each kidney of the subject.

26. The method of claim 19 or 20, wherein the administered volume of the formulation is about 10 mL larger than the volume of the renal pelvis of each kidney of the subject.

27. The method of any one of claims 24-26, wherein the administered volume of the formulation is 10 mL larger than the volume of the renal pelvis of each kidney of the subject.

28. The method of any one of claims 1 to 27, wherein the intrarenal pressure is above about 20 mm Hg and below about 200 mm Hg during the administration.

29. The method of claim 28, wherein the intrarenal pressure is above about 40 mm Hg and below about 180 mm Hg during the administration.

30. The method of any one of claims 1-28, wherein the intrarenal pressure is above baseline pressure during the administration.

31. The method of claim 30, wherein the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

32. The method of any one of claims 1 to 31, wherein the maximum intrarenal pressure during the administration is less than about 175 mm Hg, less than about 150 mm Hg, less than about 125 mm Hg, or less than about 100 mm Hg.

33. The method of any one of claims 1 to 32, wherein the maximum intrarenal pressure during the administration is less than about 125 mm Hg.

34. The method of any one of claims 1 to 33, wherein the maximum intrarenal pressure during the administration is less than about 100 mm Hg.

35. The method of any one of claims 1 to 34, wherein the ureter remains occluded for about 5 minutes to about 60 minutes after the administration.

36. The method of claim 35, wherein the ureter remains occluded for about 10 minutes after the administration37. The method of claim 35, wherein the ureter remains occluded for about 30 minutes after the administration.

38. The method of any one of claims 1-37, wherein the intrarenal pressure is below 200 mm Hg after the administration.

39. The method of any one of claims 1-38, wherein the intrarenal pressure is maintained below 100 mm Hg after the administration.

40. The method of any one of claims 1-39, wherein the intrarenal pressure is maintained above baseline pressure after the administration.

41. The method of claim 40, wherein the baseline pressure is between about 0 mm Hg and about 20 mm Hg.

42. The method of any one of claims 1-41, wherein the formulation comprises a pharmaceutically acceptable carrier or excipient.

43. The method of any one of claims 1 to 42, wherein the formulation is a therapeutic formulation.

44. The method of claim 43, wherein the therapeutic formulation is for treating a kidney disease.

45. The method of claim 43 or 44, wherein the therapeutic formulation is a gene therapy.

46. The method of any one of claims 43-45 , wherein the therapeutic formulation comprises a therapeutically effective amount of recombinant adeno-associated virus (rAAV).

47. The method of claim 46, wherein the therapeutically effective amount of rAAV is between about 108viral genomes (vg) and about 1015vg per kidney.

48. The method of claim 46 or 47, wherein the therapeutically effective amount of rAAV is between about 1012vg and about 1015vg per kidney.

49. The method of any one of claims 46-48, wherein the therapeutically effective amount of rAAV is between about 1014vg and about 1015vg per kidney.

50. The method of claim 46, wherein the therapeutically effective amount of rAAV is between about 108vg / mL and about 1015vg / mL.

51. The method of claim 46 or 50, wherein the therapeutically effective amount of rAAV is between about IO10vg / mL and about 1014vg / mL.

52. The method of claim 46, 50, or 51 , wherein the therapeutically effective amount of rAAV is between about 1012vg / mL and about 1013vg / mL.

53. The method of any one of claims 46-52, wherein the rAAV comprises a vector comprising a transgene.

54. The method of claim 53, wherein the transgene encodes a therapeutic RNA or a therapeutic protein.

55. The method of claim 54, wherein the therapeutic RNA is a circular RNA.

56. The method of claim 54 or 55, wherein the therapeutic RNA is a miRNA inhibitor.

57. The method of claim 56, wherein the miRNA inhibitor is a miRNA sponge or tough decoy.

58. The method of claim 54, wherein the therapeutic protein is polycystin-1 or a fragment thereof.

59. The method of claim 45, wherein the therapeutic protein is polycystin-2 or a fragment thereof.

60. The method of any one of claims 46-59, wherein the rAAV vector is derived from AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, or a variant thereof.

61. The method of any one of claims 46-60, wherein the rAAV comprises a capsid protein of AAV1, AAV2, AAV2i8, AAV3, AAV3-B, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAVrh8R, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAV-DJ, AAV LK03, AAVrh74, AAV44-9, AAV.kl3, AAV.k20, or a variant thereof62. The method of claim 61, wherein the capsid protein is a variant of AAV9.

63. The method of claim 61 or 62, wherein the AAV capsid protein transduces kidney cells.

64. The method of any one of claims 61-63, wherein the capsid protein is an AAV.kl3 capsid protein.

65. The method of claim 64, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO:

166. The method of claim 64 or 65, wherein the AAV.kl3 capsid protein comprises an amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452- 458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

67. The method of any one of claims 64-66, wherein the AAV.kl3 capsid protein consists of an amino acid sequence of SEQ ID NO: 2 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

68. The method of any one of claims 64-67, wherein the AAV.kl3 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wildtype AAV9 capsid.

69. The method of any one of claims 61-63, wherein the capsid protein is an AAV.k20 capsid protein.

70. The method of claim 69, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 with 1, 2, 3, 4, or more amino acid substitutions at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

71. The method of claim 69 or 70, wherein the AAV.k20 capsid protein comprises an amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452- 458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

72. The method of any one of claims 69-71, wherein the AAV.k20 capsid protein consists of an amino acid sequence of SEQ ID NO: 3 at positions 452-458, wherein positions 452-458 of the AAV capsid protein are numbered with reference to SEQ ID NO: 1.

73. The method of any one of claims 69-72, wherein the AAV.k20 capsid increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to a wildtype AAV9 capsid.

74. The method of any one of claims 46-73, wherein the method increases transduction of kidney cells by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 100%, or more, compared to transduction in kidney cells using intravenous administration.

75. The method of any one of claims 63, 68, 73, or 74, wherein the kidney cells are proximal tubule cells, distal tubule cells, or collecting duct cells.

76. The method of any one of claims 46-73, wherein the method results in the transduction of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or more of the nephrons in the kidney.

77. A method of treating a kidney disease or disorder in a subject in need thereof, the method comprising administering a therapeutic formulation comprising an rAAV to the subject by performing the method of any one of claims 46-76.

78. The method of any one of claims 1-77, wherein the subject is a human.

79. The method of any one of claims 46-78, wherein the subject is seropositive for the rAAV prior to administration of the therapeutic formulation comprising the rAAV.

80. The method of any one of claims 46-78, wherein the subject is seronegative for the rAAV prior to administration of the therapeutic formulation comprising the rAAV.

81. The method of any one of claims 1-80, wherein the subject has autosomal dominant polycystic kidney disease (ADPKD).

82. The method of claim 81, wherein the subject has ADPKD1.

83. The method of claim 81, wherein the subject has ADPKD2.

84. The method of any one of claims 1-83, wherein the method inhibits or ameliorates renal cyst development.

85. The method of any one of claims 1 to 42, wherein the formulation is a diagnostic formulation.

86. The method of claim 85, wherein the diagnostic formulation is for a kidney scan

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