Methods of recombinant adeno-associated virus kidney administration

Abstract

The technology described herein relates to methods of administering a recombinant adeno-associated virus (rAAV) to the kidney of a subject using a retrograde ureteral pathway. Such methods of administration may be used to treat kidney-related conditions in a subject in need thereof. Also described herein are pharmaceutical compositions comprising a recombinant adeno-associated virus (rAAV) for administration to the kidney.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202480048893.9 (22) Application Date 2024.05.30 (30) Priority Data 63 / 469,766 2023.05.30 US 63 / 578,838 2023.08.25 US (85) PCT International Application Entering National Phase Date 2026.01.23 (86) PCT International Application Application Data PCT / US2024 / 031768 2024.05.30 (87) PCT International Application Publication Data WO2024 / 249692 EN 2024.12.05 (71) Applicant: Asco Biotech, Inc. Address: North Carolina, USA (72) Inventors: P. Mullier, S. Gabriel, P. Dickin, K. Borner (74) Patent Agency: King & Wood Mallesons, Beijing 11256 Patent Attorneys: Tai Hongshi, Mengni (51) Int.Cl. A61K 48 / 00 (2006.01) (54) Invention Title: Method for Recombinant Adeno-Associated Virus Kidney Administration (57) Abstract: The technology described herein relates to a method for administering recombinant adeno-associated virus (rAAV) to the kidneys of a subject using a retrograde ureteral route. Such administration method can be used to treat kidney-related conditions in subjects with such needs. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) for kidney administration is also described herein. Claims (10 pages), Description (145 pages), Sequence Listing (electronic publication), Drawings (56 pages), CN 121752299 A, 2026.03.27, CN 1 21 75 22 99 A. 1. A method for transducing nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: guiding a catheter through the subject's urethra, bladder, and ureter; and administering a solution containing the rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight, wherein the nephrons in the kidney are transduced by the rAAV with high efficiency. 2. The method of claim 1, wherein the administration of the solution containing the rAAV to the kidney is carried out over about 0.5 minutes to about 60 minutes. 3. The method of claim 1, wherein the administration of the solution containing the rAAV to the kidney is carried out over about 1 minute to about 2 minutes. 4. The method of claim 1, wherein the solution containing the rAAV is administered at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. 5. The method of claim 1, wherein the method results in at least about 5%, at least about 10%, and to [amount missing] in the kidney.At least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more of the nephrons are transduced by the rAAV. 6. The method of claim 1, wherein the rAAV transduction efficiency of the nephrons in said kidney is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, or at least 3500-fold higher than the transduction efficiency of a corresponding nephron in another kidney treated by intravenous administration of the solution containing said rAAV. 7. The method of claim 1, wherein the rAAV does not contain an AAV9 capsid, and the rAAV transduction efficiency in the nephron of the kidney is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, or at least 3500-fold higher than the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner. 8. The method of claim 1, wherein the rAAV does not contain an AAV9 capsid, and the rAAV transduction efficiency in the proximal tubular cells of the nephron of the kidney is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, or at least 3500-fold higher than the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner. 9. The method of claim 1, further comprising the step of occluding renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney prior to administration of the solution containing the rAAV. 10. The method of claim 9, further comprising the step of unoccluding the renal vessels after a period of approximately 10 minutes to approximately 60 minutes following administration of the solution containing the rAAV. 11. The method of claim 1, wherein the kidney is not isolated from the systemic circulation. 12. The method of claim 1, wherein renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney are not occluded during the conduct of the method. 13. The method of claim 1, wherein the solution containing the rAAV is administered to the kidney at an intrarenal pressure of approximately 27 cm H2O to approximately 80 cm H2O. 14. The method of claim 1, wherein the subject is a human, non-human primate, horse, dog, or pig. 15. The method of claim 1, wherein at least approximately 30% of the nephrons in the kidney are transduced by the rAAV. 16.17. The method of claim 1, wherein the amount of the solution containing the rAAV administered to the subject is from about 0.13 mL / kg to about 0.33 mL / kg. 18. The method of claim 1, wherein the amount of the solution containing the rAAV administered to the subject is from about 0.27 mL / kg to about 0.33 mL / mg. 19. The method of claim 9, wherein the solution containing the rAAV is administered using a balloon catheter. 20. The method of claim 9, wherein the renal vessels are closed using a balloon catheter. 21. The method of claim 9, wherein the renal vessels are closed using clamps. 22. The method of claim 1, wherein only one of the renal artery or renal vein is closed. 23. The method of claim 1, wherein the renal vein of the kidney is not closed. 24. The method of claim 1, wherein the method does not include continuous perfusion of the isolated kidney. 25. The method of claim 1, wherein the method does not include a closed loop containing the kidney. 25. The method of claim 1, wherein the method does not include a basic closed system comprising the kidney. 26. The method of claim 1, wherein the method does not include shunting circulation from the kidney. 27. The method of claim 1, wherein the method does not include bypassing the kidney. 28. The method of claim 1, wherein the method is performed in vivo. 29. The method of claim 1, wherein the method is not performed outside the body. 30. The method of claim 10, wherein the time period for occluding the at least one renal vessel is 15-45 minutes after the occlusion. 31. The method of claim 10, wherein the time period for occluding the at least one renal vessel is 20-40 minutes after the occlusion. 32. The method of claim 10, wherein the time period for occluding the at least one renal vessel is about 15-30 minutes after the occlusion. 33. The method of claim 10, wherein the amount of the solution comprising the rAAV is about 0.13 mL / kg to about 0.33 mL / kg, and wherein the time period for occluding the renal vessel is about 15-30 minutes after the occlusion. 34. The method of claim 1, wherein the rAAV comprises an AAV capsid protein selected from Table 1. 35. The method of claim 1, wherein the rAAV comprises a capsid protein selected from AAV2G9, AAV2.5, AAVDJ, and AAV2. 36. The method of claim 35, wherein the capsid protein is AAV2G9.37. The method of claim 1, wherein the rAAV comprises a reasonable polyploid. 38. The method of claim 1, wherein the solution comprises the rAAV at a concentration of 10⁸ viral genomes / mL (vg / mL) to 10¹⁵ vg / mL. 39. The method of claim 1, wherein the solution comprises the rAAV at a concentration of 10⁸ vg / mL to 10¹³ vg / mL. 40. The method of claim 1, wherein the solution comprises a total of 1 x 10¹³ to 2 x 10¹³ rAAV viral genomes. 41. The method of claim 1, wherein the solution comprises a total of 5 x 10¹³ to 6 x 10¹³ rAAV viral genomes. 42. The method of claim 1, wherein the solution comprises a total of 1 x 10¹⁰ viral genomes. 43. The method of claim 1, wherein the rAAV comprises a transgene. 44. The method according to claim 43, wherein the transgene is selected from: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome in infants with sensorineural deafness (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); potassium inward rectifier channel subfamily J member 1 (KCNJ1); MAGED2 (V type); Mucin 1 (MUC1); Nephroticin 1 (NPHP1); Nephroticin (NPHS1); Nephroticin 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL); Polycystic protein 1 (PKD1); Polycystic protein 2 (PKD2); Polycystic kidney and liver disease 1 (PKHD1); Protein transporter Sec61 subunit α isoform 1 (SEC61A1); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 7 member 9 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and combinations thereof. 45. The method according to claim 43, wherein the transgene is selected from: aquaporin 2 (AQP2); ATPaseNa+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport V0 subunit A4 (ATP6V0A4); ATPase H+ transport V1 subunit B1 (ATP6V1B1); Arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); Carbonic anhydrase 2 (CA2); Calcium-sensitive receptor (CaSR); Chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); Chloride voltage-gated channel Kb (CLCNKB); Tight junction protein 16 (CLDN16); Tight junction protein 19 (CLDN19); Cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; Alpha stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; Pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); Sodium channel epithelial subunit 1 α (SCNN1A); Sodium channel epithelial subunit 1 β (SCNN1B); Sodium channel epithelial subunit 1 γ (SCNN1G); Solute carrier family 12 members 1(SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 1 member 1 (SLC1A1); Solute carrier family 2 member 2 (SLC2A2); Solute carrier family 34 member 1 (SLC34A1); Solute carrier family 34 member 3 (SLC34A3); Solute carrier family 36 member 2 (SLC36A2); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 4 member 1 (SLC4A1); Solute carrier family 6 member 19 (SLC6A19); Solute carrier family 6 member 20 (SLC6A20); Solute carrier family 7 member 7 (SLC7A7); Solute carrier family 7 member 9 (SLC7A9); Transient receptor potential cation channel subfamily M member 6 (TRPM6); WD repeat domain 72 (WDR72); Lysine-free (WNK, Lysine-deficient protein kinase 1 (WNK1); lysine-free (WNK, lysine-deficient) protein kinase 4 (WNK4); and combinations thereof. 46. The method of claim 43, wherein the transgene comprises an inhibitor of a gene or protein selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD). Claims 3 / 10 pages 4 CN 121752299 A 47. The method of claim 1, wherein the circulating serum of the subject does not neutralize the rAAV after administration. 48. The method of claim 1, wherein the circulating serum of the subject contains an antibody that neutralizes the rAAV to be administered and the antibody does not neutralize the rAAV in the kidney after administration. 49. The method of claim 1, wherein subsequent administration of the rAAV according to claim 1 is performed without causing a substantial inflammatory response in the kidney. 50. The method of claim 49, wherein the subsequent administration is performed at least one day later. 51. The method of claim 49, wherein the subsequent administration is performed at least one month later. 52. The method of claim 1, wherein the method transduces the proximal tubules of the kidney with the rAAV. 53. The method of claim 1, wherein the method transduces at least one cell population of the glomerulus, Bowman's capsule, proximal convoluted tubule, loop of Henry, distal convoluted tubule, or collecting duct of the kidney with the rAAV. 54. The method of claim 1, wherein the rAAV comprises a kidney-specific promoter. 55. The method of claim 54, wherein the kidney-specific promoter is selected from: kidney-specific cadherin.56. The method of claim 54, wherein the kidney-specific promoter is a synthetic promoter. 57. The method of claim 1, wherein the rAAV has a genome containing promoters specific to the proximal convoluted tubule and / or collecting duct. 58. A method for treating kidney-related conditions in a subject with this need, the method comprising administering recombinant adeno-associated virus (rAAV) to the subject by performing the method of claim 1. 59. The method of claim 58, wherein the kidney-related condition is selected from: autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome; autosomal dominant tubulointerstitial nephropathy (ADTKD); medullary cystic nephropathy; nephronial tuberculosis; Bart syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; thin basement membrane nephropathy; cystinuria; Liddle syndrome; papillary kidney syndrome; and cystin storage disease. 60. The method of claim 58, wherein the kidney-related condition is selected from: episodic mineralocorticoid hyperplasia syndrome, autosomal dominant hypocalcemia, autosomal dominant hypomagnesemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, type A cystinuria, type B cystinuria, type 1 Dent disease, type 2 Dent disease / Lowe syndrome, dicarboxyaminoaciduria, distal RTA, EAST / SeSAME syndrome, Fanconi Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2, Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Gitelman syndrome, glucocorticoid-suppressible aldosteronism, Hartnup syndrome, hereditary hypophosphatemic rickets with hypercalciuria, HNF1B-related nephropathy, BH4 deficiency hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 4 hypomagnesemia, type 5 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, hypomagnesemia, seizures and intellectual disability type 1, hypomagnesemia.Seizures and intellectual disability type 2, iminoglycineuria, type 2 Kenny-Caffey syndrome, Liddle syndrome, lysineuria protein intolerance, type 2 neonatal inflammatory skin and bowel disease, nephrogenic diabetes insipidus, nephrogenic antidiuretic hormone secretion disorder syndrome, type 1 pseudoketoalbuminemia, type 1A pseudoketoalbuminemia, type 2b pseudoketoalbuminemia, type 2c pseudoketoalbuminemia, type 2d pseudoketoalbuminemia, type 2e pseudoketoalbuminemia, type 3 renal tubular acidosis, and X-linked hypophosphatemic rickets. 61. The method according to claim 58, wherein the kidney-related condition is cystinuria, and the transgene is SLC3A1 and / or SLC7A9. 62. The method of claim 58, wherein the kidney-related condition is autosomal dominant polycystic kidney disease (ADPKD), and the transgene is PKD1, PKD2, and / or GANAB. 63. A method for transducing at least about 10% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) blocking renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney; b) guiding a catheter through the subject's urethra, bladder, and ureter; c) administering a solution containing the rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; and d) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, wherein the method results in at least about 10% of the nephrons in the kidney being transduced by the rAAV. 64. A method for transducing at least about 25% of the nephrons in a subject's kidney using recombinant adeno-associated virus (rAAV), the method comprising: a) occluding renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney; b) guiding a catheter through the subject's urethra, bladder, and ureter; c) administering a solution containing the rAAV to the renal pelvis of the kidney via the catheter at a rate of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; and d) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV. 65. A method for transducing at least about 25% of the nephrons in a subject's kidney using recombinant adeno-associated virus (rAAV), the method comprising: a) occluding the renal arteries of the kidney without occluding the renal veins of the kidney; b) guiding a catheter through the subject's urethra, bladder, and ureter; c)d) administering a certain amount of a solution containing the rAAV to the renal pelvis of the kidney via the catheter; and d) unblocking the renal artery after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV. 66. A method for transducing nephrons in the kidney of a subject, the method comprising: a) occluding a renal vessel selected from the renal artery, renal vein, and combinations thereof in the kidney; b) guiding a catheter through the urethra, bladder, and ureter of the subject; c) administering a volume of a solution containing rAAV, not rAAV9, into the renal pelvis of the kidney via the catheter; and d) unoccluding the renal vessel after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, (Claims 5 / 10, page 6, CN 121752299 A) wherein the method results in a transduction efficiency at least twice as high as that obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner. 67. The method of claim 66, wherein the rAAV comprises a capsid protein selected from Table 1. 68. The method of claim 66, wherein the rAAV has at least 2-fold higher transduction efficiency in the kidney compared to the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner. 69. The method of claim 66, wherein the rAAV has 400-fold higher transduction efficiency in the kidney compared to the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner. 70. A method of transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: a) isolating the kidney from systemic circulation; b) guiding a catheter through the subject's urethra, bladder, and ureter; c) administering a solution containing the rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; and d) re-introducing the kidney into systemic circulation after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV. 71. A method of treating a kidney condition in a subject with this need, the method comprising: administering a first recombinant adeno-associated virus (rAAV) encoding a genetically modified virus having a therapeutic effect on the kidney condition to the kidney of the subject; and, after administering the first rAAV, administering a transgenic virus encoding a genetically modified virus having a therapeutic effect on the kidney condition to the kidney or a different kidney of the subject.72. The method of claim 71, wherein at least one solution containing the first and / or second rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. 73. The method of claim 71, wherein the solution containing the second rAAV is administered to the kidney after about one week. 74. The method of claim 71, wherein the first and / or second rAAV is administered by a method comprising: guiding a catheter through the urethra, bladder, and ureter of the subject; and administering a solution containing the first or second rAAV to the renal pelvis of the kidney through the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject. 75. The method of claim 71, wherein the first and / or second rAAV is administered by an administration method comprising: a) occluding renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney; b) guiding a catheter through the subject's urethra, bladder, and ureter; c) administering a solution containing the first or second rAAV into the kidney or the renal pelvis of a different kidney at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight, through the catheter; and d) unoccluding the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the first or second rAAV. 76. The method of claim 71, wherein the administration method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV. 77. The method of claim 71, wherein the subject has neutralizing antibodies against the first rAAV therapeutic agent prior to the administration. 78. The method of claim 71, wherein the capsid protein of the first rAAV has the same serotype as the capsid protein of the second rAAV. 79. The method of claim 71, wherein the capsid protein of the first rAAV has a different serotype than the capsid protein of the second rAAV. 80. The method of claim 71, wherein the time period for subsequent administration of the second rAAV is determined based on the efficacy or persistence of the administration of the first rAAV. 81. The method of claim 71, wherein the first rAAV is administered to the subject's first kidney, and the second rAAV is administered to the subject's second kidney.82. The method of claim 71, wherein the first rAAV is administered to the first kidney of the subject, and the second rAAV is administered to the first kidney of the subject. 83. The method of claim 71, wherein the first rAAV is administered to both kidneys of the subject, and the second rAAV is administered to both kidneys of the subject. 84. A method of treating kidney disease in a subject with this need, the subject being seropositive for a recombinant adeno-associated virus (rAAV) therapeutic agent, the method comprising: administering to the kidney of the subject a transgenic rAAV therapeutic agent encoding a therapeutic effect on the kidney disease, wherein the subject does not elicit a significant immune response to the rAAV therapeutic agent in the kidney. 85. The method of claim 84, wherein the subject has neutralizing antibodies against the rAAV therapeutic agent prior to the administration. 86. The method of claim 84, wherein the rAAV is administered by means of: guiding a catheter through the urethra, bladder, and ureter of the subject; and administering a solution containing the first or second rAAV into the renal pelvis of the kidney through the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject. 87. The method of claim 84, wherein the rAAV is administered by means of: a) occluding renal vessels selected from renal arteries, renal veins, and combinations thereof; b) guiding a catheter through the urethra, bladder, and ureter of the subject; c) administering a solution containing the rAAV into the renal pelvis of the kidney at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject; and d) unoccluding the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV, wherein the administration method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV. 88. A method for transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: a) blocking renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney; b) guiding a catheter through the subject's urethra, bladder, and ureter; c) administering a solution containing the rAAV into the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight, wherein the rAAV contains capsid proteins selected from Table 1; and d)The renal vessels are unblocked after a period of approximately 10 to approximately 60 minutes following the administration of the solution containing the rAAV, wherein the method results in at least approximately 25% of the nephrons in the kidney being transduced by the rAAV. 89. A method of treating a kidney-related condition in a subject with this need, the method comprising administering the rAAV to the subject by performing the method according to claim 88. 90. The method of claim 89, wherein the rAAV is administered to the kidney at an intrarenal pressure of approximately 25 cm H2O to approximately 55 cm H2O. 91. The method of claim 89, wherein the volume of the solution is from 0.27 mL / kg to 0.33 mL / kg. 92. The method of claim 89, wherein the time period is 30–60 minutes after the administration of the solution containing the rAAV. 93. The method of claim 89, wherein the subject is seropositive for the rAAV prior to the administration of the solution containing the rAAV. 94. The method according to any one of claims 1, 58, 63-66, 70, 71, 84, 88, or 89, wherein the rAAV is administered in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, or vesicles. 95. The method according to any one of claims 1, 58, 63-66, 70, 71, 84, 88, or 89, wherein the rAAV is administered in lipid nanoparticles (LNPs). 96. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV), said pharmaceutical composition comprising: a) an AAV capsid protein selected from Table 1; b) a transgene comprising: i) a gene selected from the following: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome with sensorineural deafness (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); Potassium inward rectifying channel subfamily J member 1 (KCNJ1); MAGED2 (type V); Mucin 1 (MUC1); Nephroticin 1 (NPHP1); Nephroticin (NPHS1); Nephroticin 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL); Polycystin 1 (PKD1); Polycystin 2(PKD2); Polycystic Kidney Hepatitis 1 (PKHD1); Protein Transporter Sec61 Subunit α Isotype 1 (SEC61A1); Solute Transporter Family 12 Member 1 (SLC12A1); Solute Transporter Family 12 Member 3 (SLC12A3); Solute Transporter Family 3 Member 1 (SLC3A1); Solute Transporter Family 7 Member 9 (SLC7A9); Von Hippel-Lindau Tumor Suppressor (VHL); and combinations thereof; or ii) an inhibitor of a gene or protein selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD); and c) a pharmaceutically acceptable carrier. 97. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV), the pharmaceutical composition comprising: a) an AAV capsid protein selected from Table 1; b) a transgene comprising a gene selected from: aquaporin 2 (AQP2); ATPase Na+ / K+ transporter A subunit α1 (ATP1A1); ATPase H+ transporter V0 subunit A4 (ATP6V0A4); ATPase H+ transporter V1 subunit B1 (ATP6V1B1); arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); carbonic anhydrase 2 (CA2); calcium-sensitive receptor (CaSR); chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); chloride voltage-gated channel Kb (CLCNKB); tight junction protein 16 (CLDN16); Tight junction protein 19 (CLDN19); Cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enzyme-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator containing FXYD domain / motif 2(FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; α-stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); sodium channel epithelial subunit 1 α (SCNN1A); sodium channel epithelial subunit 1 β (SCNN1B); sodium channel epithelial subunit 1 γ (SCNN1G); solute carrier family 12 member 1 (SLC12A1); solute carrier family 12 member 3 (SLC12A3); solute carrier family 1 member 1 (SLC1A1); solute carrier family 2 member 2 (SLC2A2); solute carrier family 34 member 1 (SLC34A1); solute carrier family 34 member 3 (SLC34A3); solute carrier family 36 member 2 (SLC36A2); solute carrier family 3 member 1 (SLC3A1); solute carrier family 4 member 1 (SLC4A1); member 19 of solute carrier family 6 (SLC6A19); member 20 of solute carrier family 6 (SLC6A20); member 7 of solute carrier family 7 (SLC7A7); member 9 of solute carrier family 7 (SLC7A9); member 6 of transient receptor potential cation channel subfamily M (TRPM6); WD repeat domain 72 (WDR72); lysine-free (WNK, lysine-deficient) protein kinase 1 (WNK1); lysine-free (WNK, lysine-deficient) protein kinase 4 (WNK4); and combinations thereof; and c) a pharmaceutically acceptable carrier. 98. The pharmaceutical composition of claim 96 or 97, wherein the pharmaceutically acceptable carrier comprises mannitol. 99. The pharmaceutical composition of claim 96 or 97, wherein the AAV comprises the capsid protein of AAV2G9. 100. The pharmaceutical composition according to claim 96 or 97, wherein the solution containing the rAAV is at a concentration of 10⁸ viral genomes / mL (vg / mL) to 10¹⁵ vg / mL. 101.102. The pharmaceutical composition of claim 96 or 97, wherein the solution containing the rAAV has a concentration of 108 vg / mL to 1013 vg / mL. 103. The pharmaceutical composition of claim 96 or 97, wherein the pharmaceutical composition contains a total of 1 x 1013 to 2 x 1013 rAAV viral genomes. 104. The pharmaceutical composition of claim 96 or 97, wherein the pharmaceutical composition is a unit dose of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject. 105. The pharmaceutical composition of claim 96 or 97, wherein the pharmaceutical composition is a unit dose of about 0.27 mL / kg to about 0.33 mL / kg. 106. The pharmaceutical composition of claim 96 or 97, wherein the transgene comprises a reporter protein. 107. The pharmaceutical composition of claim 96 or 97, wherein the genome of the rAAV comprises a kidney-specific promoter. 108. The pharmaceutical composition of claim 107, wherein the kidney-specific promoter is selected from: a kidney-specific cadherin (KSPC) gene promoter; a Na+ / glucose cotransporter (SGLT2) gene promoter; a sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and an E-cadherin (ECAD) gene promoter. 109. The pharmaceutical composition of claim 107, wherein the kidney-specific promoter is a synthetic promoter. 110. The pharmaceutical composition of claim 96 or 97, wherein the genome of the rAAV comprises a promoter specific to the proximal convoluted tubule and / or collecting duct. 111. The pharmaceutical composition of claim 96 or 97, wherein the rAAV is formulated for delivery in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, or vesicles. 112. The pharmaceutical composition of claim 96 or 97, wherein the rAAV is formulated for delivery in lipid nanoparticles (LNPs). 113. A method of transducing nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: guiding a catheter through the urethra, bladder, and ureter of the subject; and administering a solution containing the rAAV to the renal pelvis of the kidney at an amount of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject, wherein the rAAV contains AAV2G9, and wherein the nephrons in the kidney are transduced by the rAAV with high efficiency. (Claims 10 / 10 pages)11 CN 121752299 A Method for Kidney Administration of Recombinant Adeno-Associated Virus Cross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 469,766, filed May 30, 2023, and U.S. Provisional Application No. 63 / 578,838, filed August 25, 2023, pursuant to 35 USC § 119(e), the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application contains a sequence list, which has been filed in XML format via the Patent Centre and is incorporated herein by reference in its entirety. The XML copy was created on May 30, 2024, named 046192-000108WOPT_SL.xml, and is 51,444 bytes in size. Technical Field

[0003] The technology described herein relates to methods for administering recombinant adeno-associated virus (rAAV) to the kidney and to pharmaceutical compositions comprising rAAV for administration to the kidney. Background Art

[0004] The primary function of the kidneys is to filter blood, thereby removing waste and excess fluid. The kidneys maintain a healthy balance of water, salts, and minerals. The kidneys also control blood pressure. In humans, the kidneys are perfused with approximately 1500 L of blood daily, which translates to 180 L of glomerular filtrate (primary urine) and 1 to 2 L of final urine daily.

[0005] The kidneys consist of over one million filtration units called nephrons. Nephrons, in order of filtrate passage, comprise the following areas: glomerulus, Bowman's capsule, proximal tubule, loop of Henry, distal tubule, and collecting duct. The glomerulus is the site of blood filtration; its small fenestrations allow fluids and small molecules to pass through while keeping blood cells and proteins outside the tubules; the filtrate is captured by Bowman's capsule. The proximal tubule (also interchangeably called the proximal convoluted tubule (PCT)) filters 65% of the primary urine; reabsorbs glucose, amino acids, solutes, and low molecular weight proteins; and maintains acid-base balance by reabsorbing bicarbonate. The loop of Henry reabsorbs water and salt; its thin descending limb reabsorbs water to concentrate urine, and its thick ascending limb allows for ion exchange. The distal tubule (also interchangeably called the distal convoluted tubule (DCT)) regulates extracellular fluid volume and electrolyte homeostasis. The collecting duct reabsorbs more water, finely regulating the final urine products. Urine flows out of the collecting duct and into the smaller and larger calyces of the kidney. The urine then travels through the renal pelvis and ureter, is stored in the bladder, and is expelled through the urethra.

[0006] Kidney-related conditions can be treated with gene vectors such as recombinant adeno-associated virus (rAAV). However, intravenous (IV) administration of rAAV is not beneficial because the virus can be transduced to other organs (e.g., the liver). An alternative administration method is multiple renal biopsies; however, this method is clinically unfavorable. Urologists are likely to rule out such procedures.Kidney biopsy is an option, but because the kidney is highly vascularized, puncture can lead to excessive bleeding. Since the kidney is accessible via a vascular tract, another approach is renal artery cannulation, renal vein cannulation, connecting a cannula to a pump, and circulating an agent (e.g., rAAV) through the vascular system, which is thus isolated and cannulated. However, this is a complex procedure that can lead to complications and discomfort. There is an urgent need for a clinically meaningful method of administration to efficiently deliver rAAV to the kidney. There is also a need for rAAV that exhibits tropism and / or high efficiency in the kidney. [Specification 1 / 145 pages 12 CN 121752299 A Summary of the Invention

[0007] Embodiments of the technology described herein relate to a method of administering recombinant adeno-associated virus (rAAV) to the kidney of a subject using a retrograde ureteral route. Such an administration method can be used to treat kidney-related conditions in subjects with this need. Pharmaceutical compositions comprising recombinant adeno-associated virus (rAAV) for administration to the kidney are also described herein.]

[0008] This document also describes specific rAAVs that exhibit high transduction in the kidneys after retrograde ureteral administration, including but not limited to rAAVs containing a capsid selected from Table 1 or Figures 4-5. Furthermore, exemplary parameters for retrograde ureteral administration are described herein, including dosage, timing, and / or renal vascular occlusion.

[0009] In several aspects, this document describes methods for transducing a sufficient number of nephrons in the kidneys of a subject with recombinant adeno-associated virus (rAAV) to achieve an effective expression level in the kidneys (e.g., the expression level of rAAV, or the expression level of the transgene contained in the rAAV).

[0010] In one aspect, this document describes a method for transducing nephrons in the kidneys of a subject with recombinant adeno-associated virus (rAAV), the method comprising: guiding a catheter through the subject's urethra, bladder, and ureter; and administering a solution containing rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight), wherein the renal nephrons containing nephron cells are transduced with high efficiency by rAAV.

[0011] In some embodiments of any aspect, the solution containing rAAV is administered to the kidney for about 0.5 minutes to about 60 minutes.

[0012] In some embodiments of any aspect, the solution containing rAAV is administered to the kidney for about 1 minute to about 2 minutes.

[0013] In some embodiments of any aspect, the solution containing rAAV is administered at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments of any aspect, the solution containing rAAV is administered at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

[0014] In some embodiments of any aspect, the method results in at least about 5%, at least about 10%, and at leastAbout 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 nephrons are transduced by rAAV.

[0015] In some embodiments of any aspect, the rAAV transduction efficiency of nephrons in a kidney is increased by at least 2, at least 5, at least 10, at least 50, at least 100, at least 400, at least 1000, or at least 3500 times compared to the corresponding transduction efficiency of a corresponding nephron in another kidney treated by intravenous administration of a solution containing rAAV.

[0016] In some embodiments of any aspect, rAAV is not AAV9, and the rAAV transduction efficiency of nephrons in the kidney is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, or at least 3500-fold higher than the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

[0017] In some embodiments, nephrons are transduced with an efficiency index greater than 1.

[0018] In some embodiments of any aspect, rAAV does not contain an AAV9 capsid, and the rAAV transduction efficiency in proximal tubular cells of nephrons in the kidney is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, at least 400-fold, at least 1000-fold, or at least 3500-fold higher than the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

[0019] In some embodiments of any aspect, the method further includes the step of blocking renal vessels selected from the renal arteries, renal veins, and combinations thereof before administering the solution containing rAAV.

[0020] In some embodiments of any aspect, the method further includes the step of unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing rAAV.

[0021] In some embodiments of any aspect, the method further includes not isolating the kidney from the systemic circulation.

[0022] In some embodiments of any aspect, the method further includes not blocking renal vessels selected from the renal arteries, renal veins, and combinations thereof during the conduct of the method.

[0023] In some embodiments of any aspect, the solution containing rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments of any aspect, the solution containing rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

[0024] In some embodiments of any aspect, the subject is a human, a non-human primate, a horse, a dog, or a pig.

[0025] In some embodiments of any aspect, at least about 30% of the renal nephrons are transduced by rAAV.

[0026] In some embodiments of any aspect, the amount of rAAV-containing solution administered to the subject is about 0.2 mL / kg to about 0.27 mL / kg.

[0027] In some embodiments of any aspect, the amount of rAAV-containing solution administered to the subject is about 0.27 mL / kg to about 0.33 mL / mg.

[0028] In some embodiments of any aspect, the rAAV-containing solution is administered using a balloon catheter.

[0029] In some embodiments of any aspect, the renal vessels are closed using a balloon catheter.

[0030] In some embodiments of any aspect, the renal vessels are closed using clamps, for example, after laparoscopy.

[0031] In some embodiments of any aspect, only one of the renal arteries or renal veins of the kidney is closed.

[0032] In some embodiments of any aspect, the renal veins of the kidney are not blocked.

[0033] In some embodiments of any aspect, the method does not include continuous perfusion of the isolated kidney.

[0034] In some embodiments of any aspect, the method does not include a closed loop containing the kidney.

[0035] In some embodiments of any aspect, the method does not include a basic closed system containing the kidney.

[0036] In some embodiments of any aspect, the method does not include shunting circulation from the kidney.

[0037] In some embodiments of any aspect, the method does not include bypassing the kidney.

[0038] In some embodiments of any aspect, the method is performed in vivo.

[0039] In some embodiments of any aspect, the method is performed without leaving the body.

[0040] In some embodiments of any aspect, the time period for blocking at least one renal vessel is 15-45 minutes after blocking.

[0041] In some embodiments of any aspect, the time period for blocking at least one renal vessel is 20-40 minutes after blocking.

[0042] In some embodiments of any aspect, the time period for occluding at least one renal vessel is about 15-30 minutes after occlusion.

[0043] In some embodiments of any aspect, the time period for occluding at least one renal vessel is about 30 minutes after occlusion.

[0044] In some embodiments of any aspect, the amount of the solution containing rAAV is about 0.13 mL / kg to about 0.33 mL / kg, and the time period for occluding the renal vessel is about 15-30 minutes after occlusion.

[0045] In some embodiments of any aspect, the amount of the solution containing rAAV is about 0.2 mL / kg to about 0.27 mL / kg.mL / kg, and wherein the time period for blocking the renal vessels is approximately 15-30 minutes after blocking.

[0046] In some embodiments of any aspect, the amount of solution containing rAAV is approximately 0.2 mL / kg to approximately 0.27 mL / kg, and wherein the time period for blocking the renal vessels is approximately 30 minutes after blocking. Specification 3 / 145 pages 14 CN 121752299 A

[0047] In some embodiments of any aspect, rAAV comprises AAV capsid protein selected from the serotypes provided in Table 1. In some embodiments of any aspect, rAAV comprises AAV capsid protein selected from serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV2.5G9, AAV2.5, AAVrh8, AAVrh10, AAVrh74, AAV10, AAV11, and AAVDJ.

[0048] In some embodiments of any aspect, rAAV comprises a capsid protein selected from AAV2G9, AAV2.5, AAVDJ, and AAV2.

[0049] In some embodiments of any aspect, the capsid protein is AAV2G9.

[0050] In some embodiments of any aspect, rAAV comprises a rational polyploid.

[0051] In some embodiments of any aspect, the solution contains rAAV at a concentration of 10⁸ viral genomes / mL (vg / mL) to 10¹⁵ vg / mL.

[0052] In some embodiments of any aspect, the solution contains rAAV at a concentration of 10⁸ vg / mL to 10¹³ vg / mL.

[0053] In some embodiments of any aspect, the solution contains a total of 1 x 10¹³ to 2 x 10¹³ rAAV viral genomes.

[0054] In some embodiments of any aspect, the solution contains a total of 5 x 10¹³ to 6 x 10¹³ rAAV viral genomes.

[0055] In some embodiments of any aspect, the solution contains a total of 1 x 10¹⁰ viral genomes.

[0056] In some embodiments of any aspect, rAAV contains a transgene.

[0057] In some embodiments of any aspect, the transgene is selected from: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome with sensorineural deafness (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II.α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); potassium inward rectifier channel subfamily J member 1 (KCNJ1); MAGED2 (type V); mucin 1 (MUC1); nephroticin 1 (NPHP1); nephrotic protein (NPHS1); nephrotic 2 (NPHS2; Podocin); inositol polyphosphate-5-phosphatase (OCRL); polycystic protein 1 (PKD1); polycystic protein 2 (PKD2); polycystic kidney and liver disease 1 (PKHD1); protein transporter Sec61 subunit α isoform 1 (SEC61A1); solute carrier family 12 member 1 (SLC12A1); solute carrier family 12 member 3 (SLC12A3); solute carrier family 3 member 1 (SLC3A1); member 9 of the solute carrier family 7 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and combinations thereof.

[0058] In some embodiments in any aspect, the transgene is selected from: aquaporin 2 (AQP2); ATPase Na+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport VO subunit A4 (ATP6VOA4); ATPase H+ transport VO subunit B1 (ATP6V1B1); arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); carbonic anhydrase 2 (CA2); calcium-sensitive receptor (CaSR); chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); chloride voltage-gated channel Kb (CLCNKB); tight junction protein 16 (CLDN16); tight junction protein 19 (CLDN19); cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enzyme-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); GlycineAcid amidotransferase (GATM); Guanine nucleotide-binding protein; α-stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); sodium channel epithelial subunit 1 α (SCNN1A); sodium channel epithelial subunit 1 β (SCNN1B); sodium channel epithelial subunit 1 γ (SCNN1G); solute carrier family 12 member 1 (SLC12A1); solute carrier family 12 member 3 (SLC12A3); solute carrier family 1 member 1 (SLC1A1); solute carrier family 2 member 2 (SLC2A2); solute carrier family 34 member 1 (SLC34A1); solute carrier family 34 member 3 (SLC34A3); solute carrier family 36 member 2 (SLC36A2); solute carrier family 3 member 1 (SLC3A1); solute carrier family 4 member 1 (SLC4A1); Solute carrier family 6 member 19 (SLC6A19); Solute carrier family 6 member 20 (SLC6A20); Solute carrier family 7 member 7 (SLC7A7); Solute carrier family 7 member 9 (SLC7A9); Transient receptor potential cation channel subfamily M member 6 (TRPM6); WD repeat domain 72 (WDR72); Lysine-free (WNK, lysine-deficient) protein kinase 1 (WNK1); Lysine-free (WNK, lysine-deficient) protein kinase 4 (WNK4); and combinations thereof.

[0059] In some embodiments of any aspect, the transgene comprises an inhibitor of a gene or protein selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD).

[0060] In some embodiments of any aspect, the circulating serum of the subject is not neutralized with rAAV after administration.

[0061] In some embodiments of any aspect, the subject's circulating serum contains antibodies that neutralize the rAAV to be administered and the antibodies do not neutralize rAAV in the kidney after administration.

[0062] In some embodiments of any aspect, subsequent administration of rAAV is performed without causing a substantial inflammatory response in the kidney.

[0063] In some embodiments of any aspect, subsequent administration is performed at least one day later. In some embodiments of any aspect, subsequent administration is performed at least one month later.

[0064] In some embodiments of any aspect, the method transduces the proximal tubules of the kidney with rAAV.

[0065] In some embodiments of any aspect, the method transduces at least one cell population of the glomerulus, Bowman's capsule, proximal convoluted tubule, loop of Henry, distal convoluted tubule, or collecting duct of the kidney with rAAV. In some embodiments of any aspect, the method transduces at least one of the glomerulus, Bowman's capsule, proximal convoluted tubule, loop of Henry, or distal convoluted tubule of the kidney with rAAV.

[0066] In some embodiments of any aspect, rAAV contains a kidney-specific promoter.

[0067] In some embodiments of any aspect, the kidney-specific promoter is selected from: the kidney-specific cadherin (KSPC) gene promoter; the Na+ / glucose cotransporter (SGLT2) gene promoter; the sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and the E-cadherin (ECAD) gene promoter.

[0068] In some embodiments of any aspect, the kidney-specific promoter is a synthetic promoter.

[0069] In some embodiments of any aspect, rAAV has a genome containing promoters specific to the proximal convoluted tubule and / or collecting duct.

[0070] In one aspect, a method for treating kidney-related conditions in a subject with such need is described herein, the method comprising administering recombinant adeno-associated virus (rAAV) to the subject by performing the method described herein.

[0071] In some embodiments of any aspect, the kidney-related condition is selected from: autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome; autosomal dominant tubulointerstitial nephropathy (ADTKD); medullary cystic nephropathy; nephron tuberculosis; Bart syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; thin basement membrane nephropathy; cystinuria; Liddle syndrome; papillary kidney syndrome; and cystine storage disease.

[0072] In some embodiments of any aspect, the kidney-related condition is selected from: episodic mineralocorticoid hyperparalysis syndrome, autosomal dominant hypocalcemia, autosomal dominant hypomagnesemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, and type 4 Bart syndrome.Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, type A cystinuria, type B cystinuria, type 1 Dent disease, type 2 Dent disease / Lowe syndrome, dicarboxyaminoaciduria, distal RTA, EAST / SeSAME syndrome, Fanconi Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2 Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Gitelman syndrome, glucocorticoid-suppressible aldosteronism, Hartnup syndrome, hereditary hypophosphatemic rickets with hypercalciuria, HNF1B-related nephropathy, BH4 deficiency hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 4 hypomagnesemia, type 5 hypomagnesemia / familial hypomagnesemia Family-related hypomagnesemia with hypercalciuria and nephrocalcinosis, hypomagnesemia, seizures and intellectual disability type 1, hypomagnesemia, seizures and intellectual disability type 2, iminoglycinuria, type 2 Kenny-Caffey syndrome, Liddle syndrome, lysineuria protein intolerance, type 2 neonatal inflammatory skin and bowel disease, nephrogenic diabetes insipidus, nephrogenic syndrome of abnormal antidiuretic hormone secretion, type 1 pseudoketoalbuminuria, type 1A pseudoketoalbuminuria, type 2b pseudoketoalbuminuria, type 2c pseudoketoalbuminuria, type 2d pseudoketoalbuminuria, type 2e pseudoketoalbuminuria, type 3 renal tubular acidosis, and X-linked hypophosphatemic rickets.

[0073] In some embodiments in any aspect, the kidney-related condition is cystinuria, and the transgene is SLC3A1 and / or SLC7A9.

[0074] In some embodiments of any aspect, the kidney-related condition is autosomal dominant polycystic kidney disease (ADPKD), and the transgene is PKD1, PKD2, and / or GANAB.

[0075] In one aspect, a method is described herein for transducing at least about 10% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder, and ureter; (c) administering a solution containing rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight); and (d) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the rAAV-containing solution, wherein the method results in the kidney...At least about 10% of the nephrons in the kidney are transduced by rAAV.

[0076] In one aspect, this article describes a method for transducing at least about 25% of the nephrons in the kidney of a subject with recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder and ureter; (c) administering a volume of rAAV-containing solution to the renal pelvis of the kidney through the catheter at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight); and (d) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the blocking and / or the administration of the rAAV-containing solution, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0077] In one aspect, this document describes a method for transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) occluding the renal artery of the kidney without occluding the renal vein; (b) guiding a catheter through the subject's urethra, bladder, and ureter; (c) administering a volume of rAAV-containing solution to the renal pelvis of the kidney via the catheter; and (d) unoccluding the renal artery after a period of about 10 minutes to about 60 minutes following occlusion and / or administration of the rAAV-containing solution, as described in page 6 / 145 of the specification, 17 CN 121752299 A, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0078] In one aspect, this document describes a method for transducing nephrons in the kidney of a subject, the method comprising: (a) occluding renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder, and ureter; (c) administering a volume of a solution containing rAAV, not rAAV9, into the renal pelvis of the kidney via the catheter; and (d) unoccluding the renal vessels after a period of approximately 10 minutes to approximately 60 minutes following occlusion and / or administration of the solution containing rAAV, wherein the method results in a transduction efficiency at least twice as high as that obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

[0079] In some embodiments of any aspect, the rAAV comprises a capsid protein selected from Table 1, but excluding AAV9.

[0080] In some embodiments of any aspect, the rAAV has at least 2-fold higher transduction efficiency in the kidney compared to the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

[0081] In some embodiments of any aspect, the rAAV has 400-fold higher transduction efficiency in the kidney compared to AAV9.Conduction efficiency. In some embodiments of any aspect, the rAAV has a 3500-fold higher transduction efficiency in the kidney compared to the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

[0082] In one aspect, a method for transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV) is described herein, the method comprising: (a) isolating the kidney from systemic circulation; (b) guiding a catheter through the subject's urethra, bladder, and ureter; (c) administering a solution containing rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight); and (d) re-introducing the kidney into systemic circulation after isolation and / or after a period of about 10 minutes to about 60 minutes following the administration of the solution containing rAAV, wherein the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0083] In one aspect, this document describes a method of treating kidney disease in a subject with this need, the method comprising: administering to the kidney of the subject a first recombinant adeno-associated virus (rAAV) encoding a transgene that has a therapeutic effect on kidney disease; and, following the administration of the first rAAV, administering to the subject's kidney, or a different kidney, a second rAAV encoding the transgene or a different transgene that has a therapeutic effect on kidney disease, wherein the first rAAV and the second rAAV are cross-reactive, and wherein the subject does not elicit a significant immune response to the second rAAV in the kidney.

[0084] In some embodiments of any aspect, at least one solution containing the first and / or the second rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments of any aspect, at least one solution containing the first and / or the second rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

[0085] In some embodiments of any aspect, the solution containing the second rAAV is administered about one week later.

[0086] In some embodiments of any aspect, the first and second rAAVs are administered by a method comprising: guiding a catheter through the subject's urethra, bladder, and ureter; and administering a solution containing the first or second rAAV into the renal pelvis of the kidney through the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight).

[0087] In some embodiments of any aspect, the first and / or second rAAVs are administered by a method comprising: (a) occluding a renal vessel selected from the renal artery, renal vein, and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder, and ureter; and (c) administering a solution containing the first or second rAAV into the renal pelvis of the kidney through the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight).The rAAV-containing solution is administered to the kidney or the renal pelvis of a different kidney at an amount of approximately 0.33 mL / kg to approximately 0.33 mL / kg (kg being the subject's body weight); and (d) the renal vessels are unblocked after the blocking and / or after a period of approximately 10 minutes to approximately 60 minutes following the administration of the rAAV-containing solution.

[0088] In some embodiments of any aspect, the administration method results in at least approximately 25% of the nephrons in the kidney being transduced by rAAV. Specification 7 / 145 pages 18 CN 121752299 A

[0089] In some embodiments of any aspect, the subject has neutralizing antibodies against the first rAAV therapeutic agent prior to administration.

[0090] In some embodiments of any aspect, the capsid protein of the first rAAV has the same serotype as the capsid protein of the second rAAV.

[0091] In some embodiments of any aspect, the capsid protein of the first rAAV has a different serotype than the capsid protein of the second rAAV.

[0092] In some embodiments of any aspect, the duration of subsequent administration of the second rAAV is determined based on the efficacy or persistence of the first rAAV administration.

[0093] In some embodiments of any aspect, the first rAAV is administered to the first kidney of the subject, and the second rAAV is administered to the second kidney of the subject.

[0094] In some embodiments of any aspect, the first rAAV is administered to the first kidney of the subject, and the second rAAV is administered to the first kidney of the subject.

[0095] In some embodiments of any aspect, the first rAAV is administered to both kidneys of the subject, and the second rAAV is administered to both kidneys of the subject.

[0096] In one aspect, a method of treating kidney disease in a subject who is seropositive for a recombinant adeno-associated virus (rAAV) therapeutic agent is described herein, the method comprising: administering to the kidney of the subject a transgenic rAAV therapeutic agent encoding a therapeutic effect on kidney disease, wherein the subject does not elicit a significant immune response to the rAAV therapeutic agent in the kidney.

[0097] In some embodiments of any aspect, the subject has neutralizing antibodies against the rAAV therapeutic agent prior to administration.

[0098] In some embodiments of any aspect, rAAV is administered by a method comprising: guiding a catheter through the subject's urethra, bladder, and ureter; and administering a solution containing rAAV into the renal pelvis of the kidney through the catheter at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight).

[0099] In some embodiments of any aspect, rAAV is administered by a method comprising: (a) blocking renal vessels selected from the renal artery, renal vein, and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder, and ureter.(c) administering a solution containing rAAV to the renal pelvis of the kidney via a catheter at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight); and (d) unblocking the renal vessels after closure and / or after administration of the solution containing rAAV for a period of about 10 minutes to about 60 minutes, wherein the administration method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0100] In one aspect, this document describes a method for transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) guiding a catheter through the subject's urethra, bladder, and ureter; (c) administering a solution containing rAAV to the renal pelvis of the kidney via the catheter at an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight), wherein the rAAV contains capsid proteins selected from Table 1; and (d) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the blocking and / or the administration of the rAAV-containing solution, wherein the method results in at least about 25% of nephrons in the kidney being transduced by rAAV.

[0101] In one aspect, this document describes a method for treating kidney-related conditions in a subject with such need, the method comprising administering 2G9 rAAV to the subject by performing a retrograde ureteral administration method as described herein.

[0102] In some embodiments of any aspect, rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments of any aspect, rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

[0103] In some embodiments of any aspect, the volume of the solution is from 0.13 mL / kg to 0.33 mL / kg.

[0104] In some embodiments of any aspect, the time period is 30-60 minutes after blocking and / or after administration of the solution containing rAAV.

[0105] In some embodiments of any aspect, the subject is seropositive for rAAV prior to administration of the solution containing rAAV.

[0106] In some embodiments of any aspect, rAAV is administered in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, or vesicles.

[0107] In some embodiments of any aspect, rAAV is administered in lipid nanoparticles (LNPs).

[0108] In one aspect, a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) is described herein, said pharmaceutical composition comprising: (a) an AAV capsid protein selected from Table 1; (b)The transgene comprises: (i) genes selected from the following: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome with sensorineural deafness in infants (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nucleus factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); potassium inward rectifier channel subfamily J member 1 (KCNJ1); MAGED2 (type V); mucin 1 (MUC1); renin 1 (NPHP1); Nephrotic protein (NPHS1); Nephrotic protein 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL); Polycystic protein 1 (PKD1); Polycystic protein 2 (PKD2); Polycystic kidney and liver disease 1 (PKHD1); Protein transporter Sec61 subunit α isoform 1 (SEC61A1); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 7 member 9 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and combinations thereof; or (ii) inhibitors of genes or proteins selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD); and (c) Pharmaceutically acceptable carrier.

[0109] In one aspect, a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) is described herein, the pharmaceutical composition comprising: (a) an AAV capsid protein selected from Table 1; (b) a transgene comprising a gene selected from: aquaporin 2 (AQP2); ATPase Na+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport VO subunit A4 (ATP6VOA4); ATPase H+ transport VO subunit B1 (ATP6V1B1); arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); carbonic anhydrase 2 (CA2); calcium-sensitive receptor (CaSR); chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); chloride voltage-gated channel Kb(CLCNKB); Tight junction protein 16 (CLDN16); Tight junction protein 19 (CLDN19); Cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; α-stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; Pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); Sodium channel epithelial subunit 1 α (SCNN1A); Sodium channel epithelial subunit 1 β (SCNN1B); Sodium channel epithelial subunit 1 γ (SCNN1G); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 1 member 1 (SLC1A1); Solute carrier family 2 member 2 (SLC2A2); Solute carrier family 34 member 1 (SLC34A1); Solute carrier family 34 member 3 (SLC34A3); Solute carrier family 36 member 2 (SLC36A2); Solute carrier family 3 member 1 (SLC3A1); Member 1 of Solute Carrier Family 4 (SLC4A1); Member 19 of Solute Carrier Family 6(SLC6A19); member 20 of solute carrier family 6 (SLC6A20); member 7 of solute carrier family 7 (SLC7A7); member 9 of solute carrier family 7 (SLC7A9); member 6 of transient receptor potential cation channel subfamily M (TRPM6); WD repeat domain 72 (WDR72); lysine-free (WNK, lysine-deficient) protein kinase 1 (WNK1); lysine-free (WNK, lysine-deficient) protein kinase 4 (WNK4); and combinations thereof; and (c) pharmaceutically acceptable carriers.

[0110] In some embodiments of any aspect, a pharmaceutically acceptable carrier comprises mannitol.

[0111] In some embodiments of any aspect, AAV comprises the capsid protein of AAV2G9.

[0112] In some embodiments of any aspect, the solution containing rAAV is at a concentration of 108 viral genomes / mL (vg / mL) to 1015 vg / mL.

[0113] In some embodiments of any aspect, the solution containing rAAV has a concentration of 10⁸ viral genomes / mL (vg / mL) to 10¹⁴ vg / mL.

[0114] In some embodiments of any aspect, the solution containing rAAV has a concentration of 10⁸ vg / mL to 10¹³ vg / mL.

[0115] In some embodiments of any aspect, the pharmaceutical composition contains a total of 1 x 10¹³ to 2 x 10¹³ rAAV viral genomes.

[0116] In some embodiments of any aspect, the pharmaceutical composition contains a total of 5 x 10¹³ to 6 x 10¹³ rAAV viral genomes.

[0117] In some embodiments of any aspect, the pharmaceutical composition is a unit dose of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject.

[0118] In some embodiments of any aspect, the pharmaceutical composition is a unit dose of about 0.27 mL / kg to about 0.33 mL / kg.

[0119] In some embodiments of any aspect, the transgene comprises a reporter protein.

[0120] In some embodiments of any aspect, the rAAV genome comprises a kidney-specific promoter.

[0121] In some embodiments of any aspect, the kidney-specific promoter is selected from: the kidney-specific cadherin (KSPC) gene promoter; the Na+ / glucose cotransporter (SGLT2) gene promoter; the sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and the E-cadherin (ECAD) gene promoter.

[0122] In some embodiments of any aspect, the kidney-specific promoter is a synthetic promoter.

[0123] In some embodiments of any aspect, the rAAV genome comprises a promoter specific to the proximal convoluted tubule and / or collecting duct.

[0124] In some embodiments of any aspect, rAAV is formulated for delivery in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, or vesicles.

[0125] In some embodiments of any aspect, rAAV is formulated for delivery in lipid nanoparticles (LNPs).

[0126] In one aspect, a method for transducing nephrons in the kidneys of a subject with recombinant adeno-associated virus (rAAV) is described herein as described in specification 10 / 145 pages 21 CN 121752299 A, the method comprising: guiding a catheter through the urethra, bladder, and ureter of a subject; and administering a solution containing rAAV to the renal pelvis of the kidney in an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight), wherein the rAAV contains AAV2G9, and wherein the nephrons of the kidney are transduced by rAAV with high efficiency. Brief Description of the Drawings

[0127] Figure 1 shows a tissue section of a rat kidney after retrograde ureteral administration of an rAAV library. The top inset shows YFP expression (brown staining) in the left kidney with the library exposed, and the bottom inset shows no detectable YFP expression in the unexposed right kidney.

[0128] Figure 2 shows magnified images from Figure 1. These images show that the renal tubules are heavily transduced by the library compared to the negative control.

[0129] Figure 3 shows a schematic diagram of the nephrons and images illustrating various transduction pathways.

[0130] Figure 4 is a bar chart showing the efficiency index of rAAV normalized to AAV9 in the renal medulla (e.g., mainly comprising the glomeruli, proximal tubules, and distal tubules) or renal cortex (e.g., mainly comprising the glomeruli, proximal tubules, and distal tubules). The efficiency index of rAAV normalized to AAV9 for each test is calculated as follows according to Formula I: (cDNA reads [%] / input [%]) / (cDNA AAV9 reads [%] / input AAV9 [%]).

[0131] Figure 5 is a dot plot showing the efficiency index of selected rAAVs normalized to AAV9 in the renal medulla or renal cortex from Figure 4. The efficiency index of rAAV normalized to AAV9 for each test was calculated as follows: (cDNA reads [%] / input [%]) / (cDNA AAV9 reads [%] / input AAV9 [%]).

[0132] Figures 6A-6X are a series of images and graphs showing the results of retrograde ureteral administration of AAV2G9 in pigs (see, for example, Example 2). Figure 6A shows the immunohistochemistry (IHC) of GFP after administration of AAV2G9 (1.41E+13VG). The images are shown in high power field (HPF), which is the standard measurement for histopathological diagnostic evaluation; HPF corresponds to 400x magnification. Figure 6B is a bar chart showing the quantitative results of the samples shown in Figure 6A, indicating the percentage of positive tubules. Figures 6C-6H are bar charts.Graphical and quantitative tables show the renal AAV gDNA (Fig. 6C), renal AAV protein (Fig. 6D), renal AAV cDNA (Fig. 6E), liver AAV gDNA (Fig. 6F), liver AAV protein (Fig. 6G), and liver AAV cDNA (Fig. 6H) in pig subjects RU13 and RU14 after retrograde ureteral administration of AAV2G9 to the treated kidney (“2G9”) or the contralateral kidney (“con”). “NoRT” is an RT-PCR negative control, indicating the absence of reverse transcriptase. Figures 6I-6L show images of the puncture sites of kidney samples 1-26 in the contralateral control kidney of subject RU13 (Figure 6I), the AAV2G9-treated kidney of subject RU13 (Figure 6J), the contralateral control kidney of subject RU14 (Figure 6K), and the AAV2G9-treated kidney of subject RU14 (Figure 6L). White circles indicate AAV protein levels below 1.00E+04 RLU / mg protein, light gray circles indicate AAV protein levels between 1.00E+04 and 1.00E+05 RLU / mg protein, dark gray circles indicate AAV protein levels between 1.00E+05 and 1.00E+06 RLU / mg protein, and black circles indicate AAV protein levels above 1.00E+06 RLU / mg protein. See also Tables 4-7 in Example 2, which correspond to Figures 6I-6L respectively. Figures 6M-6R are bar graphs showing the viral quantification results of the treated kidney (“2G9”) or the contralateral kidney (“con”) after retrograde administration of AAV2G9 to the treated kidney (“2G9”) in pig subject RU13: kidney AAV viral copy number (VCN) (Figure 6M), kidney AAV cDNA (Figure 6N), kidney AAV protein (Figure 6O), liver AAV VCN (Figure 6P), liver AAV cDNA (Figure 6Q), and liver AAV protein (Figure 6R). Figures 6S-6X are bar graphs showing the viral quantification results of the treated kidney (“2G9”) or the contralateral kidney (“con”) after retrograde administration of AAV2G9 to the treated kidney (“2G9”) in pig subject RU14: kidney AAV viral copy number (VCN) (Figure 6S), kidney AAV cDNA (Figure 6T), kidney AAV protein (Figure 6U), liver AAV VCN (Figure 6V), liver AAV cDNA (Figure 6W), and liver AAV protein (Figure 6X).

[0133] Figures 7A-7M are a series of images and graphs showing the results of retrograde ureteral administration in nonhuman primates (see, for example, Example 3). In Figures 7A-7F, kidney slices from NHP-RU1 were subjected to periodic acid Schiff.(PAS) staining and immunohistochemistry of green fluorescent protein GFP were performed (brown staining; indicating successful rAAV transduction in renal cells). Figure 7A shows a histological image of the contralateral kidney from NHP-RU1. Figure 7B shows a histological image of the kidney from NHP-RU1 after rAAV administration. Figure 7C shows a high-resolution image of a designated location in the renal cortex from anterior inferior section of Figure 7A. Figure 7D shows a high-resolution image of a designated location in the renal cortex from anterior inferior section of Figure 7B. Figure 7E shows multiple high-resolution images of designated locations in the renal medulla from anterior superior section of Figure 7B. Figure 7F shows multiple high-resolution images of designated locations in the renal cortex from anterior superior section of Figure 7B; in the high-resolution images shown, near 100% transduction is observed in the proximal convoluted tubules. The scale bar in Figures 7A-7B is 2 mm. The scale bar in the magnified images in Figures 7C-7F is 200 µm. Figure 7G is a bar chart showing the vector copy number (VCN) of each diploid genome in specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 8. Figure 7H is a bar chart showing the eGFP cDNA levels (relative fold change (log10) 2^ddCT) in specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 9. Figure 7I shows the RNA analysis for NHP_RU2, comparing the eGFP cDNA levels in specified samples (left bar in each sample) with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control levels (right bar in each sample); "NoRT" indicates no reverse transcriptase and "NTC" indicates no template control. Figure 7J is a bar chart showing the relative light units (RLU) per mg of protein in specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 10. Figures 7K-7M are a series of tables showing the localization of AAV2G9 administered to the kidneys from NHP-RU1 and NHP-RU2; biopsies 1, 2, 3, and 10 are anterior superior; biopsies 7, 8, 9, and 11 are anterior inferior; biopsies 4, 5, and 6 are midline kidneys. Figure 7K shows viral DNA (vg / dg), eGFP RNA (cDNA fold change), luciferase protein (RLU / mg), and %GFP+ in the proximal convoluted tubule (PCT). Each column of the tables (DNA / RNA / protein) was analyzed independently. Each biopsy showed consistency between molecular assays and histological staining. Figure 7L shows the biopsy ranking based on transduction (viral DNA vg / dg). Figure 7M shows the biopsy ranking based on functional transduction (luciferase protein RLU / mg).

[0134] Figures 8A-8E are a series of images showing non-human primates administered AAV2G9 via the retrograde ureteral route.Co-staining of the kidneys (see, for example, Example 3). Figure 8A illustrates the analysis procedure using VISIOPHARM®. Figures 8B-8E show counterstaining of AAV2G9 (GFP – purple staining), proximal convoluted tubules (CD13 – yellow staining), and distal convoluted tubules and collecting ducts (CK19 – blue staining). Figure 8B shows histological co-staining images of the contralateral kidney from NHP-RU1, and Figure 8C shows histological co-staining images of the kidney from NHP-RU1 treated with AAV-2G9. Figure 8D shows histological co-staining images of the contralateral kidney from NHP-RU2, and Figure 8E shows histological co-staining images of the kidney from NHP-RU2 treated with AAV-2G9. The results from Figures 8B-8E are quantified in Tables 11-12.

[0135] Figures 9A-9D are a series of graphs and tables showing the quantitative results of AAV2G9 neutralizing antibody levels in the serum of non-human primates NHP-RU1 and NHP-RU2. Figure 9A shows a schematic diagram of the transduction inhibition assay. Figure 9B shows the neutralizing antibody levels in the serum of NHP-RU1 before and after administration of AAV2G9. Figure 9C shows the neutralizing antibody levels in the serum of NHP-RU2 before and after administration of AAV2G9. Figure 9D shows the neutralizing antibody levels in the serum of both NHP-RU1 and NHP-RU2 before and after administration of AAV2G9.

[0136] Figures 10A-10E are a series of images and graphs showing retrograde ureteral administration of AAV2G9 to seropositive pig subject RU26 (see, for example, Example 5). In Figures 10A-10D, kidney sections from RU26 were stained with periodic acid Schiff (PAS) and subjected to immunohistochemistry for green fluorescent protein GFP (brown staining; indicating successful rAAV transduction in renal cells). Figure 10A shows a histological image of the contralateral kidney (anterior inferior (AI) region 1) from RU26; the scale bar is 2 mm in the low-magnification image on the left, 200 µm in the high-magnification image on the upper right, and 100 µm in the high-magnification image on the lower right. Figures 10B-10D show histological images of the rAAV-treated kidney from RU26 (AI3 in Figure 10B, AI4 in Figure 10C, and AI1 in Figure 10D). The scale bar is 2 mm in the low-magnification image on the left and 100 µm in the high-magnification image on the right. Figure 10E is a bar graph showing luciferase activity in a specified sample from RU26 in relative optical units (RLU) per mg of protein; the results are quantified in a table next to the bar graph. Detailed Description

[0137] Implementations of the technology described herein involve administering a large dose of the drug to the kidneys of a subject using a retrograde ureteral route.Methods of administration of recombinant adeno-associated virus (rAAV). As used herein, “retroureteral route” or “retroureter (RU)” or “retrograde route” or “retrograde route to ureter” or “retrograde injection via ureter” or “retrograde administration” or “retroureteral administration” are interchangeable and refer to the administration of a solution against the direction of urine outflow from the kidney, i.e., by injecting the solution into the ureter (or renal pelvis) and into the nephrons of the kidney (including into the renal tubules). Such administration methods can be used to treat kidney-related conditions in subjects in need. This document also describes pharmaceutical compositions comprising recombinant adeno-associated virus (rAAV) for administration to the kidney.

[0138] This document also describes specific rAAVs that exhibit high transduction in the kidney after retrograde ureteral administration, including but not limited to rAAVs comprising a capsid selected from those described in Table 1 and Figures 4-5. Compared to AAV9, certain types of rAAV (e.g., AAV2G9, AAV2.5, AAVDJ, AAV2, AAVKP1, AAVKP2, AAVKP3, and AAV2.7m8) exhibit improved renal transduction levels. Furthermore, exemplary parameters for retrograde ureteral administration, including dosage, timing, and renal vascular occlusion, are described herein. Retrograde ureteral administration results in improved renal transduction levels compared to other routes of administration (e.g., intravenous).

[0139] Administration and Treatment Methods In several aspects, this document describes methods for administering recombinant adeno-associated virus (rAAV) to the kidneys of subjects and methods for treating kidney-related conditions in subjects in need of such administration. In one aspect, this article describes a method for transducing a sufficient number of nephrons in the kidney of a subject with recombinant adeno-associated virus (rAAV) to obtain an effective expression level in the kidney, the method comprising administering a certain amount of an rAAV-containing solution to the ureter of the kidney via a retrograde route, wherein the rAAV-containing solution is administered to the kidney for a sufficient time and / or under intrarenal pressure sufficient to result in a pharmaceutically effective level of rAAV transduction in the renal nephrons.

[0140] In one aspect, this article describes a method for transducing nephrons in the kidney of a subject with recombinant adeno-associated virus (rAAV), the method comprising: guiding a catheter through the urethra, bladder and ureter of the subject; and administering an rAAV-containing solution to the renal pelvis of the kidney in an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight), wherein the renal nephrons containing nephron cells are transduced by rAAV with high efficiency.

[0141] In one aspect, this document describes a method for transducing at least about 15% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: administering a volume of a solution containing rAAV into the ureter of the kidney via a retrograde route, wherein said volume is about 0.13 mL / kg to about 0.33 mL / kg.mL / kg, where kg is the subject's body weight. In some embodiments, the administration of the rAAV-containing solution lasts for a period of approximately 0.5 minutes to approximately 2 minutes. In some embodiments, the administration of the rAAV-containing solution lasts for a period of approximately 1 minute to approximately 60 minutes. In some embodiments, the method further includes the step of blocking renal vessels selected from the renal artery, renal vein, and combinations thereof prior to the administration of rAAV. In some embodiments, the method further includes unblocking the renal vessels after blocking and / or after the administration of the rAAV-containing solution, for a period of approximately 10 minutes to approximately 60 minutes.

[0142] In one aspect, this document describes a method for transducing at least about 15% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) administering a volume of a solution containing rAAV into the ureter of the kidney via a retrograde route, wherein the volume is about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; and (c) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the blocking and / or the administration of the solution containing rAAV.

[0143] In one aspect, this document describes a method for transducing at least about 20% of nephrons in a subject's kidney with rAAV.

[0144] In one aspect, this document describes a method for transducing at least about 25% of nephrons in a subject's kidney with rAAV.

[0145] In one aspect, this document describes a method for transducing at least about 25% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) occluding the renal artery of the kidney without occluding the renal vein; (c) administering a volume of rAAV-containing solution into the ureter of the kidney via a retrograde route; and (c) unoccluding the renal artery after a period of about 10 minutes to about 60 minutes following the occlusion and / or the administration of the rAAV-containing solution. In some embodiments, the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0146] In one aspect, this document describes a method for transducing nephrons in the kidney of a subject, the method comprising: (a) occluding a renal vessel selected from the renal artery, renal vein, and combinations thereof; (b) administering a volume of a solution containing rAAV, not rAAV9, into the ureter of the kidney via a retrograde route; and (c) unoccluding the renal vessel after a period of approximately 10 minutes to approximately 60 minutes following occlusion and / or administration of the rAAV-containing solution. In some embodiments, the method results in at least twice the transduction efficiency compared to a corresponding administration using rAAV9 instead of the rAAV. In some embodimentsIn this context, the rAAV has a transduction efficiency at least 400 times or at least 3500 times higher than AAV9.

[0147] In one aspect, this document describes a method for transducing at least about 30% of nephrons in the kidney of a subject using rAAV. In another aspect, this document describes a method for administering recombinant adeno-associated virus (rAAV) to the kidney of a subject and / or for treating kidney-related conditions in a subject in need of this, the method comprising: (a) occluding at least one renal vessel of the kidney selected from the renal artery, renal vein, and combinations thereof; (b) administering a certain amount of a solution containing rAAV into the ureter of the kidney via a retrograde route, wherein the amount does not exceed 0.33 mL / kg; and (c) unoccluding the at least one renal vessel after a certain period of time following occlusion and / or administration of the solution containing rAAV.

[0148] In one aspect, this document describes a method for transducing at least about 15% of nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) administering a volume of a solution containing rAAV into the ureter of the kidney via a retrograde route, wherein the volume is about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight, wherein the rAAV contains a capsid protein from serotype AAV2G9; and (c) unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the blocking and / or administration of the rAAV-containing solution. In some embodiments, the method results in at least about 25% of nephrons in the kidney being transduced by 2G9 rAAV.

[0149] In one aspect, this document describes a method of administering recombinant adeno-associated virus (rAAV) to the kidney of a subject and / or treating kidney-related conditions in a subject in need of doing so, the method comprising: (a) blocking at least one renal vessel of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) administering a solution containing rAAV in an amount of about 0.13 mL / kg to about 0.33 mL / kg into the ureter of the kidney via a retrograde route, wherein the rAAV contains a capsid protein derived from serotype AAV2G9; and (c) unblocking the at least one renal vessel after blocking and / or after a certain period of time following the administration of the solution containing rAAV.

[0150] In some embodiments of any aspect, the steps of “blocking at least one renal vessel of the kidney selected from renal arteries, renal veins, and combinations thereof” or “blocking the renal vessel of the kidney selected from renal arteries, renal veins, and combinations thereof” or “blocking the renal vessel of the kidney selected from renal arteries, renal veins, and combinations thereof” are replaced by the step of “isolating the kidney from the systemic circulation”.

[0151] In some embodiments in any aspect, the renal vessels are unsealed "after a certain period of time" after sealing.The steps described in the instruction manual (pages 14 / 145, CN 121752299 A) such as "one less" or "unblocking the renal vessels after a certain period of time" are replaced with the step of "reintroducing the kidney into the systemic circulation after a certain period of time" after isolation.

[0152] In one aspect, this document describes a method for transducing at least about 25% of the nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) isolating the kidney from the systemic circulation; (b) administering a certain amount of a solution containing rAAV into the ureter of the kidney via a retrograde route, wherein the amount is about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; and (c) reintroducing the kidney into the systemic circulation after a period of about 10 minutes to about 60 minutes following isolation. In some embodiments, the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0153] In some aspects, this document describes a method for transducing nephrons of a subject's kidney using rAAV containing an AAV2g9 capsid. The method includes guiding a catheter through the subject's urethra, bladder, and ureter, and administering a solution containing rAAV to the renal pelvis of the kidney at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight). In various embodiments, one end of the catheter through which the solution is delivered is located in the ureter, and the solution is administered in the ureter and flushed into the renal pelvis of the kidney. In other embodiments, one end of the catheter through which the solution is delivered is located in the renal pelvis, and the solution is administered directly to the renal pelvis of the kidney. As a result of the administration, the nephrons of the kidney are transduced with rAAV at high efficiency.

[0154] In some aspects, this document describes a method for transducing nephrons of a subject's kidney using rAAV. The method includes guiding a catheter through the subject's urethra, bladder, and ureter, and administering a solution containing rAAV at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight). A solution containing rAAV is administered to the renal pelvis of the kidney at a rate of mL / kg (kg being the subject's body weight). In various embodiments, one end of the catheter through which the solution is delivered is located in the ureter, and the solution is administered in the ureter and flushed into the renal pelvis of the kidney. In other embodiments, one end of the catheter through which the solution is delivered is located in the renal pelvis, and the solution is administered directly to the renal pelvis of the kidney. As a result of the administration, the nephrons of the kidney are transduced with rAAV at a high efficiency.

[0155] As used herein, "high efficiency" refers to the transduction level that results in detectable and / or measurable levels of rAAV in the cells of the kidney, such as nephron transduction efficiency or nephron component transduction efficiency. In various embodiments, high efficiency corresponds to at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% orHigher nephron transduction efficiency or nephron component transduction efficiency. In some embodiments, high efficiency corresponds to an efficiency index greater than 1 (as defined herein; see, for example, Equation I), e.g., higher efficiency than that obtained by administering rAAV containing an AAV9 capsid. For example, efficiency indices greater than 1, greater than 2, greater than 3, greater than 4, greater than 5, greater than 6, greater than 7, greater than 8, greater than 9, greater than 10, greater than 20, greater than 30, greater than 40, greater than 50, greater than 60, greater than 70, greater than 80, greater than 50, greater than 600, greater than 700, greater than 800, greater than 900, greater than 1000, greater than 2000, greater than 3000, greater than 4000, greater than 5000. It should be understood that each of the individual efficiency indices described herein can be used to define a lower and upper limit for the range of efficiency indices. In some implementations, the efficiency index ranges from 1 to 6000, 1 to 5000, 1 to 4000, 1 to 3000, 1 to 2000, 1 to 1000, 1 to 900, 1 to 800, 1 to 700, 1 to 600, 1 to 500, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 90, 1 to 80, 1 to 70, 1 to 60, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. Such high efficiency corresponds to clinically significant transduction levels. In various embodiments, a clinically significant level is a therapeutically and / or pharmaceutically effective level.

[0156] In some embodiments, the method results in a therapeutically and / or pharmaceutically effective level of rAAV transduction in the nephrons of the kidney, which may vary depending on the specific disease and / or the location of the transduction in the kidney. The terms “therapeutically effective” or “pharmaceutically effective” transduction level refer to a transduction level sufficient to provide transgene expression (e.g., a transgene encoded by the rAAV genome) in the transduced kidney cells, which is sufficient to treat or improve at least one symptom caused or resulting from kidney-related disease in the subject, or to inhibit, mitigate, minimize, or reverse the progression of kidney-related disease in the subject. In various embodiments, a therapeutically and / or pharmaceutically effective level corresponds to a nephron transduction efficiency or nephron component transduction efficiency of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0157] In some embodiments, the method results in at least about 25% of the nephrons in the kidney being transduced by rAAV. For example, by blocking (e.g., clamping) the renal artery or isolating the kidney from the systemic circulation, administering 20 mL to the kidney via the ureter, holding for 15–30 minutes and then unblocking (e.g., releasing the clamp) or re-introducing the kidney into the systemic circulation, the rAAV transduction of the nephron will be at least 30%, which has not been previously reported.

[0158] As used herein, the terms “transducing a nephron” or “transducing nephrons” can be used interchangeably to refer to at least one cell of a transducing nephron, said at least one cell being: glomerular cells (including cells of Bowman's capsule); proximal tubule (also called proximal convoluted tubule) cells; cells of the loop of Henry (including the descending and / or ascending limbs, and their thick and / or thin segments); distal tubule (also called distal convoluted tubule) cells; collecting duct cells; or combinations thereof. Similarly, the terms “transducing a nephron 'component'” or “transducing nephron 'components'” can be used interchangeably to refer to at least one cell of a transducing nephron 'component', which is: a glomerulus (including Bowman's capsule); a proximal tubule (also called a proximal convoluted tubule); a loop of Henry (including the descending and / or ascending limbs, and their thick and / or thin segments); a distal tubule (also called a distal convoluted tubule); or a collecting duct.

[0159] The terms “nephron transduction efficiency” and “nephron component transduction efficiency” refer to the proportion of transduced nephrons or nephron components relative to the total number of nephrons or the total number of nephron components in the kidney. Therefore, a 10% nephron transduction efficiency means that 10% of the total number of nephrons in the kidney are transduced. For example, in a hypothetical kidney with exactly 1 million (1,000,000) nephrons, a 10% nephron transduction efficiency means that 100,000 out of those 1,000,000 nephrons are transduced. In other words, in this hypothetical kidney with exactly 1 million (1,000,000) nephrons, a 10% nephron transduction efficiency means that at least one cell in each of the 100,000 individual nephrons out of the total 1,000,000 individual nephrons is transduced. Similarly, a 10% proximal tubule transduction percentage means that 10% of the total number of proximal tubules in the kidney are transduced. For example, in a hypothetical kidney with exactly 1 million (1,000,000) proximal tubules (e.g., one proximal tubule per nephron), a 10% proximal tubule transduction efficiency means that 100,000 of those 1,000,000 proximal tubules are transduced.In other words, in a hypothetical kidney with exactly 1 million (1,000,000) nephrons (and therefore 10,000,000 proximal tubules), a 10% proximal tubule transduction efficiency means that at least one cell is transduced in each of the 100,000 individual proximal tubules out of the total 1,000,000 individual proximal tubules.

[0160] Because a transduced nephron component may have more than one transduced cell, this aspect can be discussed from the perspective of “cell transduction efficiency.” For example, a single nephron may have a 30% nephron cell transduction efficiency, meaning that 30% of the total number of cells constituting that single nephron is transduced. Similarly, a single proximal tubule may have a 30% proximal tubule cell transduction efficiency, meaning that 30% of the total number of cells in that single proximal tubule is transduced. It should be understood that even if 30% of the total number of cells in the proximal tubule is transduced, this does not preclude transduction of other components of the same nephron. For example, cells may have a 30% proximal tubule cell transduction efficiency as well as specified or unspecified cell efficiencies associated with cells of the loop of Henry (ascending and / or descending limbs, and their thick and / or thin segments), cells of the distal tubule, and / or cells of the corresponding collecting duct.

[0161] Thus, in some aspects, the prior art provides both nephron transduction efficiency and cell transduction efficiency. As an example, the terms "at least 20% proximal tubule transduction efficiency and at least 30% proximal tubule cell transduction efficiency" mean that at least 20% of the total number of proximal tubules in the kidney are transduced and at least 30% of the proximal tubule cells in each transduced proximal tubule are transduced, or in other words, at least 30% of the proximal tubule cells in at least 20% of the total number of proximal tubules in the kidney are transduced.

[0162] For example, the description herein of a certain percentage of nephron transduction in the kidney may refer to nephron transduction efficiency, nephron component transduction efficiency, and / or cell transduction efficiency.

[0163] In some embodiments, the method results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or higher proximal tubule transduction efficiency.

[0164] In some embodiments, the method results in at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or higher proximal tubule cell transduction efficiency. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0165] In some embodiments, the method results in at least 30% proximal tubule transduction efficiency and at least 30% proximal tubule cell transduction efficiency. In some embodiments, the method results in at least 20% proximal tubule transduction efficiency and at least 30% proximal tubule cell transduction efficiency. In some embodiments, the method results in at least 30% proximal tubule transduction efficiency and at least 20% proximal tubule cell transduction efficiency. In some embodiments, the method results in at least 20% proximal tubule transduction efficiency and at least 20% proximal tubule cell transduction efficiency. In some embodiments, the method results in at least 25% proximal tubule transduction efficiency and at least 25% proximal tubule cell transduction efficiency.

[0166] In some embodiments, the method results in about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more of the nephrons in the kidney being transduced by rAAV, or in other words, about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55- 65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more were converted by rAAVIn some implementations, the transduced cells are epithelial cells. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0167] In some embodiments, the method results in about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more of the nephrons in the kidney being transduced by rAAV, or in other words, about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25% of the total number of “nephron cells” (e.g., proximal convoluted tubule cells, loop of Henry cells, distal convoluted tubule cells, collecting duct cells, or any combination thereof) in the kidney. 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more are transduced by rAAV. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range. In some embodiments, the transduced cells are epithelial cells from the proximal tubule (see, for example, Example 3). In some embodiments, the transduced cells are epithelial cells from the loop of Henle. In some embodiments, the transduced cells are epithelial cells from the distal convoluted tubule. In some embodiments, the transduced cells are epithelial cells from the collecting duct. In some embodiments, the transduced cells are epithelial cells from the distal convoluted tubule and collecting duct (see, for example, Example 3).

[0168] In some embodiments, the transduced nephron cells are epithelial cells from the proximal tubule. Therefore, the method may include transducing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more (see, for example, Table 11). It should be understood that each of the individual percentages described herein can be used to define the lower and upper limits of the percentage range. Transduction of the proximal tubule or its epithelial cells can be measured using a variety of methods known in the art. As a non-limiting example, co-localization of the thick brush border (typical and unique to the proximal tubule) with the rAAV reporter gene can be quantified by comparing it to the total number of thick brush border cells. Other non-limiting examples of proximal tubule epithelial cell markers include macroprotein, cuboprotein, sodium-glucose cotransporter 1 (SGLT1), sodium-glucose cotransporter 2 (SGLT2), vimentin, Kim-1, Na / Pi, PDZ domain protein 1 (PDZK1), solute carrier family 3 member 1 (SLC3A1; also known as NaS1), stem cell antigen-1 (Sca-1), CD13 and / or water channels; see, for example, Agarwal et al., “Renal cell markers: lighthouses for managing renal diseases,” Am J Physiol Renal Physiol. 1 Dec 2021;321(6):F715-F739, the contents of which are incorporated herein by reference in their entirety.

[0169] In some embodiments, the transduced nephron cells are epithelial cells derived from the loop of Henle. Therefore, the method may include at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more of the percentages transduced in epithelial cells of the loop of Henry. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0170] In some embodiments, the method results in at least 30% Henry's loop transduction efficiency and at least 30% Henry's loop cell transduction efficiency. In some embodiments, the method results in at least 20% Henry's loop transduction efficiency and at least 30%Henry's loop cell transduction efficiency. In some embodiments, the method results in at least 30% Henry's loop transduction efficiency and at least 20% Henry's loop cell transduction efficiency. In some embodiments, the method results in at least 20% Henry's loop transduction efficiency and at least 20% Henry's loop cell transduction efficiency. In some embodiments, the method results in at least 25% Henry's loop transduction efficiency and at least 25% Henry's loop cell transduction efficiency.

[0171] In some embodiments, the transduced nephron cells are epithelial cells from the distal convoluted tubule. Therefore, the method may include transducing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more of the percentages described herein. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0172] In some embodiments, the method results in at least 30% distal tubule transduction efficiency and at least 30% distal tubule cell transduction efficiency. In some embodiments, the method results in at least 20% distal tubule transduction efficiency and at least 30% distal tubule cell transduction efficiency. In some embodiments, the method results in at least 30% distal tubule transduction efficiency and at least 20% distal tubule cell transduction efficiency. In some embodiments, the method results in at least 20% distal tubule transduction efficiency and at least 20% distal tubule cell transduction efficiency. In some embodiments, the method results in at least 25% distal tubule transduction efficiency and at least 25% distal tubule cell transduction efficiency.

[0173] In some embodiments, the transduced nephron cells are epithelial cells from collecting ducts. Therefore, the method may include transducing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0174] In some embodiments, the method results in at least 30% collecting duct transduction efficiency and at least 30% collecting duct cell transduction efficiency. In some embodiments, the method results in at least 20% collecting duct transduction efficiency and at least 30% collecting duct cell transduction efficiency. In some embodiments, the method results in at least 30% collecting duct transduction efficiency and at least 20% collecting duct cell transduction efficiency. In some embodiments, the method results in at least 20% collecting duct transduction efficiency and at least 20% collecting duct cell transduction efficiency. In some embodiments, the method results in at least 25% collecting duct transduction efficiency and at least 25% collecting duct cell transduction efficiency.

[0175] In some embodiments, the transduced nephron cells are epithelial cells from the distal convoluted tubule and / or collecting duct. Therefore, the method may include transducing at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%, 45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more of the percentages described herein in the epithelial cells of the distal convoluted tubules and / or collecting ducts of the kidney (see, for example, Table 12). It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0176] In some embodiments, the method results in at least 30% distal convoluted tubule and / or collecting duct transduction efficiency and at least 30% distal convoluted tubule and / or collecting duct cell transduction efficiency. In some embodiments, the method results in at least 20% distal convoluted tubule and / or collecting duct transduction efficiency and at least 30% distal convoluted tubule and / or collecting duct cell transduction efficiency. In some embodiments, the method results in at least 30% distal convoluted tubule and / or collecting duct transduction efficiency and at least 20% distal convoluted tubule and / or collecting duct cell transduction efficiency.Transduction efficiency of distal convoluted tubules and / or collecting duct cells. In some embodiments, the method results in at least 20% transduction efficiency of distal convoluted tubules and / or collecting ducts and at least 20% transduction efficiency of distal convoluted tubules and / or collecting duct cells. In some embodiments, the method results in at least 25% transduction efficiency of distal convoluted tubules and / or collecting ducts and at least 25% transduction efficiency of distal convoluted tubules and / or collecting duct cells.

[0177] In some embodiments, the transduction percentage of nephrons may represent the transduction percentage of all proximal tubules in the kidney. As a non-limiting example, 30% transduction of nephrons means the transduction of 30% of the proximal tubules or proximal tubule cells in each treated kidney. In some embodiments, at least 30% of nephrons or nephron cells in each treated kidney are transduced, and other nephrons may be transduced at locations other than the proximal tubules. In some embodiments, the percentage of nephron transduction in the kidney can be higher than the percentage of transduction in the proximal tubules, for example, when considering transduction in other locations of the proximal tubules and nephrons. Specification 19 / 145 pages 30 CN 121752299 A

[0178] In some embodiments, the method results in transduction of cells and / or nephrons in at least one renal pyramid and / or associated cortical region of the kidney. The renal pyramid is cone-shaped renal tissue in the medulla; a kidney may contain 7 to 18 renal pyramids. Each renal pyramid terminates in a renal papilla, where collecting ducts drain urine into the minor and major calyces, which converge into the renal pelvis and then into the ureter. The superficial cortical region includes proximal and distal tubules, which connect to the loops of Henry and collecting ducts in the deeper medullary renal pyramids. In some embodiments, the method results in the transduction of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more of the cells and / or nephrons in at least one renal pyramid and / or related cortical region of the kidney. In some embodiments, the method results in at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18 kidneys, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 40-50%,45-55%, 50-60%, 55-65%, 60-70%, 65-75%, 70-80%, 75-85%, 80-90%, 85-95%, 90-100%, 95-100% or more of transduction in at least one cell or at least one nephron in the renal pyramidal and / or associated cortical region. It should be understood that each of the individual percentages described herein can be used to define a lower and upper limit for the percentage range.

[0179] In some embodiments, the amount of solution containing rAAV is from about 0.13 mL / kg subject to about 0.33 mL / kg subject. “kg” (kilogram) represents the weight of the subject. In a 75 kg subject, the amount from 0.13 mL / kg to about 0.33 mL / kg is equivalent to 10 mL to 25 mL. In some embodiments, the amount may be adjusted according to the weight of the subject. For example, a maximum dose of 30 mL can be administered to a 90 kg subject instead of the maximum dose of 25 mL for a 75 kg subject. In some embodiments, the subject's weight is between 60 kg and 90 kg. Amounts below 0.13 mL / kg may be insufficient to induce adequate transduction in the kidneys. On the other hand, amounts above 0.33 mL / kg may impair the kidneys.

[0180] In some embodiments, the amount of the solution containing rAAV is from about 0.13 mL / kg to about 0.35 mL / kg. In some embodiments, the amount of the solution containing rAAV is from about 0.13 mL / kg to about 0.33 mL / kg, from about 0.15 mL / kg to about 0.30 mL / kg, or from about 0.2 mL / kg to about 0.25 mL / kg. In some embodiments, the amount of the solution containing rAAV is from about 0.27 mL / kg to about 0.33 mL / kg. In some embodiments, the amount of the solution containing rAAV is from about 0.2 mL / kg to about 0.27 mL / kg. In some embodiments, the amount of the solution containing rAAV is about 0.24 mL / kg. In some embodiments, the amount of the solution containing rAAV is about 0.13 mL / kg to about 0.35 mL / kg, about 0.15 mL / kg to about 0.35 mL / kg, about 0.2 mL / kg to about 0.35 mL / kg, about 0.25 mL / kg to about 0.35 mL / kg, about 0.3 mL / kg to about 0.35 mL / kg, about 0.13 mL / kg to about 0.30 mL / kg, about 0.13 mL / kg to about 0.25 mL / kg, or about 0.13 mL / kg to about 0.2 mL / kg. In some embodiments, the amount of the solution containing rAAV is 0.13 mL / kg, 0.14 mL / kg, 0.15 mL / kg, 0.16 mL / kg, etc.mL / kg, 0.17 mL / kg, 0.18 mL / kg, 0.19 mL / kg, 0.2 mL / kg, 0.21 mL / kg, 0.22 mL / kg, 0.23 mL / kg, 0.24 mL / kg, 0.25 mL / kg, 0.26 mL / kg, 0.27 mL / kg, 0.28 mL / kg, 0.29 mL / kg, 0.3 mL / kg, 0.31 mL / kg, 0.32 mL / kg, 0.33 mL / kg, 0.34 mL / kg, 0.35 mL / kg, 0.05-0.15 mL / kg, 0.10-0.20 mL / kg, 0.15-0.25 mL / kg, 0.20-0.30 mL / kg, 0.25-0.35 mL / kg, 0.05- 0.10 mL / kg, 0.10-0.15 mL / kg, 0.15–0.20 mL / kg, 0.20–0.25 mL / kg, 0.25–0.30 mL / kg, 0.30–0.35 mL / kg, 0.05–0.35 mL / kg, 0.10–0.35 mL / kg, 0.15–0.35 mL / kg, 0.20–0.35 mL / kg, 0.05–0.20 mL / kg, 0.05–0.25 mL / kg, or 0.05–0.30 mL / kg. It should be understood that each of the individual quantities described herein can be used to define the lower and upper limits of the quantity range. Specification 20 / 145 pages 31 CN 121752299 A

[0181] In some embodiments, the amount of solution containing rAAV is selected from the following amounts: at least 0.05 mL / kg, at least 0.1 mL / kg, at least 0.13 mL / kg, at least 0.14 mL / kg, at least 0.15 mL / kg, at least 0.16 mL / kg, at least 0.17 mL / kg, at least 0.18 mL / kg, at least 0.19 mL / kg, at least 0.20 mL / kg, at least 0.21 mL / kg, at least 0.22 mL / kg, at least 0.23 mL / kg, at least 0.24 mL / kg, at least 0.25 mL / kg, at least 0.26 mL / kg, at least 0.27 mL / kg, at least 0.28 mL / kg, at least 0.29 mL / kg, at least 0.30 mL / kg, at least 0.31 mL / kg, at least 0.32 mL / kg. The amounts are: at least 0.33 mL / kg, at least 0.34 mL / kg, and at least 0.35 mL / kg. In some embodiments, the amount of solution containing rAAV is selected from the following amounts: up to 0.30 mL / kg, up to 0.31 mL / kg, and at least 0.35 mL / kg.mL / kg, up to 0.32 mL / kg, up to 0.33 mL / kg, up to 0.34 mL / kg, or up to 0.35 mL / kg. It should be understood that each of the individual quantities described herein can be used to define the lower and upper limits of the quantity range.

[0182] In some embodiments, heparin is administered to the subject before, during, or after retrograde rAAV administration, for example, to prevent blood clotting during surgery.

[0183] In some embodiments, a solution containing rAAV is administered to the kidney under intrarenal pressure that causes tubular reflux and venous return without causing rupture of the fornix. The term “tubular reflux” (which may also be interchangeably referred to as “intrarenal reflux”) refers to the reflux (i.e., retrograde or backflow) of urine from the renal pelvis and calyces into the collecting ducts; in voiding cystourethrography, tubular reflux may manifest as a blush of the renal pyramids. The term “renal pelvic venous return” (also known as “renal pelvic reflux”) refers to the flow of fluid from the renal pelvis of the kidney into the renal venous system; renal pelvic venous return may occur when an abnormal amount of intrarenal pressure occurs in the opposite direction to normal. The term “renal fornix” (or “fornix”) refers to a fine, dot-like projection that extends from the lateral side of each renal calyx and a short distance into the renal column; each fornix contacts the renal pyramid on its inner surface. Rupture of the renal fornix may be due to increased renal pelvic pressure; rupture of one or more renal fornixes may result in perirenal or retroperitoneal extravasation (leakage) of urine.

[0184] Normal intrarenal pressure (IRP) ranges from 0 cm H2O to 20 cm H2O. IRP between 27 cm H2O and 41 cm H2O can cause renal pelvic tubular reflux. IRP between 41 cm H2O and 68 cm H2O can cause renal pelvic venous return. IRPs below 27 cm H2O do not cause tubular reflux or venous return in the renal pelvis and are therefore not effective for the method described herein. IRPs between 81 cm H2O and 95 cm H2O may cause fornix rupture. Therefore, the method described herein uses IRPs of 80 cm H2O or less. IRPs above normal (e.g., lasting for a longer period, such as several days) may be associated with infectious and hemorrhagic complications and kidney damage. See, for example, Pauchard et al., “A Practical Guide for Intra-Renal Temperature and Pressure Management during Rirs: What Is the Evidence Telling Us,” J Clin Med. 2022 Jun; 11(12): 3429; the contents of which are incorporated herein by reference in their entirety.

[0185] In some embodiments, the dosage and the resulting intrarenal pressure are determined in subjects such as humans, non-human primates, or pigs, which are relevant animal models for human translational studies due to their anatomical and physiological similarities to humans. Intrarenal pressure can be measured by methods known in the art, such as sensor leads (e.g., leads containing pressure sensors; e.g., placed in the renal cavity).

[0186] When the perfusion flow rate (i.e., the flow velocity) is greater than 6 mL / min, the ureter behaves like an open tube, resulting in a linear relationship between flow rate and pressure. In some embodiments, the administration of approximately 0.13 mL / kg to approximately 0.35 mL / kg of rAAV solution takes place over a period of approximately 0.5 minutes (30 seconds) to approximately 2 minutes (120 seconds). For subjects with smaller doses and / or smaller body size, the administration time of rAAV can be shorter (e.g., for subjects weighing 8–12 kg, administer 2.5 mL at 5 mL / min over approximately 0.5–1.0 minutes); for subjects with larger doses and / or larger body size, the administration time of rAAV can be longer (e.g., for subjects weighing 60–80 kg, administer 18 mL at 9 mL / min over approximately 1–2 minutes). Shorter rAAV administration times, as per the instructions (page 21 / 145, 32 CN 121752299, paragraph A), can reduce the risk of damage to the renal fornix structure. In some embodiments, the rAAV-containing solution is administered for a time sufficient to achieve the desired intrarenal pressure (e.g., approximately 25 cm H2O to approximately 55 cm H2O or approximately 27 cm H2O to approximately 80 cm H2O). In some embodiments, the rAAV solution is administered at a rate of about 0.13 mL / kg to about 0.35 mL / kg over a period of about 10 minutes to about 60 minutes, resulting in a flow rate between about 0.167 mL / min and about 2.5 mL / min. In some embodiments, the flow rate is less than 6 mL / min. In some embodiments, the ureter does not behave like an open tube during the administration of the rAAV solution. In some embodiments, the amount of solution administered significantly affects the efficacy of the treatment, while the flow rate does not significantly affect the efficacy of the treatment.

[0187] In some embodiments, the rAAV-containing solution is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments, the rAAV-containing solution is administered to the kidney at an intrarenal pressure of about 46 cm H2O. In some embodiments, the rAAV-containing solution is administered to the kidney at an intrarenal pressure of about 45 cm H2O. In some embodiments, a solution containing rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O. In some embodiments, the intrarenal pressure is about 27 cm H2O to about 41 cm H2O.A solution containing rAAV is administered to the kidney at an intrarenal pressure of approximately 41 cm H2O to approximately 68 cm H2O. In some embodiments, the solution is administered to the kidney at an intrarenal pressure of approximately 68 cm H2O to approximately 80 cm H2O. In some embodiments, the solution is administered to the kidney at an intrarenal pressure of at least 20 cm H2O, at least 25 cm H2O, at least 30 cm H2O, at least 35 cm H2O, at least 40 cm H2O, at least 45 cm H2O, at least 50 cm H2O, at least 55 cm H2O, at least 60 cm H2O, at least 65 cm H2O, at least 70 cm H2O, or at least 75 cm H2O. In some implementations, a solution containing rAAV is administered to the kidney at an intrarenal pressure of approximately 25 cm H2O, 30 cm H2O, 35 cm H2O, 40 cm H2O, 45 cm H2O, 50 cm H2O, 55 cm H2O, 60 cm H2O, 65 cm H2O, 70 cm H2O, 75 cm H2O, or 80 cm H2O. In some embodiments, a solution containing rAAV is administered to the kidney at intrarenal pressures ranging from approximately 20–30 cm H2O, 25–35 cm H2O, 30–40 cm H2O, 35–45 cm H2O, 40–50 cm H2O, 45–55 cm H2O, 50–60 cm H2O, 55–65 cm H2O, 60–70 cm H2O, 65–75 cm H2O, 70–80 cm H2O, 20–40 cm H2O, 20–50 cm H2O, 20–60 cm H2O, 20–70 cm H2O, 20–80 cm H2O, 25–85 cm H2O, 30–80 cm H2O, 40–80 cm H2O, 50–80 cm H2O, or 60–80 cm H2O. It should be understood that each of the individual intrarenal pressures described herein can be used to define the lower and upper limits of the intrarenal pressure range.

[0188] In some embodiments, a catheter or cannula is used to administer a solution containing rAAV to the ureter via a retrograde route. As used herein, the terms “catheter” and “cannula” are used interchangeably to refer to a hollow tube that can be inserted into, for example, a body cavity, blood vessel, urethra, ureter, etc.; in some embodiments, the catheter is thin (e.g., with a diameter of up to 5, 6, 7, 8, 9, 10, 11, 12 mm).(mm) and / or soft. In some embodiments, the catheter is made of a material that does not trigger an immune response (e.g., latex, silicone, TEFLON, polyvinyl chloride, etc.) and / or treated to reduce infection (e.g., silver-coated catheter). In some embodiments, any solution or pharmaceutical composition described herein (e.g., containing at least one rAAV) may be manually delivered into the ureter using a plunger associated with the catheter or cannula. In some embodiments, any solution or pharmaceutical composition described herein (e.g., containing at least one rAAV) may be manually delivered into the ureter using an automated infusion device such as a pump (injection pump, peristaltic pump, etc.).

[0189] In some embodiments, a catheter may be used to administer an rAAV solution to the kidney. The catheter may be inserted transurethral, ​​transbladder, and up the ureter of the kidney to a target location within the ureter, such as the renal pelvis, near the renal pelvis, or within the renal pelvis. In some embodiments, the catheter used for administering the rAAV-containing solution is a balloon catheter. In some embodiments, the balloon catheter is inflated before, during, and / or after administration of the rAAV-containing solution to close the ureter and prevent, inhibit, or minimize the backflow of urine and / or rAAV downstream of the balloon (e.g., from the bladder toward the kidney). In some embodiments, such as page 22 / 145 of CN 121752299 A, the balloon catheter is deflated to unclose the ureter, for example, after closure and / or after administration of the rAAV-containing solution for a certain period of time (e.g., 10–60 minutes). In some embodiments, the catheter used for administration of the rAAV-containing solution is a non-balloon catheter and / or the ureter is not closed during administration of the rAAV-containing solution.

[0190] In some embodiments, the amount of the rAAV-containing solution is administered retrogradely into the ureter of the kidney by injecting rAAV (e.g., a composition containing rAAV) into the ureter. The injection can be performed using a syringe connected to and in fluid communication with the catheter. The duration of the injection ranges from approximately 1 second (s) to approximately 5 minutes (min), approximately 1 second to approximately 4 minutes, approximately 1 second to approximately 3 minutes, approximately 1 second to approximately 2 minutes, approximately 1 second to approximately 1 minute, approximately 0.5 minutes (30 seconds) to approximately 0.75 minutes (45 seconds), approximately 0.5 minutes (30 seconds) to approximately 1 minute (60 seconds), approximately 1 minute (60 seconds) to approximately 2 seconds (120 seconds), approximately 0.5 minutes (30 seconds) to approximately 2 minutes (120 seconds), for example, approximately 1 s, approximately 2 s, approximately 3 s, approximately 4 s, approximately 5 s, approximately 6 s, approximately 7 s, approximately 8 s, approximately 9 s, approximately 10 s, approximately 11 s, approximately 12 s, approximately 13 s, approximately 14 s, approximately 15 s, approximately 16 s, approximately 17 s, approximately 18 s, approximately 19 s, approximately 20 s, approximately 21 s, approximately 22 s, approximately 23 s, approximately 24 s, approximately 25 s, approximately 26 s, approximately 27 ss, approximately 28 s, approximately 29 s, approximately 30 s, approximately 31 s, approximately 32 s, approximately 33 s, approximately 34 s, approximately 35 s, approximately 36 s, approximately 37 s, approximately 38 s, approximately 39 s, approximately 40 s, approximately 41 s, approximately 42 s, approximately 43 s, approximately 44 s, approximately 45 s, approximately 46 s, approximately 47 s, approximately 48 s, approximately 49 s, approximately 50 s, approximately 51 s, approximately 52 s, approximately 53 s, approximately 54 s, approximately 55 s, approximately 56 s, approximately 57 s, approximately 58 s, approximately 59 s, approximately 1 min, approximately 1.25 min, approximately 1.5 min, approximately 1.75 min, approximately 2 min, approximately 2.25 min, approximately 2.5 min, approximately 2.75 min, approximately 3 Injection time periods of approximately 3.25 min, 3.5 min, 3.75 min, 4 min, 4.25 min, 4.5 min, 4.75 min, or 5 min. In some embodiments, the injection time period is less than or equal to about 5 min, less than or equal to about 4 min, less than or equal to about 3 min, less than or equal to about 2 min, or less than or equal to about 1 min. It should be understood that each of the individual times described herein can be used to define the lower and upper limits of the time range.

[0191] In other embodiments, the administration of the amount of rAAV-containing solution does not include continuous perfusion of the rAAV-containing composition for more than about 5 minutes. In some embodiments, the rAAV-containing solution is administered to a kidney reversibly, i.e. temporarily, isolated from the systemic circulation using a retrograde ureteral route (e.g., using a syringe connected and in fluid communication with a catheter in the ureter) for a period of up to 5 minutes, and the isolated kidney is reintroduced into the systemic circulation after a period of about 10 minutes to about 60 minutes following isolation. In some implementations, the isolation period is replaced by a closure period of approximately 10 to 60 minutes after closure (e.g., using a balloon catheter or clamp), and the rAAV-containing solution is administered to the closed kidney via a retrograde ureteral route (e.g., using a syringe connected to and in fluid communication with a catheter in the ureter) for a period of up to 5 minutes. This up to 5-minute administration of rAAV can be performed at any time during the 10- to 60-minute isolation or closure period of the kidney. For example, rAAV administration may be performed at approximately 0-5 minutes, approximately 5-10 minutes, approximately 10-15 minutes, approximately 15-20 minutes, approximately 20-25 minutes, approximately 25-30 minutes, approximately 30-35 minutes, approximately 35-40 minutes, approximately 40-45 minutes, approximately 45-50 minutes, approximately 50-55 minutes, or approximately 55-60 minutes during the 10- to 60-minute kidney isolation or closure period. In some implementations, the rAAV-containing solution is administered in discontinuous incremental increments.The solution, for example, is injected for about 1 minute, then isolated or blocked for about 1 minute without injection, and this process is repeated until the entire amount of rAAV has been administered.

[0192] As described herein, the kidneys are reversibly isolated from the subject's systemic circulation before and during administration of the rAAV-containing solution. As used herein, the term "systemic circulation" refers to the flow of blood through the subject's vascular system from the heart to all organs and tissues (including the kidneys) and back to the heart. Oxygenated blood is carried by the heartbeat through arteries to organs and tissues (where arteries convert into arterioles) and then to capillaries where gas exchange occurs. Deoxygenated blood is then transferred from the capillaries to venules, which convert into veins and then back to the heart. Specifically for the kidneys, the vascular system includes the renal arteries (which supply oxygenated blood to the kidneys) and the renal veins (which carry deoxygenated blood away from the kidneys). "Isolating the kidneys from the systemic circulation" means slowing, minimizing, substantially stopping, or stopping the blood flow through a particular kidney. Isolating the kidney from the systemic circulation (Instructions for use, pages 23 / 145, CN 121752299 A) can be achieved by blocking at least one renal vessel of the kidney (i.e., the renal artery and / or renal vein) and / or by diverting circulation from the kidney (e.g., through an external circuit) so that systemic blood flow discontinuously enters the "isolated kidney" via the renal artery. Therefore, when the kidney is "isolated," any agent delivered to the systemic circulation will not enter the kidney and / or circulate through the kidney. Isolating the kidney from the systemic circulation results in reduced or absent urine production in the nephrons of the kidney (and thus in the kidney itself). Therefore, urine production is minimized or reduced relative to a kidney that is not isolated from or has stopped from the systemic circulation, which allows for more efficient backflow of retrograde ureteral rAAV administration into the urinary tract.

[0193] In some embodiments, the method includes blocking at least one renal vessel of the kidney. By blocking at least one renal vessel, the kidney is isolated from the systemic circulation. In some embodiments, only the renal artery is blocked and not the renal vein. In other embodiments, only the renal vein is blocked and not the renal artery. In other embodiments, both the renal artery and renal vein are blocked. Therefore, only one renal vessel may be blocked, or both renal vessels may be blocked. Blocking a renal vessel can be done by methods known in the art, including by occlusion or clamping. Blocking a renal vessel by occlusion or clamping isolates the kidney from the systemic circulation without forming or introducing an external or auxiliary circuit. Therefore, in some embodiments, the at least one kidney is isolated from the systemic circulation without an external or auxiliary circuit. In some embodiments, neither the renal artery nor the renal vein is blocked.

[0194] Occlusion of at least one renal vessel includes introducing an occlusive agent into at least one vessel such that blood flow through the at least one renal vessel is slowed, minimized, substantially stopped, or stopped. Such an occlusive agent can be introduced using a catheter.Examples of non-limiting examples include dilation catheters, balloon catheters, or perfusion catheters. In some embodiments, the catheter is inserted into an accessible artery or vein, such as the femoral artery, femoral vein, internal jugular vein, etc., as determined by a medical professional. In some embodiments, the catheter is inserted percutaneously. Thus, the catheter can be internally guided to the desired location of the target vessel (such as the renal artery or renal vein). For example, in some embodiments, a balloon catheter is percutaneously guided to the desired renal vessel and then inflated to close the renal vessel, thereby slowing, minimizing, substantially stopping, or stopping blood supply to the kidney and isolating the kidney from the systemic circulation. When both renal vessels are to be closed, balloon catheters are guided separately to each individual renal vessel. Closure of at least one renal vessel is performed before administration of a solution containing rAAV. As discussed herein, the at least one renal vessel is unblocked after a period of approximately 10 minutes to approximately 60 minutes following closure and / or administration of the rAAV-containing solution. Both the closure period and the isolation period (whichever is implied) are measured from the moment of closure or isolation, i.e., after closure or isolation and / or after administration of a solution containing rAAV. Unblocking at least one renal vessel is performed, for example, by deflating a balloon in the catheter, which restores blood flow to the kidney and reintroduces the kidney into the systemic circulation.

[0195] Clamping at least one renal vessel includes clamping at least one vessel such that blood flow through said at least one renal vessel is slowed, minimized, substantially stopped, or stopped. Non-limiting examples of suitable clamps include renal artery clamps, artery clamps, vascular clamps, arterial clips, vascular clips, renal vein clamps, renal vein clips, Dieffenbach clamps, and hemostatic clamps. Such clamps can be introduced through an incision made in the subject. For example, in some embodiments, the clamp is guided to and placed on the desired renal vessel. The clamp squeezes and closes the renal vessel, thereby slowing, minimizing, substantially stopping, or stopping blood supply to the kidney and isolating the kidney from the systemic circulation. When both renal vessels are to be closed, the clamp is guided to each individual renal vessel separately. At least one renal vessel is blocked before administration of a solution containing rAAV. As discussed herein, the at least one renal vessel is unblocked after a period of approximately 10 to approximately 60 minutes following the blocking and / or administration of the solution containing rAAV. Unblocking the at least one renal vessel is performed, for example, by releasing the clamp or releasing the clamp, which restores blood flow to the kidney and reintroduces the kidney into the systemic circulation.

[0196] In some embodiments, the method includes introducing an external or auxiliary circuit to isolate the kidney from the systemic circulation. In some embodiments, the method of using an external circuit to isolate the kidney includes: (a) positioning an infusion catheter in the renal artery of the kidney; (b) positioning a recovery catheter in the renal vein of the kidney, wherein the infusion catheter andThe retrieval catheter, together with the membrane oxygenation device, forms a closed external perfusion circuit through the kidney; and (c) perfusion fluid is allowed to flow through this external circuit, wherein the external circuit isolates the perfusion through the kidney from the subject's systemic circulation. Positioning the perfusion catheter and retrieval catheter may include closing the renal artery and / or renal vein, respectively, for example, by using a balloon catheter. See, for example, International Patent Publication WO2022175546A1, the contents of which are incorporated herein by reference in their entirety. The perfusion fluid may be, for example, blood donated by the subject or another subject prior to the implementation of this method. Such use of an external circuit to isolate the kidney from the systemic circulation may also be referred to as "isolated perfused kidney (IPK)". In some embodiments, the IPK is maintained in a physiological environment free from ischemia. In some embodiments, to avoid ischemia and / or underperfusion, the flow rate of the perfusion fluid circulating through the closed circuit does not deviate significantly from the patient's own blood flow rate. In some embodiments, blood from the retrieval catheter in the renal vein is recirculated back into the perfusion catheter in the renal artery to form the external circuit. While isolating the kidney from systemic circulation via an external circuit, rAAV can be administered via a retrograde route through the ureter.

[0197] In some embodiments, a method for reintegrating the kidney into systemic circulation includes disassembling the external circuit, for example by: (a) removing the perfusion catheter from the renal artery of the kidney; (b) removing the retrieval catheter from the renal vein of the kidney; and (c) allowing blood from systemic circulation to flow into the renal artery and out through the renal vein back into systemic circulation. Because the external circuit allows for continuous oxygen supply to the kidney, reintegration of the kidney into systemic circulation can be performed over a longer period than kidney isolation methods that cause renal ischemia.

[0198] In some embodiments, the method does not include continuous perfusion of the kidney. In some embodiments, the method does not include a closed circuit containing the kidney. In some embodiments, the method does not include a basic closed system containing the kidney. In some embodiments, the method does not include shunting circulation from the kidney. In some embodiments, the method does not include bypassing the kidney. In some embodiments, the method is performed in vivo. In some embodiments, the method is performed without leaving the body.

[0199] In some embodiments, the method includes continuous perfusion of the kidney. In some embodiments, the method includes a closed circuit comprising the kidney. In some embodiments, the method includes a basic closed system comprising the kidney. In some embodiments, the method includes shunting circulation away from the kidney. In some embodiments, the method includes bypassing the kidney. In some embodiments, the method is not performed in vivo. In some embodiments, the method is performed ex vivo.

[0200] In some embodiments, the kidney is isolated from systemic circulation (e.g., by closure of at least one renal vessel).During the time period, the isolated kidney was maintained at a normal temperature "warm" condition (i.e., body temperature). Non-limiting examples of normal temperature conditions for the isolated kidney include approximately 36°C, approximately 36.1°C, approximately 36.2°C, approximately 36.3°C, approximately 36.4°C, approximately 36.5°C, approximately 36.6°C, approximately 36.7°C, approximately 36.8°C, approximately 36.9°C, approximately 37°C, approximately 37.1°C, approximately 37.2°C, approximately 37.3°C, approximately 37.4°C, approximately 37.5°C, approximately 37.6°C, approximately 37.7°C, approximately 37.8°C, approximately 37.9°C, or approximately 36.0°C–38.0°C.

[0201] In some embodiments, the period of isolation of the kidney from the systemic circulation (e.g., by occluding at least one renal vessel) is such that the kidney does not suffer severe ischemic damage (e.g., accumulation of metabolic waste, cell membrane failure, mitochondrial damage, and / or leakage of autolytic proteolytic enzymes into the renal cells and surrounding renal tissue). In some embodiments, the period of isolation of the kidney from the systemic circulation is approximately 15 minutes after isolation. In some embodiments, the period of isolation of the kidney from the systemic circulation is 10–60 minutes after isolation. In some embodiments, the period of isolation of the kidney from the systemic circulation is 30–60 minutes after isolation. In some embodiments, the period of isolation of the kidney from the systemic circulation is 30–45 minutes after isolation. In some embodiments, the period of isolation of the kidney from the systemic circulation is 15–45 minutes after isolation. In some embodiments, the period of isolation of the kidney from the systemic circulation is 20–40 minutes after isolation. In some embodiments, the kidney is isolated from the systemic circulation for approximately 15-30 minutes after isolation. In some embodiments, the kidney is isolated from the systemic circulation for approximately 30 minutes after isolation. In some embodiments, the kidney is isolated from the systemic circulation for no more than 45 minutes after isolation. In some implementations, the period of isolation of the kidney from the systemic circulation is at least 10 min, at least 11 min, at least 12 min, at least 13 min, at least 14 min, at least 15 min, at least 16 min, at least 17 min, at least 18 min, at least 19 min, at least 20 min, at least 21 min, at least 22 min, at least 23 min, at least 24 min, at least 25 min, at least 26 min, at least 27 min, at least 28 min, at least 29 min, at least 30 min, at least 31 min, at least 32 min, at least 33 min, at least 34 min, at least 35 min, at least 36 min, at least 37 min, at least 38 min, at least 39 min, at least 40 min, at least 41 min, at least 42 min after isolation.The time ranges are: at least 43 min, at least 44 min, at least 45 min, at least 46 min, at least 47 min, at least 48 min, at least 49 min, at least 50 min, at least 51 min, at least 52 min, at least 53 min, at least 54 min, at least 55 min, at least 56 min, at least 57 min, at least 58 min, or at least 59 min. It should be understood that each of the individual times described herein can be used to define the lower and upper limits of the time range.

[0202] In some embodiments, the period of isolation of the kidney from the systemic circulation is at most 10 min, at most 11 min, at most 12 min, at most 13 min, at most 14 min, at most 15 min, at most 16 min, at most 17 min, at most 18 min, at most 19 min, at most 20 min, at most 21 min, at most 22 min, at most 23 min, at most 24 min, at most 25 min, at most 26 min, at most 27 min, at most 28 min, at most 29 min, at most 30 min, at most 31 min, at most 32 min, at most 33 min, at most 34 min, at most 35 min, at most 36 min, at most 37 min, at most 38 min, at most 39 min, at most 40 min, at most 41 min, at most 42 min, at most 43 min, at most 44 min, at most 45 min, at most 46 min after isolation. The time range is defined as 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min. It should be understood that each of the individual times described in this document can be used to define the lower and upper limits of the time range.

[0203] In some embodiments, the time period for isolating the kidney from the systemic circulation is approximately 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, 21 min, 22 min, 23 min, 24 min, 25 min, 26 min, 27 min, 28 min, 29 min, 30 min, 31 min, 32 min, 33 min, 34 min, 35 min, and 36 min after isolation.Approximately 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, 47 min, 48 min, 49 min, 50 min, 51 min, 52 min, 53 min, 54 min, 55 min, 56 min, 57 min, 58 min, 59 min, or 60 min. It should be understood that each of the individual times described herein can be used to define the lower and upper limits of a time range.

[0204] In some embodiments, the amount of solution containing rAAV is 0.13 mL / kg to about 0.35 mL / kg, about 0.2 mL / kg to about 0.35 mL / kg, about 0.25 mL / kg to about 0.35 mL / kg, about 0.3 mL / kg to about 0.35 mL / kg, about 0.2 mL / kg to about 0.30 mL / kg, or about 0.2 mL / kg to about 0.25 mL / kg; and the period of time for isolating the kidney from the systemic circulation (e.g., by blocking at least one renal vessel or creating an external circuit containing the kidney) is 10-60 minutes after isolation. In some embodiments, the amount of solution containing rAAV is about 0.13 mL / kg to about 0.35 mL / kg, about 0.2 mL / kg to about 0.35 mL / kg, about 0.25 mL / kg to about 0.35 mL / kg, about 0.3 mL / kg to about 0.35 mL / kg, about 0.2 mL / kg to about 0.30 mL / kg, or about 0.2 mL / kg to about 0.25 mL / kg; and the period of isolation of the kidney from the systemic circulation (e.g., by occluding at least one renal vessel or creating an external circuit containing the kidney) is about 30 minutes after isolation or about 15-30 minutes after isolation.

[0205] In some embodiments, the treatment method may include first diagnosing a subject or patient who may benefit from treatment using the methods described herein and / or the pharmaceutical compositions described herein. In some embodiments, such diagnosis includes detecting or measuring abnormal levels of analytes related to kidney-related conditions in samples from the subject or patient. In some embodiments, the method further includes administering rAAV to the kidney of the subject.

[0206] In some embodiments, the subject has previously been identified as having abnormal levels of the analyte or biomarker described herein relative to a reference value. In some embodiments, the reference level may be one with similar cell types to the sample / subject.The level of this type of sample, the sample processed, and / or samples obtained from subjects with similar age, sex, and other demographic parameters to the sample / subject. In some embodiments, the test sample and the control reference sample are of the same type, i.e., obtained from the same biological source and containing the same composition, e.g., the same number and type of cells.

[0207] As used herein, the term “sample” or “test sample” means a sample collected or isolated from a living organism, e.g., a blood or plasma sample from a subject. In some embodiments of any aspect, the techniques described herein cover several instances of biological samples. In some embodiments of any aspect, the biological sample is cells or tissue or peripheral blood or body fluid. Exemplary biological samples include, but are not limited to, biopsies (e.g., from the kidney), blood, serum, plasma, or urine. The term also includes mixtures of the above samples. The term “test sample” also includes unprocessed or pre-processed (or pre-treated) biological samples. In some embodiments of any aspect, the test sample may contain cells from a subject.

[0208] In some embodiments, the reference value may be the level of the analyte in a group of subjects who do not have or are not diagnosed with kidney-related disease and / or do not exhibit signs or symptoms of kidney-related disease. In some embodiments, the reference value may also be the expression level of the analyte in a control sample, a pooled sample of control individuals, or a numerical value or range of values ​​based thereon. In some embodiments of any aspect, the reference value may be the level of the analyte in a sample obtained from the same subject at a previous time point, for example, the methods described herein may be used to determine whether a subject's sensitivity or response to rAAV therapy has changed over time.

[0209] In some embodiments of any aspect, the step of determining whether a subject has an abnormal level of the analyte described herein may include: i) obtaining or having obtained a sample from the subject; and ii) performing or having performed a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether a subject has an abnormal level of the analyte described herein may include performing or having performed a determination on the sample obtained from the subject to determine / measure the level of the analyte in the subject. In some embodiments of any aspect, the step of determining whether a subject has abnormal levels of the analyte described herein may include arranging or requesting a determination of the analyte level in the subject based on a sample obtained from the subject. In some embodiments of any aspect, the step of determining whether a subject has abnormal levels of the analyte described herein may include receiving the results of a determination of the analyte level in the subject based on a sample obtained from the subject. In some embodiments of any aspect, the step of determining whether a subject has abnormal levels of the analyte described herein may include receiving identification of the subject as having decreased analyte levels.Low subject reports, results, or other means.

[0210] In one aspect of any embodiment, a method of treating kidney-related conditions in a subject with such need is described herein, the method comprising: a) determining whether the subject has an abnormal level of the analyte described herein; and b) if the level of the analyte is abnormal relative to a reference value, instructing or directing the subject to administer a solution or pharmaceutical composition containing rAAV as described herein. In some embodiments of any aspect, the step of instructing or directing the subject to administer a specific treatment may include providing a report of the measurement results. In some embodiments of any aspect, the step of instructing or directing the subject to administer a specific treatment may include providing a report of the measurement results and / or treatment recommendations based on the measurement results.

[0211] In some embodiments of any aspect, the solution (or pharmaceutical composition) containing rAAV described herein is administered as a monotherapy, for example, without administering other treatments for kidney-related conditions to the subject.

[0212] In some embodiments of any aspect, the method described herein may also include administering a second dose and / or treatment to the subject, for example, as part of a combination therapy. Non-limiting examples of second-dose and / or treatment may include: treatments for kidney-related conditions, such as blood pressure medications (e.g., angiotensin-converting enzyme (ACE) such as ramipril, enalapril, and lisinopril); medications for diabetes or high albumin-to-creatinine ratio (ACR) (e.g., dapagliflozin); medications for cardiovascular diseases (e.g., statins such as atorvastatin or simvastatin); potassium-lowering medications (e.g., sodium zirconium cyclosilicate); fluid reduction; diuretics (e.g., furosemide); and medications for anemia (e.g., erythropoietin). Calcium supplements; steroids (e.g., cyclophosphamide); dialysis (e.g., hemodialysis, peritoneal dialysis); lifestyle modifications (e.g., smoking cessation; a healthy, balanced diet; limiting salt intake, e.g., at least 6 g / day; regular exercise, e.g., at least 150 minutes / week; reducing alcohol intake, e.g., not exceeding the recommended limit of 14 alcohol units / week; weight loss if overweight or obese; avoiding over-the-counter nonsteroidal anti-inflammatory drugs (NSAIDs), such as ibuprofen); and / or kidney transplantation (e.g., transplantation of a kidney not transduced by rAAV).

[0213] As a non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject may also be given a second dose and / or treatment known to be beneficial to subjects experiencing pain or inflammation. Examples of such agents and / or treatments include, but are not limited to: nonsteroidal anti-inflammatory drugs (NSAIDs, such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g., corticosteroids, prednisone, prednisolone, methylprednisolone, dexamethasone).Betamethasone, triamcinolone, and beclomethasone); methotrexate; sulfasalazine; leflunomide; anti-TNF drugs; cyclophosphamide; pro-resolving drugs; mycophenolate mofetil; or opioids (e.g., endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, etc.

[0214] In some embodiments in any aspect, the methods described herein may also include administering a second dose and / or treatment to the subject, for example, as part of an immunosuppressive therapy. In some embodiments, at least one immunosuppressant is DEPO-MEDROL® (methylprednisolone acetate) and / or tacrolimus (calcineurin inhibitor). In some embodiments, the at least one immunosuppressant is selected from: prednisone, cyclosporine, tacrolimus, azathioprine, morphifenproxetine, sirolimus, everolimus, alemtuzumab, and is DEPO-MEDROL® (methylprednisolone acetate). In some embodiments, the at least one immunosuppressant is administered approximately 7 days, approximately 6 days, approximately 5 days, approximately 4 days, approximately 3 days, approximately 2 days, approximately 1 day, on the day of rAAV administration via retrograde ureter, approximately 1 day later, approximately 2 days later, approximately 3 days later, approximately 4 days later, approximately 5 days later, approximately 6 days later, approximately 7 days later, approximately 8 days later, approximately 9 days later, approximately 10 days later, approximately 11 days later, approximately 12 days later, approximately 13 days later, approximately 14 days later, approximately 15 days later, approximately 16 days later, approximately 17 days later, approximately 18 days later, approximately 19 days later, approximately 20 days later, or longer after administration. In some embodiments, if at least one transgene is immunogenic, at least one immunosuppressant is administered; as a non-limiting example, GFP, mCherry, HA1, and luciferase and some variants thereof are known to be immunogenic. In some embodiments, the transgene is not immunogenic and does not contain an immunogenicity marker, therefore at least one immunosuppressant is not administered.

[0215] Recombinant Adeno-Associated Virus (rAAV) This document describes a method of administering recombinant adeno-associated virus (rAAV) to the kidneys of a subject. Recombinant AAV (rAAV) vectors typically consist of at least a transgene and its regulatory sequence, as well as 5' and 3' AAV inverted terminal repeat (ITR) sequences. As further described herein, the transgene may contain one or more regions encoding one or more repressive RNAs (e.g., miRNAs) containing nucleic acids targeting the subject's endogenous mRNA. The transgene may also contain regions encoding, for example, proteins and / or expression control sequences (e.g., poly-A tails), as further described herein. Isolated nucleic acids (e.g., recombinant AAV vectors) can be packaged into capsid proteins and administered to a subject (e.g., via retrograde ureteral administration) and / or delivered to selected target cells, such as kidney cells.

[0216] In some embodiments, rAAV includes a caption selected from those described in Table 1. In other embodiments, rAAV includes a caption selected from those described in Table 1, but does not include AAV9. The exemplary captions provided in Table 1 include representative AAV VP1 sequences, which also contain corresponding VP2 and VP3 sequences, as known in the art. Each reference (including non-patent and patent documents) recorded in Table 1 is incorporated herein by reference in its entirety. Instruction manual 29 / 145 pages 40 CN 121752299 A Instruction manual 30 / 145 pages 41 CN 121752299 A Instruction manual 31 / 145 pages 42 CN 121752299 A Instruction manual 32 / 145 pages 43 CN 121752299 A Instruction manual 33 / 145 pages 44 CN 121752299 A Instruction manual 34 / 145 pages 45 CN 121752299 A Instruction manual 35 / 145 pages 46 CN 121752299 A Instruction manual 36 / 145 pages 47 CN 121752299 A Instruction manual 37 / 145 pages 48 CN 121752299 A Instruction manual 38 / 145 pages 49 CN 121752299 A Instruction manual 39 / 145 pages 50 CN 121752299 A Instruction Manual 40 / 145 pages 51 CN 121752299 A Instruction Manual 41 / 145 pages 52 CN 121752299 A Instruction Manual 42 / 145 pages 53 CN 121752299 A Instruction Manual 43 / 145 pages 54 CN 121752299 A Instruction Manual 44 / 145 pages 55 CN 121752299 A Instruction Manual 45 / 145 pages 56 CN 121752299 A Instruction Manual 46 / 145 pages 57 CN 121752299 A Instruction Manual 47 / 145 pages 58 CN 121752299 A Instruction Manual 48 / 145 pages 59 CN 121752299 A Instruction Manual 49 / 145 pages 60 CN 121752299 A Instruction manual 50 / 145 pages 61 CN 121752299 A Instruction manual 51 / 145 pages 62 CN 121752299 A Instruction manual 52 / 145 pages 63 CN 121752299 A Instruction manual 53 / 145Page 64 CN 121752299 A Specification 54 / 145 Page 65 CN 121752299 A Specification 55 / 145 Page 66 CN 121752299 A Specification 56 / 145 Page 67 CN 121752299 A Specification 57 / 145 Page 68 CN 121752299 A

[0217] In some embodiments, rAAV comprises one or more AAV capsid proteins selected from serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV2.5G9, AAV2.5, AAVrh8, AAVrh10, AAVrh74, AAV10, AAV11, and AAVDJ.

[0218] In some embodiments, rAAV comprises one or more AAV capsid proteins selected from serotypes AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV2G9, AAV2.5G9, AAV2.5, AAVrh8, AAVrh10, AAVrh74, AAV10, AAV11, and AAVDJ. Instruction manual, pages 58 / 145, 69, CN 121752299 A

[0219] In some embodiments, rAAV comprises serotypes selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-PHP.eB, AAVLK03, AAVANc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV2 3xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5, AAV4E, and AAV4A are one or more AAV capsid proteins.

[0220] In some embodiments, rAAV comprises one or more AAV capsid proteins selected from AAV2, AAV6, AAVLK03, AAVDJ, AAV9A2i, AAV9A6i,AAV capsid proteins of AAVrh10A2i, AAV2g9, or AAV2.5 (see, for example, Figure 4). In some embodiments, rAAV comprises an AAV capsid protein selected from AAV2, AAVDJ, AAVJEA (minimal), AAV2g9, or AAV2.5 (see, for example, Figure 5).

[0221] In some embodiments, rAAV comprises a capsid selected from AAV2G9, AAV2.5, AAVDJ, and AAV2. In some embodiments, rAAV does not contain a capsid protein derived from serum AAV9. In some embodiments, rAAV is not rAAV9. In some embodiments, rAAV exhibits tropism for the kidney, i.e., it has the ability to preferentially and efficiently infect kidney cells and / or tissues.

[0222] In some embodiments, rAAV comprises an AAV capsid protein selected from AAV2, AAV6, AAVLK03, AAVDJ, AAV9A2i, AAV9A6i, AAVrh10A2i, AAV2g9, AAV2.5, AAVKP1, AAVKP2, AAVKP3, and AAV2.7m8.

[0223] In some embodiments, rAAV does not contain an AAV9 capsid.

[0224] In some embodiments, rAAV comprises a capsid selected from AAV2G9, AAV2.5, AAVDJ, AAV2, AAVKP1, AAVKP2, AAVKP3, and AAV2.7m8. In some embodiments, rAAV comprises a capsid selected from AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, and AAV2.7m8. In some embodiments, rAAV comprises a capsid selected from AAVKP1, AAVKP2, and AAVKP3. In some embodiments, rAAV comprises a capsid of AAV2.7m8. In some embodiments, rAAV comprises a capsid of AAV-DJ.

[0225] In some embodiments, rAAV administered via the retrograde ureteral route has at least 10-fold higher transduction efficiency in the kidneys (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct) compared to transduction efficiency in the kidneys via other routes such as intravenous, intraperitoneal, intrarenal artery or intrarenal vein, retroorbital, direct renal injection, or subcapsular administration. In some embodiments, rAAV administered via the retrograde ureteral route has at least 2-fold, at least 5-fold, at least 10-fold, and at least 25-fold higher transduction efficiency in the kidneys (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct) compared to rAAV administered via other routes (e.g., IV).At least 50 times, at least 75 times, at least 100 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1000 times, at least 1100 times, at least 1200 times, at least 1300 times, at least 1400 times, at least 1500 times, at least 1600 times, at least 1700 times, at least 1800 times, at least 1900 times, at least 2000 times, at least 2100 times, at least 2200 times, at least 2300 times, at least 2400 times, at least 2500 times, at least 2600 times, at least 2700 times, at least 2800 times, at least 2900 times, at least 3000 times, at least 3100 times, at least 3200 times, at least 3300 times, at least 3400 times The rAAV has a transduction efficiency at least 3500 times higher than that of AAV9 (e.g., administered via the retrograde route as described herein). In some embodiments, the rAAV has a transduction efficiency at least 3500 times higher than that of AAV9 (e.g., administered via the retrograde route as described herein). In some embodiments, the rAAV has a transduction efficiency at least 100 times higher than that of AAV9 in the kidney (e.g., nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct cells). In some embodiments, the rAAV has a transduction efficiency at least 800 times higher than that of AAV9 in the kidney (e.g., nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct cells). In some embodiments, the rAAV has a transduction efficiency at least 800 times higher than that of AAV9 in the kidney (e.g., nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct cells). Cells in tubules and / or collecting ducts (containing cells) with at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1000-fold, at least 1100-fold, at least 1200-fold, at least 1300-fold, at least 1400-fold, at least 1500-fold, at least At least 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, or 3500 times the transduction efficiency.

[0227] In some embodiments, rAAV comprises a reasonable polyploid. As used herein, the term "reasonable polyploid" refers to a transduction efficiency of at least 1600 times, 1700 times, 1800 times, 1900 times, 2000 times, 2100 times, 2200 times, 2300 times, 2400 times, 2500 times, 2600 times, 2700 times, 2800 times, 2900 times, 3000 times, 3100 times, 3200 times, 3300 times, 3400 times, or 3500 times.AAV vectors composed of capsids derived from two or more AAV serotypes can leverage the advantages of different serotypes to alter their behavior, such as tropism, transduction, or antigenicity. Some of these polyploid viruses possess the ability to alter tropism and transduction efficiency, as well as evade neutralization by neutralizing antibodies (Nabs). The previously described methods allow for the rational design and production of virions. Such virions are sometimes referred to as “rationally polyploid” virions to describe the fact that the capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes rather than all from the same serotype. The term “haploid” is sometimes used to refer to virions in which the capsid proteins VP1, VP2, and VP3 are derived from at least two different serotypes, while the term “triploid” is generally used to refer to virions in which the capsid proteins VP1, VP2, and VP3 are derived from three different serotypes. In particular, such rationally polyploid (e.g., rationally haploid) virions and methods for producing them are disclosed in U.S. Patent No. 10,550,405, which is incorporated herein by reference in its entirety.

[0228] In some embodiments, rAAV comprises a capsid protein derived from serotype AAV2G9 or a variant thereof. The AAV2G9 capsid comprises amino acid substitutions that introduce novel glycan binding sites into the AAV capsid protein. The AAV2G9 capsid protein is generated by replacing the Gal binding footprint from AAV9 onto AAV2 VP3. The AAV2G9 capsid protein is generated by replacing amino acid residues on the AAV9 VP3 capsid protein subunit that are directly involved in or have Gal recognition sites attached to them with corresponding residues on the VP3 subunit of AAV2 (e.g., AAV2 VP3 numbers: A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F and / or S501A). See, for example, Shen et al., “Engraftment of a Galactose Receptor Footprint onto Adeno-associated Viral Capsids Improves Transduction Efficiency,” *The Journal of Biological Chemistry*, Vol. 288, no. 40, pp. 28814–28823, October 4, 2013; Shen, “Understanding And Manipulating AAV-Glycan Interactions,” University of North Carolina at Chapel Hill dissertation (2013).(Available at cdr.lib.unc.edu / concern / dissertations / pc289j203); International Patent Publication WO2014144229A1; US ​​Patent 10,077,291 B2; US Patent 11,059,862 B2; US Patent Publication 20210115091 A1; the contents of each of these are incorporated herein by reference in their entirety.

[0229] In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof comprises the A266S variant. In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof does not comprise the A266S variant. In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof comprises the A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A variants. In some implementations, the AAV2G9 VP3 capsid protein or its variants include the variants Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A. The differences between 2G9 capsids are negligible regardless of the presence of the A266S variant. 2G9 may also contain other variants that do not affect its general properties (e.g., tropism, transduction efficacy in the kidney (e.g., PCT), etc.).

[0230] In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof comprises inserting at least one variant of the AAV9 VP3 capsid protein (see, for example, SEQ ID NO: 2) into the AAV2 VP3 capsid protein (see, for example, SEQ ID NO: 1) specification page 60 / 145 71 CN 121752299 A, at the following insertion locations: A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F and / or S501A (AAV2 VP3 number).

[0231] SEQ ID NO: 1, AAV2 VP3 capsid protein (see, for example, SEQ ID NO: 65 of WO2014144229), 735 amino acids (aa) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADN NEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHF SPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPAD VFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYL SRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQG VLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTG QVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 2, AAV9 VP3 capsid protein (see, for example, SEQ ID NO: 75 of WO2014144229), 736 aa MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAAD AAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPV EQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGS SSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQY GYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGS GQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGE DRFFPLGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQ DRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEW In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof comprises an amino acid sequence, or a functional fragment thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with and retaining the same function as the sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

[0232] SEQ ID NO: 3, exemplary AAV2G9 VP3 capsid protein, the following variants are indicated in bold and underlined text: A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A (AAV2 VP3 numbers). MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAA LEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSG NWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGSSNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNN WGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTL NNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQ SRLQFSVAGPSNMAVQGRNWLPGPCYRQQRVSKTSADNNNSEFAWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFF PQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDV YLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQK Specification 61 / 145 pages 72 CN 121752299 A ENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 4, exemplary AAV2G9 VP3 capsid protein, the following variants are highlighted in bold and underlined text: Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F and S501A (AAV2 VP3 number); see, for example, SEQ ID NO: 24 of US20220354969. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSR GLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFG YSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLP YVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQ SLDRLMMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSVAGPSNMAVQGRNWLPGPCYRQQRVSKTSADNNNSEFAWTGA TKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTN In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof contains SVAGPSNMAVQGR (SEQ ID NO: 15) at positions 464-476 corresponding to amino acids SEQ ID NO: 2 of AAV9 VP3. In some embodiments, the AAV2G9 VP3 capsid protein or a variant thereof contains EFAW (SEQ ID NO: 16) at positions 500-503 corresponding to amino acids SEQ ID NO: 2 of AAV9 VP3.

[0233] In some embodiments, rAAV contains a capsid protein derived from serum type AAV2.5. AAV2.5 is a chimera composed of AAV2 with five amino acid substitutions from AAV1 (which uses α-2,3- and α-2,6-N-linked sialic acid as the major acceptor). AAV2.5 contains four AAV1 substitutions (N705A, Q263A, V708A, and T716N, AAV2 numbered) and one T265 insertion from AAV1. See, for example, Korneyenkov et al., “Next Step in Gene Delivery: Modern Approaches and Further”.Perspectives of AAV Tropism Modification. Pharmaceutics May 2021, 13(5): 750; Hemphill et al., “Adeno-Associated Viral Vectors Show Serotype Specific Transduction of Equine Joint Tissue Explants and Cultured Monolayers,” Scientific Reports volume 4, Article number: 5861 (2014); the contents of each of these are incorporated herein by reference in their entirety.

[0234] In some embodiments, rAAV contains a capsid protein derived from serotype AAVDJ. AAV-DJ is a highly recombinant hybridization vector created by experiments involving DNA shuffling of eight AAV serotypes. AAV-DJ is an AAV Chimeras of type 2 / 8 / 9. AAV2 and AAV8 are the closest parental vectors of AAV-DJ. Mutations at ubiquitination or phosphorylation sites of 137 / 251 / 503 in the AAV2 or AAV8 capsid have been reported to lead to a dramatic enhancement of gene delivery (K137R / T251A / S503A). See, for example, Grimm et al., “In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses,” J Virol. 2008 Jun; 82(12): 5887–5911; Mao et al., “Single point mutation in adeno-associated viral vectors-DJ capsid leads to improvement for gene delivery in vivo,” BMC Biotechnology volume 16, Article number: 1 (2016); the contents of each of these are incorporated herein by reference in their entirety.

[0235] In some embodiments, rAAV contains a capsid protein derived from serotype AAV2. The molecular clone of AAV2 was isolated in 1983. In some embodiments, rAAV does not contain a capsid protein derived from serotype AAV9. AAV9 was first isolated in 2004.It was isolated from human DNA in 2000. See, for example, Samulski et al., “Rescue of adeno-associated virus, 62 / 145 pages, 73 CN 121752299 A from recombinant plasmids: gene correction within the terminal repeats of AAV,” Cell, May 1983, 33(1):135-43; Guo et al., “Clades of Adeno-associated viruses are widely disseminated in human tissues,” J Virol, June 2004, 78(12):6381-8; the contents of each of these are incorporated herein by reference in their entirety.

[0236] In some embodiments, the solution contains a concentration of rAAV effective for treating kidney-related conditions. In some embodiments, the solution contains concentrations of 10⁸ viral genomes / mL (vg / mL) to 10¹⁵ vg / mL, 10⁹ vg / mL to 10¹⁵ vg / mL, 10¹⁰ vg / mL to 10¹⁵ vg / mL, 10¹¹ vg / mL to 10¹⁵ vg / mL, 10¹² vg / mL to 10¹⁵ vg / mL, and 10¹³ vg / mL to 10¹⁵ vg / mL. rAAV concentrations of 10¹¹ vg / mL to 10¹² vg / mL, 10¹² vg / mL to 10¹³ vg / mL, 10¹³ vg / mL to 10¹⁴ vg / mL, 10¹⁴ vg / mL to 10¹⁵ vg / mL, 10⁸ vg / mL to 10¹⁴ vg / mL, 10⁸ vg / mL to 10¹³ vg / mL, 10⁸ vg / mL to 10¹² vg / mL, 10⁸ vg / mL to 10¹¹ vg / mL, 10⁸ vg / mL to 10¹⁰ vg / mL, or 10⁸ vg / mL to 10⁹ vg / mL are used. In some embodiments, the solution contains rAAV at a concentration of 10⁸ vg / mL to 10¹³ vg / mL. In some embodiments, the solution contains rAAV at a concentration of at least 10⁸ vg / mL, at least 10⁹ vg / mL, at least 10¹⁰ vg / mL, at least 10¹¹ vg / mL, at least 10¹² vg / mL, at least 10¹³ vg / mL, at least 10¹⁴ vg / mL, at least 10¹⁵ vg / mL, or higher. In some embodiments, the solution contains rAAV at a concentration of at most 10⁹ vg / mL, at most 10¹⁰ vg / mL, at most 10¹¹ vg / mL, at most 10¹² vg / mL, at most 10¹³ vg / mL, at most 10¹⁴ vg / mL, or at most 10¹⁵ vg / mL.The rAAV concentration is approximately 5.2 x 10¹⁰ vg / kg, for example, in a 75 kg subject. In some embodiments, the solution contains rAAV at concentrations of approximately 1 x 10¹⁰ vg / kg, approximately 2 x 10¹⁰ vg / kg, approximately 3 x 10¹⁰ vg / kg, approximately 4 x 10¹⁰ vg / kg, approximately 5 x 10¹⁰ vg / kg, approximately 6 x 10¹⁰ vg / kg, approximately 7 x 10¹⁰ vg / kg, approximately 8 x 10¹⁰ vg / kg, and approximately 9 x 10¹⁰ vg / kg, for example, in a 75 kg subject. It should be understood that each of the individual rAAV concentrations described herein can be used to define a lower and upper limit for the range of rAAV concentrations.

[0237] In some embodiments, the solution contains a total of 1x10¹¹ to 1x10¹⁴ vg, 1x10¹¹ to 1x10¹³ vg, 1x10¹¹ to 1x10¹² vg, 1x10¹² to 1x10¹³ vg, 1x10¹² to 1x10¹⁴ vg, 1x10¹³ to 1x10¹⁴ vg, 1x10¹³ to 6x10¹³ vg, 2x10¹³ to 5x10¹³ vg, or 1x10¹³ to 2x10¹³ rAAV viral genomes per 75 kg of subject. In some embodiments, the solution contains a total of 5x10¹³ to 6x10¹³ rAAV viral genomes. In some embodiments, the solution contains a total of at least 1x10¹³, at least 2x10¹³, at least 3x10¹³, at least 4x10¹³, at least 5x10¹³, at least 6x10¹³, at least 7x10¹³, at least 8x10¹³, at least 9x10¹³ or more rAAV viral genomes. In some embodiments, the solution contains a total of at most 1x10¹³, at most 2x10¹³, at most 3x10¹³, at most 4x10¹³, at most 5x10¹³, at most 6x10¹³, at most 7x10¹³, at most 8x10¹³, at most 9x10¹³ or more rAAV viral genomes. In some embodiments, the solution contains approximately 1 x 10¹¹ vg, approximately 2 x 10¹¹ vg, approximately 3 x 10¹¹ vg, approximately 4 x 10¹¹ vg, approximately 5 x 10¹¹ vg, approximately 6 x 10¹¹ vg, approximately 7 x 10¹¹ vg, approximately 8 x 10¹¹ vg, approximately 9 x 10¹¹ vg, approximately 10 x 10¹¹ vg, approximately 1 x 10¹² vg, approximately 2 x 10¹² vg, approximately 3 x 10¹² vg, approximately 4 x 10¹² vg, approximately 5 x 10¹² vg, approximately 6 x 10¹² vg, approximately 7 x 10¹² vg, approximately 8 x 10¹² vg, and approximately 9 x 10¹² vg.Approximately 10 x 10¹² vg, approximately 1 x 10¹³ vg, approximately 2 x 10¹³ vg, approximately 3 x 10¹³ vg, approximately 4 x 10¹³ vg, approximately 5 x 10¹³ vg, approximately 6 x 10¹³ vg, approximately 7 x 10¹³ vg, approximately 8 x 10¹³ vg, approximately 9 x 10¹³ vg, approximately 10 x 10¹³ vg, or approximately 1 x 10¹⁴ vg. It should be understood that each of the individual rAAV quantities described herein can be used to define a lower and upper limit for the range of rAAV quantities.

[0238] In some embodiments, the solution contains a total of 1 x 10¹⁰ viral genomes. In some embodiments, the solution contains a total of 1 x 10¹⁰ viral genomes (e.g., 4 x 10⁸ vg / mL to 1 x 10⁹ vg / mL).

[0239] In some embodiments, the genome of the rAAV contains a transgene. As used herein, the term “transgene” refers to a gene or other nucleic acid sequence transduced into the genome of a subject using rAAV. For transduction of cells, the rAAV vector enters the cell instruction manual 63 / 145 page 74 CN 121752299 A and delivers its single-stranded DNA genome to the cell nucleus, whereby the genome becomes double-stranded, and is then transcribed and integrated into the subject’s cellular genome.

[0240] In some embodiments, the transgene contains a reporter protein. Non-limiting examples of such reporter proteins include fluorescent proteins (e.g., GPF, mCherry, etc.), luciferases, alkaline phosphatases, β-galactosidases, β-lactamases, horseradish peroxidases, detectable tags (such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin) and variants thereof. In some embodiments, the transgene contains a barcode that can be identified by sequencing.

[0241] In some embodiments, the transgene has a therapeutic effect on kidney-related conditions. In some implementations, the kidney-related conditions for which the transgene has a therapeutic effect are selected from: autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome; autosomal dominant tubulointerstitial nephropathy (ADTKD); medullary cystic nephropathy; nephronial tuberculosis; Bart syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; thin basement membrane nephropathy; cystinuria; Liddle syndrome; papillary kidney syndrome; and cystin storage diseases, as described in Table 2A.

[0242] Table 2A: Exemplary kidney-related conditions (adapted from Rubin et al., 2020, “Improving molecular therapy in the kidney,” Mol Diagn Ther.)24(4): 375–396, the contents of which are incorporated herein by reference in their entirety. Specification 64 / 145 pages 75 CN 121752299 A Specification 65 / 145 pages 76 CN 121752299 A

[0243] In some embodiments, the transgene contains a gene that is effective in treating kidney-related conditions when expressed in a subject (e.g., at approximately physiological levels). In some implementations, the transgene includes genes selected from: alanine-glyoxylate aminotransferase (AGXT; e.g., type I); Bart syndrome with sensorineural deafness in infants (BSND; e.g., type IV); chloride voltage-gated channel 5 (CLCN5; e.g., type I); chloride voltage-gated channel Ka (CLCNKA; e.g., type IV); chloride voltage-gated channel Kb (CLCNKB; e.g., types III and IV); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR; e.g., type II); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1; e.g., type III); and potassium inward rectifier channel subfamily J member 1. (KCNJ1; e.g., type II); MAGED2 (type V); Mucin 1 (MUC1; e.g., type I); Nephroticin 1 (NPHP1); Nephroticin (NPHS1); Nephroticin 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL; e.g., type II); Polycystic protein 1 (PKD1); Polycystic protein 2 (PKD2); Polycystic kidney and liver disease 1 (PKHD1); Protein transporter Sec61 subunit α isoform 1 (SEC61A1); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 7 member 9 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and any combination thereof.

[0244] In some embodiments, the kidney-related conditions for which the transgene has a therapeutic effect are selected from: episodic mineralocorticoid hyperparalysis, autosomal dominant hypocalcemia, autosomal dominant hypomagnesemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, and type A cystitis.Cystinuria, B-type cystinuria, Dent disease type 1, Dent disease type 2 / Lowe syndrome, dicarboxyaminoaciduria, distal RTA, EAST / SeSAME syndrome, Fanconi Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2, Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Gitelman syndrome, glucocorticoids (see product manual 66 / 145, page 77, CN 121752299 A) Suppressive aldosteronism, Hartnup syndrome, hereditary hypophosphatemic rickets with hypercalciuria, HNF1B-related nephropathy, BH4 deficiency hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 4 hypomagnesemia, type 5 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, hypomagnesemia, type 1 seizures and intellectual disability, type 2 hypomagnesemia, seizures and intellectual disability. , iminoglycineuria, type 2 Kenny-Caffey syndrome, Liddle syndrome, lysineuria protein intolerance, type 2 neonatal inflammatory skin and intestinal disease, nephrogenic diabetes insipidus, nephrogenic syndrome of abnormal antidiuretic hormone secretion, type 1 pseudoketoalbuminemia, type 1A pseudoketoalbuminemia, type 2b pseudoketoalbuminemia, type 2c pseudoketoalbuminemia, type 2d pseudoketoalbuminemia, type 2e pseudoketoalbuminemia, type 3 renal tubular acidosis and X-linked hypophosphatemic rickets, as described in Table 2B.

[0245] Table 2B: Exemplary kidney-related conditions (adapted from Downie et al., 2020, “Inherited tubulopathies of the kidney,” Clin J Am Soc Nephrol. 16(4): 620-630, the contents of which are incorporated herein by reference in their entirety). The following are exemplary renal tubular diseases grouped by affected nephron segment, basic gene, encoded protein, and its Online Human Mendelian Inheritance Database (OMIM) accession number. AD, autosomal dominant; AR, autosomal recessive; XLR, X-linked recessive; RTA, renal tubular acidosis. Specification 67 / 145 pages 78 CN 121752299 A Specification 68 / 145 pages 79 CN 121752299 A Specification 69 / 145 pages 80 CN 121752299 A Specification 70 / 145 pages 81 CN 121752299 A

[0246] In some embodiments, the kidney-related disease for which the transgene has a therapeutic effect is associated with the proximal tubule andSelected from: Type A cystinuria, Type B cystinuria, Type 1 Dent disease, Type 2 Dent disease / Lowe syndrome, Dicarboxyaminoaciduria, Fanconi Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2, Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Hartnup syndrome, Hereditary hypophosphatemic rickets with hypercalciuria, Iminoglycineuria, Lysineuria protein intolerance, Type 3 renal tubular acidosis, and X-linked hypophosphatemic rickets. In some embodiments, the kidney-related conditions for which the transgene has a therapeutic effect are associated with the thick ascending limb of the loop of Henry and are selected from: autosomal dominant hypocalcemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 5 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, and type 2 Kenny-Caffey syndrome.

[0248] In some embodiments, the kidney-related conditions for which the transgene has a therapeutic effect are associated with the distal convoluted tubule and are selected from: autosomal dominant hypomagnesemia, EAST / SeSAME syndrome, Gitelman syndrome, HNF1B-related nephropathy, BH4 deficiency hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 4 hypomagnesemia, hypomagnesemia, seizures and intellectual disability type 1, hypomagnesemia, seizures and intellectual disability type 2, type 2 neonatal inflammatory skin and bowel disease, type 2b pseudoketoaldolemia, type 2c pseudoketoaldolemia, type 2d pseudoketoaldolemia, and type 2e pseudoketoaldolemia.

[0249] In some embodiments, the kidney-related condition for which the transgene has a therapeutic effect is associated with collecting ducts and is selected from: episodic mineralocorticoid hyperplasia syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, distal RTA, glucocorticoid-suppressible aldosteronism, Liddle syndrome, nephrotic diabetes insipidus, nephrotic syndrome of abnormal antidiuretic hormone secretion, type 1 pseudoketoaldosteronism, and type 1A pseudoketoaldosteronism.

[0250] In some embodiments, the transgene includes a gene that is effective in treating kidney-related conditions when expressed in a subject (e.g., at approximately physiological levels). In some embodiments, the transgene includes a gene selected from: aquaporin 2 (AQP2); ATPase Na+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport VO subunit A4.(ATP6V0A4); ATPase H+ transporter V1 subunit B1 (ATP6V1B1); Arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); Carbonic anhydrase 2 (CA2); Calcium-sensitive receptor (CaSR); Chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); Chloride voltage-gated channel Kb (CLCNKB); Tight junction protein 16 (CLDN16); Tight junction protein 19 (CLDN19); Cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enzyme-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; Alpha stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; Pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); Sodium channel epithelial subunit 1 α (SCNN1A); Sodium channel epithelial subunit 1 β (SCNN1B); Sodium channel epithelial subunit 1 γ (SCNN1G); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 1 member 1(SLC1A1); Solute carrier family 2 member 2 (SLC2A2); Solute carrier family 34 member 1 (SLC34A1); Solute carrier family 34 member 3 (SLC34A3); Solute carrier family 36 member 2 (SLC36A2); Solute carrier family 3 member 1 (SLC3A1); Solute carrier specification 72 / 145 pages 83 CN 121752299 A family 4 member 1 (SLC4A1); Solute carrier family 6 member 19 (SLC6A19); Solute carrier family 6 member 20 (SLC6A20); Solute carrier family 7 member 7 (SLC7A7); Solute carrier family 7 member 9 (SLC7A9); Transient receptor potential cation channel subfamily M member 6 (TRPM6); WD repeat domain 72 (WDR72); Lysine-deficient (WNK) protein kinase 1 (WNK1); lysine-deficient (WNK, lysine-deficient) protein kinase 4 (WNK4); and any combination thereof.

[0251] In some embodiments, the transgene comprises a gene encoding a polypeptide that is effective in treating kidney-related conditions when expressed in a subject (e.g., at approximately physiological levels). In some embodiments, the transgene comprises a gene encoding a polypeptide selected from the following: 11-β-HSD2, 11-β-hydroxylase, 17-α-hydroxylase, 21-hydroxylase, 3-β-HSD2, AE1, aldosterone synthase (ALDOS), AQP-2, ATP1A1, AVPR2, B(O)AT1, b(O,+)AT1, Barttin, calcium-sensitive receptor, carbonic anhydrase 2, Claudin16, Claudin19, CLC-5, CLC-Ka+CLC-Kb, CLC-Kb, CNNM2, CUL3, EGF, EGFR, ENaC α subunit, ENaC β subunit, ENaC γ subunit, FAM111A, forkhead box protein I1, GLUT-2, G-αs, HNF1B, HNF-4, Kir4.1, KLHL3, Kv1.1, L-Arginine: glycine amidotransferase, MAGED2, MR, Na-K-ATPase, NaPi2A, NaPi2c, NCCT, NKCC2, OCRL, PBFE, PCDB1, PHEX, rBAT, ROMK, SLC36A2+SLC6A20 / SLC6A19, TRPM6, V2R, V-ATPase subunit a4, V-ATPase subunit B1, WD repeat protein 72, WNK1, WNK4, y(+)LAT1, and any combination thereof.

[0252] In some embodiments, the kidney-related condition is cystinuria (e.g., A-type cystinuria, B-type cystinuria).Cystinuria is an autosomal recessive genetic disorder characterized by high concentrations of the amino acid cystine in the urine, leading to the formation of cystine stones in the kidneys, ureters, and bladder. Cystinuria is a type of aminoaciduria. Cystine is a dimer of cysteine. Symptoms of polycystic kidney disease include, but are not limited to: crystalluria (crystals in the urine); aminoaciduria (abnormally high levels of amino acids (e.g., cystine) in the urine); flank or back pain (e.g., usually unilateral pain); dysuria; hematuria; severe flank or back pain; groin, pelvic, or abdominal pain; nausea and vomiting; flank pain; back pain; recurrent abdominal pain; recurrent urinary tract infections; and / or fever.

[0253] In some embodiments, the transgene is SLC3A1 and / or SLC7A9. SLC3A1 and SLC7A9 are subunits of amino acid transporters (b0,+ transporter system) that function to reabsorb cystine from the urine into the renal tubules. In some embodiments, the kidney-associated condition is A-type cystinuria, and the transgene is SLC3A1. In some embodiments, the kidney-associated condition is B-type cystinuria, and the transgene is SLC7A9.

[0254] In some embodiments, the transgene comprises a nucleic acid sequence, or a functional fragment thereof, or a codon-optimized version of a nucleic acid, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 5, SEQ ID NO: 6 and retaining the same function (e.g., cystine transport) when expressed as a protein.

[0255] SEQ ID NO: 5, member 1 of solute carrier family 3 (SLC3A1), also known as: ATR1, CSNU1, D2H, NBAT, RBAT; NCBI ref NM_000341.4 position 57-2114, CCDS1819.1, human, 2058 nucleotides (nt) ATGG CTGAAGATAAAAGCAAGAGAGACTCCATCGAGATGAGTATGAAGGGATGCCAGACAAACAACGGGTTTGTCCATAAT GAAGACATTCTGGAGCAGACCCCGGATCCAGGAAGCTCAACAGACAACCTGAAGCACAGCACCAGGGGCATCCTTGG CTCCCAGGAGCCCGACTTCAAGGGCGTCCAGCCCTATGCGGGGATGCCCAAGGAGGTGCTGTTCCAGTTCTCTGGCCAGGCCCGCTACCGCATACCTCGGGAGATCCTCTTCTGGCTCACAGTGGCTTCTGTGCTGGTGCTCATCGCGGCCACC ATAGCCATCATTGCCCTCTCTCCAAAGTGCCTAGACTGGTGGCAGGAGGGGCCCATGTACCAGATCTACCCAAGGTC TTTCAAGGACAGTAACAAGGATGGGAACGGAGATCTGAAAGGTATTCAAGATAAACTGGACTACATCACAGCTTTAA DESCRIPTION Page 73 / 145 Page 84 CN 121752299 A ATATAAAAACTGTTTGGATTACTTCATTTTATAAATCGTCCCTTAAAGATTTCAGATATGGTGTTGAAGATTTCCGG GAAGTTGATCCCATTTTTGGAACGATGGAAGATTTTGAGAATCTGGTTGCAGCCATACATGATAAAGGTTTAAAATT AATCATCGATTTCATACCAAACCACACGAGTGATAAACATATTTGGTTTCAATTGAGTCGGACACGGACAGGAAAAT ATACTGATTATTATATCTGGCATGACTGTACCCATGAAAATGGCAAAACCATTCCACCCAACAACTGGTTAAGTGTG TATGGAAACTCCAGTTGGCACTTTGACGAAGTGCGAAACCAATGTTATTTTCATCAGTTTATGAAAGAGCAACCTGA TTTAAATTTCCGCAATCCTGATGTTCAAGAAGAAATAAAAGAAATTTTACGGTTCTGGCTCACAAAGGGTGTTGATG GTTTTAGTTTGGATGCTGTTAAATTCCTCCTAGAAGCAAAGCACCTGAGAGATGAGATCCAAGTAAATAAGACCCAA ATCCCGGACACGGTCACACAATACTCGGAGCTGTACCATGACTTCACCACCACGCAGGTGGGAATGCACGACATTGT CCGCAGCTTCCGGCAGACCATGGACCAATACAGCACGGAGCCCGGCAGATACAGGTTCATGGGGACTGAAGCCTATGCAGAGAGTATTGACAGGACCGTGATGTACTATGGATTGCCATTTATCCAAGAAGCTGATTTTCCCTTCAACAATTAC CTCAGCATGCTAGACACTGTTTCTGGGAACAGCGTGTATGAGGTTATCACATCCTGGATGGAAAACATGCCAGAAGG AAAATGGCCTAACTGGATGATTGGTGGACCAGACAGTTCACGGCTGACTTCGCGTTTGGGGAATCAGTATGTCAACG TGATGAACATGCTTCTTTTCACACTCCCTGGAACTCCTATAACTTACTATGGAGAAGAAATTGGAATGGGAAATATT GTAGCCGCAAATCTCAATGAAAGCTATGATATTAATACCCTTCGCTCAAAGTCACCAATGCAGTGGGACAATAGTTC AAATGCTGGTTTTTCTGAAGCTAGTAACACCTGGTTACCTACCAATTCAGATTACCACACTGTGAATGTTGATGTCC AAAAGACTCAGCCCAGATCGGCTTTGAAGTTATATCAAGATTTAAGTCTACTTCATGCCAATGAGCTACTCCTCAAC AGGGGCTGGTTTTGCCATTTGAGGAATGACAGCCACTATGTTGTGTACACAAGAGAGCTGGATGGCATCGACAGAAT CTTTATCGTGGTTCTGAATTTTGGAGAATCAACACTGTTAAATCTACATAATATGATTTCGGGCCTTCCCGCTAAAA TGAGAATAAGGTTAAGTACCAATTCTGCCGACAAAGGCAGTAAAGTTGATACAAGTGGCATTTTTCTGGACAAGGGA GAGGGACTCATCTTTGAACACAACACGAAGAATCTCCTTCATCGCCAAACAGCTTTCAGAGATAGATGCTTTGTTTC CAATCGAGCATGCTATTCCAGTGTACTGAACATACTGTATACCTCGTGTTAG SEQ ID NO: 6, Solute Carrier Family 7 Member 9 (SLC7A9), also known as B(0,+)-type amino acid transporter 1, BAT1 or CSNU3; NCBI refPosition 108‑1571 of NM_001126335.2, CCDS12425.1, Homo sapiens, 1464 nt ATGGCTGAAGATAAAAGCAAGAGAGACTCCATCGAGATGAGTATGAAGGGATGCCAGACAAACAACGGGTTTG TCCATAATGAAGACATTCTGGAGCAGACCCCGGATCCAGGAAGCTCAACAGACAACCTGAAGCACAGCACCAGGGGC ATCCTTGGCTCCCAGGAGCCCGACTTCAAGGGCGTCCAGCCCTATGCGGGGATGCCCAAGGAGGTGCTGTTCCAGTT CTCTGGCCAGGCCCGCTACCGCATACCTCGGGAGATCCTCTTCTGGCTCACAGTGGCTTCTGTGCTGGTGCTCATCG CGGCCACCATAGCCATCATTGCCCTCTCTCCAAAGTGCCTAGACTGGTGGCAGGAGGGGCCCATGTACCAGATCTAC CCAAGGTCTTTCAAGGACAGTAACAAGGATGGGAACGGAGATCTGAAAGGTATTCAAGATAAACTGGACTACATCAC AGCTTTAAATATAAAAACTGTTTGGATTACTTCATTTTATAAATCGTCCCTTAAAGATTTCAGATATGGTGTTGAAG ATTTCCGGGAAGTTGATCCCATTTTTGGAACGATGGAAGATTTTGAGAATCTGGTTGCAGCCATACATGATAAAGGT TTAAAATTAATCATCGATTTCATACCAAACCACACGAGTGATAAACATATTTGGTTTCAATTGAGTCGGACACGGAC AGGAAAATATACTGATTATTATATCTGGCATGACTGTACCCATGAAAATGGCAAAACCATTCCACCCAACAACTGGT TAAGTGTGTATGGAAACTCCAGTTGGCACTTTGACGAAGTGCGAAACCAATGTTATTTTCATCAGTTTATGAAAGAG CAACCTGATTTAAATTTCCGCAATCCTGATGTTCAAGAAGAAATAAAAGAAATTTTACGGTTCTGGCTCACAAAGGGTGTTGATGGTTTTAGTTTGGATGCTGTTAAATTCCTCCTAGAAGCAAAGCACCTGAGAGATGAGATCCAAGTAAATA AGACCCAAATCCCGGACACGGTCACACAATACTCGGAGCTGTACCATGACTTCACCACCACGCAGGTGGGAATGCAC GACATTGTCCGCAGCTTCCGGCAGACCATGGACCAATACAGCACGGAGCCCGGCAGATACAGGTTCATGGGGACTGA AGCCTATGCAGAGAGTATTGACAGGACCGTGATGTACTATGGATTGCCATTTATCCAAGAAGCTGATTTTCCCTTCA Description Page 74 / 145 Page 85 CN 121752299 A ACAATTACCTCAGCATGCTAGACACTGTTTCTGGGAACAGCGTGTATGAGGTTATCACATCCTGGATGGAAAACATG CCAGAAGGAAAATGGCCTAACTGGATGATTGGTGGACCAGACAGTTCACGGCTGACTTCGCGTTTGGGGAATCAGTA TGTCAACGTGATGAACATGCTTCTTTTCACACTCCCTGGAACTCCTATAACTTACTATGGAGAAGAAATTGGAATGG GAAATATTGTAGCCGCAAATCTCAATGAAAGCTATGATATTAATACCCTTCGCTCAAAGTCACCAATGCAGTGGGAC AATAGTTCAAATGCTGGTTTTTCTGAAGCTAGTAACACCTGGTTACCTACCAATTCAGATTACCACACTGTGAATGT TGATGTCCAAAAGACTCAGCCCAGATCGGCTTTGAAGTTATATCAAGATTTAAGTCTACTTCATGCCAATGAGCTAC TCCTCAACAGGGGCTGGTTTTGCCATTTGAGGAATGACAGCCACTATGTTGTGTACACAAGAGAGCTGGATGGCATC GACAGAATCTTTATCGTGGTTCTGAATTTTGGAGAATCAACACTGTTAAATCTACATAATATGATTTCGGGCCTTCCIn some embodiments, the transgene encodes a polypeptide comprising: SEQ ID NO: 7, SEQ ID NO: 8, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 7, SEQ ID NO: 8 and retaining the same function (e.g., cysteine ​​transport), or a functional fragment thereof.

[0256] SEQ ID NO: 7, member 1 of solute carrier family 3 (SLC3A1), NCBI ref NP_000332.2, human, 685 amino acids (aa) MAEDKSKRDSIEMSMKGCQTNNGFVHNEDILEQTPDPGSSTDNLKHSTRGILGSQEPDFK GVQPYAGMPKEVLFQFSGQARYRIPREILFWLTVASVLVLIAATIAIIALSPKCLDWWQEGPMYQIYPRSFKDSNKD GNGDLKGIQDKLDYITALNIKTVWITSFYKSSLKDFRYGVEDFREVDPIFGTMEDFENLVAAIHDKGLKLIIDFIPN HTSDKHIWFQLSRTRTGKYTDYYIWHDCTHENGKTIPPNNWLSVYGNSSWHFDEVRNQCYFHQFMKEQPDLNFRNPD VQEEIKEILRFWLTKGVDGFSLDAVKFLLEAKHLRDEIQVNKTQIPDTVTQYSELYHDFTTTQVGMHDIVRSFRQTM DQYSTEPGRYRFMGTEAYAESIDRTVMYYGLPFIQEADFPFNNYLSMLDTVSGNSVYEVITSWMENMPEGKWPNWMIGGPDSSRLTSRLGNQYVNVMNMLLFTLPGTPITYYGEEIGMGNIVAANLNESYDINTLRSKSPMQWDNSSNAGFSEA SNTWLPTNSDYHTVNVDVQKTQPRSALKLYQDLSLLHANELLLNRGWFCHLRNDSHYVVYTRELDGIDRIFIVVLNF GESTLLNLHNMISGLPAKMRIRLSTNSADKGSKVDTSGIFLDKGEGLIFEHNTKNLLHRQTAFRDRCFVSNRACYSS VLNILYTSC SEQ ID NO: 8, solute carrier family 7 member 9 (SLC7A9), NCBI ref NP_001119807.1, human, 487 aa MGDTGLRKRREDEKSIQSQEPKTTSLQKELGLISGISIIVGTIIGSGIFVSPKSVLSNTEAVGPCL IIWAACGVLATLGALCFAELGTMITKSGGEYPYLMEAYGPIPAYLFSWASLIVIKPTSFAIICLSFSEYVCAPFYVG CKPPQIVVKCLAAAAILFISTVNSLSVRLGSYVQNIFTAAKLVIVAIIIISGLVLLAQGNTKNFDNSFEGAQLSVGA ISLAFYNGLWAYDGWNQLNYITEELRNPYRNLPLAIIIGIPLVTACYILMNVSYFTVMTATELLQSQAVAVTFGDRV LYPASWIVPLFVAFSTIGAANGTCFTAGRLIYVAGREGHMLKVLSYISVRRLTPAPAIIFYGIIATIYIIPGDINSL VNYFSFAAWLFYGLTILGLIVMRFTRKELERPIKVPVVIPVLMTLISVFLVLAPIISKPTWEYLYCVLFILSGLLFY FLFVHYKFGWAQKISKPITMHLQMLMEVVPPEEDPE In some implementations, the kidney-related condition is autosomal dominant polycystic kidney disease (ADPKD). ADPKD is a genetic disorder that causes fluid-filled sacs (called cysts) to replace normal renal tubules in the kidneys. Cysts can occur in any nephron segment, but they most commonly form in the distal nephrons (such as the distal convoluted tubule) and collecting ducts (CD). Symptoms of polycystic kidney disease include, but are not limited to, abdominal pain or tenderness, hematuria, nocturia, unilateral or bilateral flank pain, somnolence, and joint pain.Pain, nail abnormalities, high blood pressure, back or flank pain, abdominal distension, hepatomegaly, heart murmurs, and / or kidney or abdominal swelling. (Instructions 75 / 145 pages, 86 CN 121752299 A block). In some embodiments, the transgene is PKD1, PKD2, and / or GANAB. The PKD1 and PKD2 genes encode the proteins polycystin-1 and polycystin-2, respectively. These two proteins interact to regulate cells in the kidneys and liver, are part of the process of forming tubular structures, and affect growth and fluid secretion functions. Mutations in the PKD1 or PKD2 genes result in dysfunctional cells and ultimately lead to cystic growth common in ADPKD. GANAB encodes the α subunit of glucosidase II and is a member of the glycosylhydrolase 31 family of proteins. The heterodimer glucosidase II plays a role in protein folding and quality control by cleaving glucose residues from immature glycoproteins in the endoplasmic reticulum. Mutations in the GANAB gene can lead to autosomal dominant polycystic kidney disease and liver disease.

[0257] In some embodiments, the transgene comprises a nucleic acid sequence, or a functional fragment thereof, having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with one of the sequences in SEQ ID NO: 9-10 and retaining the same function (e.g., regulating renal cells, influencing the formation of renal tubular structures, and influencing renal fluid secretion function) when expressed as a protein. SEQ ID NO: 9, polycystin-1 (PKD1), also known as: PBP, PC1, Pc-1, TRPP1; position 210-13118 of NCBI ref NM-000296.4, CCDS45385.1, human, 12,909 nt ATGCCGCCCGCCGCGCCCGCCCGCC TGGCGCTGGCCCTGGGCCTGGGCCTGTGGCTCGGGGCGCTGGCGGGGGGCCCCGGGCGCGGCTGCGGGCCCTGCGAG CCCCCCTGCCTCTGCGGCCCAGCGCCCGGCGCCGCCTGCCGCGTCAACTGCTCGGGCCGCGGGCTGCGGACGCTCGG TCCCGCGCTGCGCATCCCCGCGGACGCCACAGCGCTAGACGTCTCCCACAACCTGCTCCGGGCGCTGGACGTTGGGCTCCTGGCGAACCTCTCGGCGCTGGCAGAGCTGGATATAAGCAACAACAAGATTTCTACGTTAGAAGAAGGAATATTT GCTAATTTATTTAATTTAAGTGAAATAAACCTGAGTGGGAACCCGTTTGAGTGTGACTGTGGCCTGGCGTGGCTGCC GCGATGGGCGGAGGAGCAGCAGGTGCGGGTGGTGCAGCCCGAGGCAGCCACGTGTGCTGGGCCTGGCTCCCTGGCTG GCCAGCCTCTGCTTGGCATCCCCTTGCTGGACAGTGGCTGTGGTGAGGAGTATGTCGCCTGCCTCCCTGACAACAGC TCAGGCACCGTGGCAGCAGTGTCCTTTTCAGCTGCCCACGAAGGCCTGCTTCAGCCAGAGGCCTGCAGCGCCTTCTG CTTCTCCACCGGCCAGGGCCTCGCAGCCCTCTCGGAGCAGGGCTGGTGCCTGTGTGGGGCGGCCCAGCCCTCCAGTG CCTCCTTTGCCTGCCTGTCCCTCTGCTCCGGCCCCCCGCCACCTCCTGCCCCCACCTGTAGGGGCCCCACCCTCCTC CAGCACGTCTTCCCTGCCTCCCCAGGGGCCACCCTGGTGGGGCCCCACGGACCTCTGGCCTCTGGCCAGCTAGCAGC CTTCCACATCGCTGCCCCGCTCCCTGTCACTGCCACACGCTGGGACTTCGGAGACGGCTCCGCCGAGGTGGATGCCG CTGGGCCGGCTGCCTCGCATCGCTATGTGCTGCCTGGGCGCTATCACGTGACGGCCGTGCTGGCCCTGGGGGCCGGC TCAGCCCTGCTGGGGACAGACGTGCAGGTGGAAGCGGCACCTGCCGCCCTGGAGCTCGTGTGCCCGTCCTCGGTGCA GAGTGACGAGAGCCTCGACCTCAGCATCCAGAACCGCGGTGGTTCAGGCCTGGAGGCCGCCTACAGCATCGTGGCCCTGGGCGAGGAGCCGGCCCGAGCGGTGCACCCGCTCTGCCCCTCGGACACGGAGATCTTCCCTGGCAACGGGCACTGC TACCGCCTGGTGGTGGAGAAGGCGGCCTGGCTGCAGGCGCAGGAGCAGTGTCAGGCCTGGGCCGGGGCCGCCCTGGC AATGGTGGACAGTCCCGCCGTGCAGCGCTTCCTGGTCTCCCGGGTCACCAGGAGCCTAGACGTGTGGATCGGCTTCT CGACTGTGCAGGGGGTGGAGGTGGGCCCAGCGCCGCAGGGCGAGGCCTTCAGCCTGGAGAGCTGCCAGAACTGGCTG CCCGGGGAGCCACACCCAGCCACAGCCGAGCACTGCGTCCGGCTCGGGCCCACCGGGTGGTGTAACACCGACCTGTG CTCAGCGCCGCACAGCTACGTCTGCGAGCTGCAGCCCGGAGGCCCAGTGCAGGATGCCGAGAACCTCCTCGTGGGAG CGCCCAGTGGGGACCTGCAGGGACCCCTGACGCCTCTGGCACAGCAGGACGGCCTCTCAGCCCCGCACGAGCCCGTG GAGGTCATGGTATTCCCGGGCCTGCGTCTGAGCCGTGAAGCCTTCCTCACCACGGCCGAATTTGGGACCCAGGAGCT CCGGCGGCCCGCCCAGCTGCGGCTGCAGGTGTACCGGCTCCTCAGCACAGCAGGGACCCCGGAGAACGGCAGCGAGC CTGAGAGCAGGTCCCCGGACAACAGGACCCAGCTGGCCCCCGCGTGCATGCCAGGGGGACGCTGGTGCCCTGGAGCC Description Page 76 of 145 Page 87 CN 121752299 A AACATCTGCTTGCCGCTGGACGCCTCCTGCCACCCCCAGGCCTGCGCCAATGGCTGCACGTCAGGGCCAGGGCTACC CGGGGCCCCCTATGCGCTATGGAGAGAGTTCCTCTTCTCCGTTCCCGCGGGGCCCCCCGCGCAGTACTCGGTCACCCTCCACGGCCAGGATGTCCTCATGCTCCCTGGTGACCTCGTTGGCTTGCAGCACGACGCTGGCCCTGGCGCCCTCCTG CACTGCTCGCCGGCTCCCGGCCACCCTGGTCCCCGGGCCCCGTACCTCTCCGCCAACGCCTCGTCATGGCTGCCCCA CTTGCCAGCCCAGCTGGAGGGCACTTGGGCCTGCCCTGCCTGTGCCCTGCGGCTGCTTGCAGCCACGGAACAGCTCA CCGTGCTGCTGGGCTTGAGGCCCAACCCTGGACTGCGGCTGCCTGGGCGCTATGAGGTCCGGGCAGAGGTGGGCAAT GGCGTGTCCAGGCACAACCTCTCCTGCAGCTTTGACGTGGTCTCCCCAGTGGCTGGGCTGCGGGTCATCTACCCTGC CCCCCGCGACGGCCGCCTCTACGTGCCCACCAACGGCTCAGCCTTGGTGCTCCAGGTGGACTCTGGTGCCAACGCCA CGGCCACGGCTCGCTGGCCTGGGGGCAGTGTCAGCGCCCGCTTTGAGAATGTCTGCCCTGCCCTGGTGGCCACCTTC GTGCCCGGCTGCCCCTGGGAGACCAACGATACCCTGTTCTCAGTGGTAGCACTGCCGTGGCTCAGTGAGGGGGAGCA CGTGGTGGACGTGGTGGTGGAAAACAGCGCCAGCCGGGCCAACCTCAGCCTGCGGGTGACGGCGGAGGAGCCCATCT GTGGCCTCCGCGCCACGCCCAGCCCCGAGGCCCGTGTACTGCAGGGAGTCCTAGTGAGGTACAGCCCCGTGGTGGAG GCCGGCTCGGACATGGTCTTCCGGTGGACCATCAACGACAAGCAGTCCCTGACCTTCCAGAACGTGGTCTTCAATGT CATTTATCAGAGCGCGGCGGTCTTCAAGCTCTCACTGACGGCCTCCAACCACGTGAGCAACGTCACCGTGAACTACAACGTAACCGTGGAGCGGATGAACAGGATGCAGGGTCTGCAGGTCTCCACAGTGCCGGCCGTGCTGTCCCCCAATGCC ACGCTAGCACTGACGGCGGGCGTGCTGGTGGACTCGGCCGTGGAGGTGGCCTTCCTGTGGACCTTTGGGGATGGGGA GCAGGCCCTCCACCAGTTCCAGCCTCCGTACAACGAGTCCTTCCCGGTTCCAGACCCCTCGGTGGCCCAGGTGCTGG TGGAGCACAATGTCATGCACACCTACGCTGCCCCAGGTGAGTACCTCCTGACCGTGCTGGCATCTAATGCCTTCGAG AACCTGACGCAGCAGGTGCCTGTGAGCGTGCGCGCCTCCCTGCCCTCCGTGGCTGTGGGTGTGAGTGACGGCGTCCT GGTGGCCGGCCGGCCCGTCACCTTCTACCCGCACCCGCTGCCCTCGCCTGGGGGTGTTCTTTACACGTGGGACTTCG GGGACGGCTCCCCTGTCCTGACCCAGAGCCAGCCGGCTGCCAACCACACCTATGCCTCGAGGGGCACCTACCACGTG CGCCTGGAGGTCAACAACACGGTGAGCGGTGCGGCGGCCCAGGCGGATGTGCGCGTCTTTGAGGAGCTCCGCGGACT CAGCGTGGACATGAGCCTGGCCGTGGAGCAGGGCGCCCCCGTGGTGGTCAGCGCCGCGGTGCAGACGGGCGACAACA TCACGTGGACCTTCGACATGGGGGACGGCACCGTGCTGTCGGGCCCGGAGGCAACAGTGGAGCATGTGTACCTGCGG GCACAGAACTGCACAGTGACCGTGGGTGCGGCCAGCCCCGCCGGCCACCTGGCCCGGAGCCTGCACGTGCTGGTCTT CGTCCTGGAGGTGCTGCGCGTTGAACCCGCCGCCTGCATCCCCACGCAGCCTGACGCGCGGCTCACGGCCTACGTCACCGGGAACCCGGCCCACTACCTCTTCGACTGGACCTTCGGGGATGGCTCCTCCAACACGACCGTGCGGGGGTGCCCG ACGGTGACACACAACTTCACGCGGAGCGGCACGTTCCCCCTGGCGCTGGTGCTGTCCAGCCGCGTGAACAGGGCGCA TTACTTCACCAGCATCTGCGTGGAGCCAGAGGTGGGCAACGTCACCCTGCAGCCAGAGAGGCAGTTTGTGCAGCTCG GGGACGAGGCCTGGCTGGTGGCATGTGCCTGGCCCCCGTTCCCCTACCGCTACACCTGGGACTTTGGCACCGAGGAA GCCGCCCCCACCCGTGCCAGGGGCCCTGAGGTGACGTTCATCTACCGAGACCCAGGCTCCTATCTTGTGACAGTCAC CGCGTCCAACAACATCTCTGCTGCCAATGACTCAGCCCTGGTGGAGGTGCAGGAGCCCGTGCTGGTCACCAGCATCA AGGTCAATGGCTCCCTTGGGCTGGAGCTGCAGCAGCCGTACCTGTTCTCTGCTGTGGGCCGTGGGCGCCCCGCCAGC TACCTGTGGGATCTGGGGGACGGTGGGTGGCTCGAGGGTCCGGAGGTCACCCACGCTTACAACAGCACAGGTGACTT CACCGTTAGGGTGGCCGGCTGGAATGAGGTGAGCCGCAGCGAGGCCTGGCTCAATGTGACGGTGAAGCGGCGCGTGC GGGGGCTCGTCGTCAATGCAAGCCGCACGGTGGTGCCCCTGAATGGGAGCGTGAGCTTCAGCACGTCGCTGGAGGCC GGCAGTGATGTGCGCTATTCCTGGGTGCTCTGTGACCGCTGCACGCCCATCCCTGGGGGTCCTACCATCTCTTACAC CTTCCGCTCCGTGGGCACCTTCAATATCATCGTCACGGCTGAGAACGAGGTGGGCTCCGCCCAGGACAGCATCTTCGTCTATGTCCTGCAGCTCATAGAGGGGCTGCAGGTGGTGGGCGGTGGCCGCTACTTCCCCACCAACCACACGGTACAG DESCRIPTION Page 77 / 145 88 CN 121752299 A CTGCAGGCCGTGGTTAGGGATGGCACCAACGTCTCCTACAGCTGGACTGCCTGGAGGGACAGGGGCCCGGCCCTGGC CGGCAGCGGCAAAGGCTTCTCGCTCACCGTGCTCGAGGCCGGCACCTACCATGTGCAGCTGCGGGCCACCAACATGC TGGGCAGCGCCTGGGCCGACTGCACCATGGACTTCGTGGAGCCTGTGGGGTGGCTGATGGTGGCCGCCTCCCCGAAC CCAGCTGCCGTCAACACAAGCGTCACCCTCAGTGCCGAGCTGGCTGGTGGCAGTGGTGTCGTATACACTTGGTCCTT GGAGGAGGGGCTGAGCTGGGAGACCTCCGAGCCATTTACCACCCATAGCTTCCCCACACCCGGCCTGCACTTGGTCA CCATGACGGCAGGGAACCCGCTGGGCTCAGCCAACGCCACCGTGGAAGTGGATGTGCAGGTGCCTGTGAGTGGCCTC AGCATCAGGGCCAGCGAGCCCGGAGGCAGCTTCGTGGCGGCCGGGTCCTCTGTGCCCTTTTGGGGGCAGCTGGCCAC GGGCACCAATGTGAGCTGGTGCTGGGCTGTGCCCGGCGGCAGCAGCAAGCGTGGCCCTCATGTCACCATGGTCTTCC CGGATGCTGGCACCTTCTCCATCCGGCTCAATGCCTCCAACGCAGTCAGCTGGGTCTCAGCCACGTACAACCTCACG GCGGAGGAGCCCATCGTGGGCCTGGTGCTGTGGGCCAGCAGCAAGGTGGTGGCGCCCGGGCAGCTGGTCCATTTTCA GATCCTGCTGGCTGCCGGCTCAGCTGTCACCTTCCGCCTGCAGGTCGGCGGGGCCAACCCCGAGGTGCTCCCCGGGCCCCGTTTCTCCCACAGCTTCCCCCGCGTCGGAGACCACGTGGTGAGCGTGCGGGGCAAAAACCACGTGAGCTGGGCC CAGGCGCAGGTGCGCATCGTGGTGCTGGAGGCCGTGAGTGGGCTGCAGGTGCCCAACTGCTGCGAGCCTGGCATCGC CACGGGCACTGAGAGGAACTTCACAGCCCGCGTGCAGCGCGGCTCTCGGGTCGCCTACGCCTGGTACTTCTCGCTGC AGAAGGTCCAGGGCGACTCGCTGGTCATCCTGTCGGGCCGCGACGTCACCTACACGCCCGTGGCCGCGGGGCTGTTG GAGATCCAGGTGCGCGCCTTCAACGCCCTGGGCAGTGAGAACCGCACGCTGGTGCTGGAGGTTCAGGACGCCGTCCA GTATGTGGCCCTGCAGAGCGGCCCCTGCTTCACCAACCGCTCGGCGCAGTTTGAGGCCGCCACCAGCCCCAGCCCCC GGCGTGTGGCCTACCACTGGGACTTTGGGGATGGGTCGCCAGGGCAGGACACAGATGAGCCCAGGGCCGAGCACTCC TACCTGAGGCCTGGGGACTACCGCGTGCAGGTGAACGCCTCCAACCTGGTGAGCTTCTTCGTGGCGCAGGCCACGGT GACCGTCCAGGTGCTGGCCTGCCGGGAGCCGGAGGTGGACGTGGTCCTGCCCCTGCAGGTGCTGATGCGGCGATCAC AGCGCAACTACTTGGAGGCCCACGTTGACCTGCGCGACTGCGTCACCTACCAGACTGAGTACCGCTGGGAGGTGTAT CGCACCGCCAGCTGCCAGCGGCCGGGGCGCCCAGCGCGTGTGGCCCTGCCCGGCGTGGACGTGAGCCGGCCTCGGCT GGTGCTGCCGCGGCTGGCGCTGCCTGTGGGGCACTACTGCTTTGTGTTTGTCGTGTCATTTGGGGACACGCCACTGACACAGAGCATCCAGGCCAATGTGACGGTGGCCCCCGAGCGCCTGGTGCCCATCATTGAGGGTGGCTCATACCGCGTG TGGTCAGACACACGGGACCTGGTGCTGGATGGGAGCGAGTCCTACGACCCCAACCTGGAGGACGGCGACCAGACGCC GCTCAGTTTCCACTGGGCCTGTGTGGCTTCGACACAGAGGGAGGCTGGCGGGTGTGCGCTGAACTTTGGGCCCCGCG GGAGCAGCACGGTCACCATTCCACGGGAGCGGCTGGCGGCTGGCGTGGAGTACACCTTCAGCCTGACCGTGTGGAAG GCCGGCCGCAAGGAGGAGGCCACCAACCAGACGGTGCTGATCCGGAGTGGCCGGGTGCCCATTGTGTCCTTGGAGTG TGTGTCCTGCAAGGCACAGGCCGTGTACGAAGTGAGCCGCAGCTCCTACGTGTACTTGGAGGGCCGCTGCCTCAATT GCAGCAGCGGCTCCAAGCGAGGGCGGTGGGCTGCACGTACGTTCAGCAACAAGACGCTGGTGCTGGATGAGACCACC ACATCCACGGGCAGTGCAGGCATGCGACTGGTGCTGCGGCGGGGCGTGCTGCGGGACGGCGAGGGATACACCTTCAC GCTCACGGTGCTGGGCCGCTCTGGCGAGGAGGAGGGCTGCGCCTCCATCCGCCTGTCCCCCAACCGCCCGCCGCTGG GGGGCTCTTGCCGCCTCTTCCCACTGGGCGCTGTGCACGCCCTCACCACCAAGGTGCACTTCGAATGCACGGGCTGG CATGACGCGGAGGATGCTGGCGCCCCGCTGGTGTACGCCCTGCTGCTGCGGCGCTGTCGCCAGGGCCACTGCGAGGA GTTCTGTGTCTACAAGGGCAGCCTCTCCAGCTACGGAGCCGTGCTGCCCCCGGGTTTCAGGCCACACTTCGAGGTGGGCCTGGCCGTGGTGGTGCAGGACCAGCTGGGAGCCGCTGTGGTCGCCCTCAACAGGTCTTTGGCCATCACCCTCCCA GAGCCCAACGGCAGCGCAACGGGGCTCACAGTCTGGCTGCACGGGCTCACCGCTAGTGTGCTCCCAGGGCTGCTGCG GCAGGCCGATCCCCAGCACGTCATCGAGTACTCGTTGGCCCTGGTCACCGTGCTGAACGAGTACGAGCGGGCCCTGG ACGTGGCGGCAGAGCCCAAGCACGAGCGGCAGCACCGAGCCCAGATACGCAAGAACATCACGGAGACTCTGGTGTCC DESCRIPTION Page 78 / 145 89 CN 121752299 A CTGAGGGTCCACACTGTGGATGACATCCAGCAGATCGCTGCTGCGCTGGCCCAGTGCATGGGGCCCAGCAGGGAGCT CGTATGCCGCTCGTGCCTGAAGCAGACGCTGCACAAGCTGGAGGCCATGATGCTCATCCTGCAGGCAGAGACCACCG CGGGCACCGTGACGCCCACCGCCATCGGAGACAGCATCCTCAACATCACAGGAGACCTCATCCACCTGGCCAGCTCG GACGTGCGGGCACCACAGCCCTCAGAGCTGGGAGCCGAGTCACCATCTCGGATGGTGGCGTCCCAGGCCTACAACCT GACCTCTGCCCTCATGCGCATCCTCATGCGCTCCCGCGTGCTCAACGAGGAGCCCCTGACGCTGGCGGGCGAGGAGA TCGTGGCCCAGGGCAAGCGCTCGGACCCGCGGAGCCTGCTGTGCTATGGCGGCGCCCCAGGGCCTGGCTGCCACTTC TCCATCCCCGAGGCTTTCAGCGGGGCCCTGGCCAACCTCAGTGACGTGGTGCAGCTCATCTTTCTGGTGGACTCCAA TCCCTTTCCCTTTGGCTATATCAGCAACTACACCGTCTCCACCAAGGTGGCCTCGATGGCATTCCAGACACAGGCCGGCGCCCAGATCCCCATCGAGCGGCTGGCCTCAGAGCGCGCCATCACCGTGAAGGTGCCCAACAACTCGGACTGGGCT GCCCGGGGCCACCGCAGCTCCGCCAACTCCGCCAACTCCGTTGTGGTCCAGCCCCAGGCCTCCGTCGGTGCTGTGGT CACCCTGGACAGCAGCAACCCTGCGGCCGGGCTGCATCTGCAGCTCAACTATACGCTGCTGGACGGCCACTACCTGT CTGAGGAACCTGAGCCCTACCTGGCAGTCTACCTACACTCGGAGCCCCGGCCCAATGAGCACAACTGCTCGGCTAGC AGGAGGATCCGCCCAGAGTCACTCCAGGGTGCTGACCACCGGCCCTACACCTTCTTCATTTCCCCGGGGAGCAGAGA CCCAGCGGGGAGTTACCATCTGAACCTCTCCAGCCACTTCCGCTGGTCGGCGCTGCAGGTGTCCGTGGGCCTGTACA CGTCCCTGTGCCAGTACTTCAGCGAGGAGGACATGGTGTGGCGGACAGAGGGGCTGCTGCCCCTGGAGGAGACCTCG CCCCGCCAGGCCGTCTGCCTCACCCGCCACCTCACCGCCTTCGGCGCCAGCCTCTTCGTGCCCCCAAGCCATGTCCG CTTTGTGTTTCCTGAGCCGACAGCGGATGTAAACTACATCGTCATGCTGACATGTGCTGTGTGCCTGGTGACCTACA TGGTCATGGCCGCCATCCTGCACAAGCTGGACCAGTTGGATGCCAGCCGGGGCCGCGCCATCCCTTTCTGTGGGCAG CGGGGCCGCTTCAAGTACGAGATCCTCGTCAAGACAGGCTGGGGCCGGGGCTCAGGTACCACGGCCCACGTGGGCAT CATGCTGTATGGGGTGGACAGCCGGAGCGGCCACCGGCACCTGGACGGCGACAGAGCCTTCCACCGCAACAGCCTGGACATCTTCCGGATCGCCACCCCGCACAGCCTGGGTAGCGTGTGGAAGATCCGAGTGTGGCACGACAACAAAGGGCTC AGCCCTGCCTGGTTCCTGCAGCACGTCATCGTCAGGGACCTGCAGACGGCACGCAGCGCCTTCTTCCTGGTCAATGA CTGGCTTTCGGTGGAGACGGAGGCCAACGGGGGCCTGGTGGAGAAGGAGGTGCTGGCCGCGAGCGACGCAGCCCTTT TGCGCTTCCGGCGCCTGCTGGTGGCTGAGCTGCAGCGTGGCTTCTTTGACAAGCACATCTGGCTCTCCATATGGGAC CGGCCGCCTCGTAGCCGTTTCACTCGCATCCAGAGGGCCACCTGCTGCGTTCTCCTCATCTGCCTCTTCCTGGGCGC CAACGCCGTGTGGTACGGGGCTGTTGGCGACTCTGCCTACAGCACGGGGCATGTGTCCAGGCTGAGCCCGCTGAGCG TCGACACAGTCGCTGTTGGCCTGGTGTCCAGCGTGGTTGTCTATCCCGTCTACCTGGCCATCCTTTTTCTCTTCCGG ATGTCCCGGAGCAAGGTGGCTGGGAGCCCGAGCCCCACACCTGCCGGGCAGCAGGTGCTGGACATCGACAGCTGCCT GGACTCGTCCGTGCTGGACAGCTCCTTCCTCACGTTCTCAGGCCTCCACGCTGAGGCCTTTGTTGGACAGATGAAGA GTGACTTGTTTCTGGATGATTCTAAGAGTCTGGTGTGCTGGCCCTCCGGCGAGGGAACGCTCAGTTGGCCGGACCTG CTCAGTGACCCGTCCATTGTGGGTAGCAATCTGCGGCAGCTGGCACGGGGCCAGGCGGGCCATGGGCTGGGCCCAGA GGAGGACGGCTTCTCCCTGGCCAGCCCCTACTCGCCTGCCAAATCCTTCTCAGCATCAGATGAAGACCTGATCCAGCAGGTCCTTGCCGAGGGGGTCAGCAGCCCAGCCCCTACCCAAGACACCCACATGGAAACGGACCTGCTCAGCAGCCTG TCCAGCACTCCTGGGGAGAAGACAGAGACGCTGGCGCTGCAGAGGCTGGGGGAGCTGGGGCCACCCAGCCCAGGCCT GAACTGGGAACAGCCCCAGGCAGCGAGGCTGTCCAGGACAGGACTGGTGGAGGGTCTGCGGAAGCGCCTGCTGCCGG CCTGGTGTGCCTCCCTGGCCCACGGGCTCAGCCTGCTCCTGGTGGCTGTGGCTGTGGCTGTCTCAGGGTGGGTGGGT GCGAGCTTCCCCCCGGGCGTGAGTGTTGCGTGGCTCCTGTCCAGCAGCGCCAGCTTCCTGGCCTCATTCCTCGGCTG GGAGCCACTGAAGGTCTTGCTGGAAGCCCTGTACTTCTCACTGGTGGCCAAGCGGCTGCACCCGGATGAAGATGACA CCCTGGTAGAGAGCCCGGCTGTGACGCCTGTGAGCGCACGTGTGCCCCGCGTACGGCCACCCCACGGCTTTGCACTC DESCRIPTION Page 79 / 145 90 CN 121752299 A TTCCTGGCCAAGGAAGAAGCCCGCAAGGTCAAGAGGCTACATGGCATGCTGCGGAGCCTCCTGGTGTACATGCTTTT TCTGCTGGTGACCCTGCTGGCCAGCTATGGGGATGCCTCATGCCATGGGCACGCCTACCGTCTGCAAAGCGCCATCA AGCAGGAGCTGCACAGCCGGGCCTTCCTGGCCATCACGCGGTCTGAGGAGCTCTGGCCATGGATGGCCCACGTGCTG CTGCCCTACGTCCACGGGAACCAGTCCAGCCCAGAGCTGGGGCCCCCACGGCTGCGGCAGGTGCGGCTGCAGGAAGC ACTCTACCCAGACCCTCCCGGCCCCAGGGTCCACACGTGCTCGGCCGCAGGAGGCTTCAGCACCAGCGATTACGACGTTGGCTGGGAGAGTCCTCACAATGGCTCGGGGACGTGGGCCTATTCAGCGCCGGATCTGCTGGGGGCATGGTCCTGG GGCTCCTGTGCCGTGTATGACAGCGGGGGCTACGTGCAGGAGCTGGGCCTGAGCCTGGAGGAGAGCCGCGACCGGCT GCGCTTCCTGCAGCTGCACAACTGGCTGGACAACAGGAGCCGCGCTGTGTTCCTGGAGCTCACGCGCTACAGCCCGG CCGTGGGGCTGCACGCCGCCGTCACGCTGCGCCTCGAGTTCCCGGCGGCCGGCCGCGCCCTGGCCGCCCTCAGCGTC CGCCCCTTTGCGCTGCGCCGCCTCAGCGCGGGCCTCTCGCTGCCTCTGCTCACCTCGGTGTGCCTGCTGCTGTTCGC CGTGCACTTCGCCGTGGCCGAGGCCCGTACTTGGCACAGGGAAGGGCGCTGGCGCGTGCTGCGGCTCGGAGCCTGGG CGCGGTGGCTGCTGGTGGCGCTGACGGCGGCCACGGCACTGGTACGCCTCGCCCAGCTGGGTGCCGCTGACCGCCAG TGGACCCGTTTCGTGCGCGGCCGCCCGCGCCGCTTCACTAGCTTCGACCAGGTGGCGCAGCTGAGCTCCGCAGCCCG TGGCCTGGCGGCCTCGCTGCTCTTCCTGCTTTTGGTCAAGGCTGCCCAGCAGCTACGCTTCGTGCGCCAGTGGTCCG TCTTTGGCAAGACATTATGCCGAGCTCTGCCAGAGCTCCTGGGGGTCACCTTGGGCCTGGTGGTGCTCGGGGTAGCC TACGCCCAGCTGGCCATCCTGCTCGTGTCTTCCTGTGTGGACTCCCTCTGGAGCGTGGCCCAGGCCCTGTTGGTGCT GTGCCCTGGGACTGGGCTCTCTACCCTGTGTCCTGCCGAGTCCTGGCACCTGTCACCCCTGCTGTGTGTGGGGCTCTGGGCACTGCGGCTGTGGGGCGCCCTACGGCTGGGGGCTGTTATTCTCCGCTGGCGCTACCACGCCTTGCGTGGAGAG CTGTACCGGCCGGCCTGGGAGCCCCAGGACTACGAGATGGTGGAGTTGTTCCTGCGCAGGCTGCGCCTCTGGATGGG CCTCAGCAAGGTCAAGGAGTTCCGCCACAAAGTCCGCTTTGAAGGGATGGAGCCGCTGCCCTCTCGCTCCTCCAGGG GCTCCAAGGTATCCCCGGATGTGCCCCCACCCAGCGCTGGCTCCGATGCCTCGCACCCCTCCACCTCCTCCAGCCAG CTGGATGGGCTGAGCGTGAGCCTGGGCCGGCTGGGGACAAGGTGTGAGCCTGAGCCCTCCCGCCTCCAAGCCGTGTT CGAGGCCCTGCTCACCCAGTTTGACCGACTCAACCAGGCCACAGAGGACGTCTACCAGCTGGAGCAGCAGCTGCACA GCCTGCAAGGCCGCAGGAGCAGCCGGGCGCCCGCCGGATCTTCCCGTGGCCCATCCCCGGGCCTGCGGCCAGCACTG CCCAGCCGCCTTGCCCGGGCCAGTCGGGGTGTGGACCTGGCCACTGGCCCCAGCAGGACACCCCTTCGGGCCAAGAA CAAGGTCCACCCCAGCAGCACTTAG SEQ ID NO: 10, polycystin-2 (PKD2), also known as: PC2, PKD4, Pc-2, APKD2, TRPP2; positions 100-3006 of NCBI ref NM_000297.4, CCDS3627.1, human, 2907 nt ATGGTGAACTCCAGTCGCGTGC AGCCTCAGCAGCCCGGGGACGCCAAGCGGCCGCCCGCGCCCCGCGCGCCGGACCCGGGCCGGCTGATGGCTGGCTGC GCGGCCGTGGGCGCCAGCCTCGCCGCCCCGGGCGGCCTCTGCGAGCAGCGGGGCCTGGAGATCGAGATGCAGCGCATCCGGCAGGCGGCCGCGCGGGACCCCCCGGCCGGAGCCGCGGCCTCCCCTTCTCCTCCGCTCTCGTCGTGCTCCCGGC AGGCGTGGAGCCGCGATAACCCCGGCTTCGAGGCCGAGGAGGAGGAGGAGGAGGTGGAAGGGGAAGAAGGCGGAATG GTGGTGGAGATGGACGTAGAGTGGCGCCCGGGCAGCCGGAGGTCGGCCGCCTCCTCGGCCGTGAGCTCCGTGGGCGC GCGGAGCCGGGGGCTTGGGGGCTACCACGGCGCGGGCCACCCGAGCGGGAGGCGGCGCCGGCGAGAGGACCAGGGCC CGCCGTGCCCCAGCCCAGTCGGCGGCGGGGACCCGCTGCATCGCCACCTCCCCCTGGAAGGGCAGCCGCCCCGAGTG GCCTGGGCGGAGAGGCTGGTTCGCGGGCTGCGAGGTCTCTGGGGAACAAGACTCATGGAGGAAAGCAGCACTAACCG AGAGAAATACCTTAAAAGTGTTTTACGGGAACTGGTCACATACCTCCTTTTTCTCATAGTCTTGTGCATCTTGACCT ACGGCATGATGAGCTCCAATGTGTACTACTACACCCGGATGATGTCACAGCTCTTCCTAGACACCCCCGTGTCCAAA ACGGAGAAAACTAACTTTAAAACTCTGTCTTCCATGGAAGACTTCTGGAAGTTCACAGAAGGCTCCTTATTGGATGG Description Page 80 / 145 91 CN 121752299 A GCTGTACTGGAAGATGCAGCCCAGCAACCAGACTGAAGCTGACAACCGAAGTTTCATCTTCTATGAGAACCTGCTGT TAGGGGTTCCACGAATACGGCAACTCCGAGTCAGAAATGGATCCTGCTCTATCCCCCAGGACTTGAGAGATGAAATT AAAGAGTGCTATGATGTCTACTCTGTCAGTAGTGAAGATAGGGCTCCCTTTGGGCCCCGAAATGGAACCGCTTGGATCTACACAAGTGAAAAAGACTTGAATGGTAGTAGCCACTGGGGAATCATTGCAACTTATAGTGGAGCTGGCTATTATC TGGATTTGTCAAGAACAAGAGAGGAAACAGCTGCACAAGTTGCTAGCCTCAAGAAAAATGTCTGGCTGGACCGAGGA ACCAGGGCAACTTTTATTGACTTCTCAGTGTACAACGCCAACATTAACCTGTTCTGTGTGGTCAGGTTATTGGTTGA ATTCCCAGCAACAGGTGGTGTGATTCCATCTTGGCAATTTCAGCCTTTAAAGCTGATCCGATATGTCACAACTTTTG ATTTCTTCCTGGCAGCCTGTGAGATTATCTTTTGTTTCTTTATCTTTTACTATGTGGTGGAAGAGATATTGGAAATT CGCATTCACAAACTACACTATTTCAGGAGTTTCTGGAATTGTCTGGATGTTGTGATCGTTGTGCTGTCAGTGGTAGC TATAGGAATTAACATATACAGAACATCAAATGTGGAGGTGCTACTACAGTTTCTGGAAGATCAAAATACTTTCCCCA ACTTTGAGCATCTGGCATATTGGCAGATACAGTTCAACAATATAGCTGCTGTCACAGTATTTTTTGTCTGGATTAAG CTCTTCAAATTCATCAATTTTAACAGGACCATGAGCCAGCTCTCGACAACCATGTCTCGATGTGCCAAAGACCTGTT TGGCTTTGCTATTATGTTCTTCATTATTTTCCTAGCGTATGCTCAGTTGGCATACCTTGTCTTTGGCACTCAGGTCG ATGACTTCAGTACTTTCCAAGAGTGTATCTTCACTCAATTCCGTATCATTTTGGGCGATATCAACTTTGCAGAGATT GAGGAAGCTAATCGAGTTTTGGGACCAATTTATTTCACTACATTTGTGTTCTTTATGTTCTTCATTCTTTTGAATATGTTTTTGGCTATCATCAATGATACTTACTCTGAAGTGAAATCTGACTTGGCACAGCAGAAAGCTGAAATGGAACTCT CAGATCTTATCAGAAAGGGCTACCATAAAGCTTTGGTCAAACTAAAACTGAAAAAAAATACCGTGGATGACATTTCA GAGAGTCTGCGGCAAGGAGGAGGCAAGTTAAACTTTGACGAACTTCGACAAGATCTCAAAGGGAAGGGCCATACTGA TGCAGAGATTGAGGCAATATTCACAAAGTACGACCAAGATGGAGACCAAGAACTGACCGAACATGAACATCAGCAGA TGAGAGACGACTTGGAGAAAGAGAGGGAGGACCTGGATTTGGATCACAGTTCTTTACCACGTCCCATGAGCAGCCGA AGTTTCCCTCGAAGCCTGGATGACTCTGAGGAGGATGACGATGAAGATAGCGGACATAGCTCCAGAAGGAGGGGAAG CATTTCTAGTGGCGTTTCTTACGAAGAGTTTCAAGTCCTGGTGAGACGAGTGGACCGGATGGAGCATTCCATCGGCA GCATAGTGTCCAAGATTGACGCCGTGATCGTGAAGCTAGAGATTATGGAGCGAGCCAAACTGAAGAGGAGGGAGGTG CTGGGAAGGCTGTTGGATGGGGTGGCCGAGGATGAAAGGCTGGGTCGTGACAGTGAAATCCATAGGGAACAGATGGA ACGGCTAGTACGTGAAGAGTTGGAACGCTGGGAATCCGATGATGCAGCTTCCCAGATCAGTCATGGTTTAGGCACGC CAGTGGGACTAAATGGTCAACCTCGCCCCAGAAGCTCCCGCCCATCTTCCTCCCAATCTACAGAAGGCATGGAAGGT GCAGGTGGAAATGGGAGTTCTAATGTCCACGTATGA In some embodiments, the transgene encodes a polypeptide comprising: one of SEQ ID NO: 11-12 or a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at leastAn amino acid sequence, or a functional fragment thereof, that is 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and retains the same function (e.g., regulating renal cells, influencing the formation of renal tubular structures, and influencing renal fluid secretion). SEQ ID NO: 11, polycystin-1 (PKD1), NCBI ref NP_000287.4, human, 4302 aa MP PAAPARLALALGLWLGALAGGPGRGCGPCEPPCLCGPAPGAACRVNCSGRGLRTLGPALRIPADATALDVSHNLL RALDVGLLANLSALAELDISNNKISTLEEGIFANLFNLSEINLSGNPFECDCGLAWLPRWAEEQQVRVVQPEAATCA GPGSLAGQPLLGIPLLDSGCGEEYVACLPDNSSGTVAAVSFSAAHEGLLQPEACSAFCFSTGQGLAALSEQGWCLCG AAQPSSASFACLSLCSGPPPPPAPTCRGPTLLQHVFPASPGATLVGPHGPLASGQLAAFHIAAPLPVTATRWDFGDG SAEVDAAGPAASHRYVLPGRYHVTAVLALGAGGSALLGTDVQVEAAPAALELVCPSSVQSDESLDLSIQNRGGSGLEA AYSIVALGEEPARAVHPLCPSDTEIFPGNGHCYRLVVEKAAWLQAQEQCQAWAGAALAMVDSPAVQRFLVSRVTRSL Instructions 81 / 145 Page 92 CN 121752299 A DVWIGFSTVQGVEVGPAPQGEAFSLESCQNWLPGEPHPATAEHCVRLGPTGWCNTDLCSAPHSYVCELQPGGPVQDA ENLLVGAPSGDLQGPLTPLAQQDGLSAPHEPVEVMVFPGLRLSREAFLTTAEFGTQELRRPAQLRLQVYRLLSTAGT PENGSEPESRSPDNRTQLAPACMPGGRWCPGANICLPLDASCHPQACANGCTSGPGLPGAPYALWREFLFSVPAGPP AQYSVTLHGQDVLMLPGDLVGLQHDAGPGALLHCSPAPGHPGPRAPYLSANASSWLPHLPAQLEGTWACPACALRLLAATEQLTVLLGLRPNPGLRLPGRYEVRAEVGNGVSRHNLSCSFDVVSPVAGLRVIYPAPRDGRLYVPTNGSALVLQV DSGANATATARWPGGSVSARFENVCPALVATFVPGCPWETNDTLFSVVALPWLSEGEHVVDVVVENSASRANLSLRV TAEEPICGLRATPSPEARVLQGVLVRYSPVVEAGSDMVFRWTINDKQSLTFQNVVFNVIYQSAAVFKLSLTASNHVS NVTVNYNVTVERMNRMQGLQVSTVPAVLSPNATLALTAGVLVDSAVEVAFLWTFGDGEQALHQFQPPYNESFPVPDP SVAQVLVEHNVMHTYAAPGEYLLTVLASNAFENLTQQVPVSVRASLPSVAVGVSDGVLVAGRPVTFYPHPLPSPGGV LYTWDFGDGSPVLTQSQPAANHTYASRGTYHVRLEVNNTVSGAAAQADVRVFEELRGLSVDMSLAVEQGAPVVVSAA VQTGDNITWTFDMGDGTVLSGPEATVEHVYLRAQNCTVTVGAASPAGHLARSLHVLVFVLEVLRVEPAACIPTQPDA RLTAYVTGNPAHYLFDWTFGDGSSNTTVRGCPTVTHNFTRSGTFPLALVLSSRVNRAHYFTSICVEPEVGNVTLQPE RQFVQLGDEAWLVACAWPPFPYRYTWDFGTEEAAPTRARGPEVTFIYRDPGSYLVTVTASNNISAANDSALVEVQEP VLVTSIKVNGSLGLELQQPYLFSAVGRGRPASYLWDLGDGGWLEGPEVTHAYNSTGDFTVRVAGWNEVSRSEAWLNV TVKRRVRGLVVNASRTVVPLNGSVSFSTSLEAGSDVRYSWVLCDRCTPIPGGPTISYTFRSVGTFNIIVTAENEVGS AQDSIFVYVLQLIEGLQVVGGGRYFPTNHTVQLQAVVRDGTNVSYSWTAWRDRGPALAGSGKGFSLTVLEAGTYHVQLRATNMLGSAWADCTMDFVEPVGWLMVAASPNPAAVNTSVTLSAELAGGSGVVYTWSLEEGLSWETSEPFTTHSFPT PGLHLVTMTAGNPLGSANATVEVDVQVPVSGLSIRASEPGGSFVAAGSSVPFWGQLATGTNVSWCWAVPGGSSKRGP HVTMVFPDAGTFSIRLNASNAVSWVSATYNLTAEEPIVGLVLWASSKVVAPGQLVHFQILLAAGSAVTFRLQVGGAN PEVLPGPRFSHSFPRVGDHVVSVRGKNHVSWAQAQVRIVVLEAVSGLQVPNCCEPGIATGTERNFTARVQRGSRVAY AWYFSLQKVQGDSLVILSGRDVTYTPVAAGLLEIQVRAFNALGSENRTLVLEVQDAVQYVALQSGPCFTNRSAQFEA ATSPSPRRVAYHWDFGDGSPGQDTDEPRAEHSYLRPGDYRVQVNASNLVSFFVAQATVTVQVLACREPEVDVVLPLQ VLMRRSQRNYLEAHVDLRDCVTYQTEYRWEVYRTASCQRPGRPARVALPGVDVSRPRLVLPRLALPVGHYCFVFVVS FGDTPLTQSIQANVTVAPERLVPIIEGGSYRVWSDTRDLVLDGSESYDPNLEDGDQTPLSFHWACVASTQREAGGCA LNFGPRGSSTVTIPRERLAAGVEYTFSLTVWKAGRKEEATNQTVLIRSGRVPIVSLECVSCKAQAVYEVSRSSYVYL EGRCLNCSSGSKRGRWAARTFSNKTLVLDETTTSTGSAGMRLVLRRGVLRDGEGYTFTLTVLGRSGEEEGCASIRLS PNRPPLGGSCRLFPLGAVHALTTKVHFECTGWHDAEDAGAPLVYALLLRRCRQGHCEEFCVYKGSLSSYGAVLPPGF RPHFEVGLAVVVQDQLGAAVVALNRSLAITLPEPNGSATGLTVWLHGLTASVLPGLLRQADPQHVIEYSLALVTVLNEYERALDVAAEPKHERQHRAQIRKNITETLVSLRVHTVDDIQQIAAALAQCMGPSRELVCRSCLKQTLHKLEAMMLI LQAETTAGTVTPTAIGDSILNITGDLIHLASSDVRAPQPSELGAESPSRMVASQAYNLTSALMRILMRSRVLNEEPL TLAGEEIVAQGKRSDPRSLLCYGGAPGPGCHFSIPEAFSGALANLSDVVQLIFLVDSNPFPFGYISNYTVSTKVASM AFQTQAGAQIPIERLASERAITVKVPNNSDWAARGHRSSANSANSVVVQPQASVGAVVTLDSSNPAAGLHLQLNYTL LDGHYLSEEPEPYLAVYLHSEPRPNEHNCSASRRIRPESLQGADHRPYTFFISPGSRDPAGSYHLNLSSHFRWSALQ VSVGLYTSLCQYFSEEDMVWRTEGLLPLEETSPRQAVCLTRHLTAFGASLFVPPSHVRFVFPEPTADVNYIVMLTCA VCLVTYMVMAAILHKLDQLDASRGRAIPFCGQRGRFKYEILVKTGWGRGSGTTAHVGIMLYGVDSRSGHRHLDGDRA FHRNSLDIFRIATPHSLGSVWKIRVWHDNKGLSPAWFLQHVIVRDLQTARSAFFLVNDWLSVETEANGGLVEKEVLA ASDAALLRFRRLLVAELQRGFFDKHIWLSIWDRPPRSRFTRIQRATCCVLLICLFLGANAVWYGAVGDSAYSTGHVS RLSPLSVDTVAVGLVSSVVVYPVYLAILFLFRMSRSKVAGSPSPTPAGQQVLDIDSCLDSSVLDSSFLTFSGLHAEA FVGQMKSDLFLDDSKSLVCWPSGEGTLSWPDLLSDPSIVGSNLRQLARGQAGHGLGPEEDGFSLASPYSPAKSFSAS DESCRIPTION Page 82 / 145 93 CN 121752299 A DEDLIQQVLAEGVSSPAPTQDTHMETDLLSSLSSTPGEKTETLALQRLGELGPPSPGLNWEQPQAARLSRTGLVEGLRKRLLPAWCASLAHGLSLLLVAVAVAVSGWVGASFPPGVSVAWLLSSSASFLASFLGWEPLKVLLEALYFSLVAKRL HPDEDDTLVESPAVTPVSARVPRVRPPHGFALFLAKEEARKVKRLHGMLRSLLVYMLFLLVTLLASYGDASCHGHAY RLQSAIKQELHSRAFLAITRSEELWPWMAHVLLPYVHGNQSSPELGPPRLRQVRLQEALYPDPPGPRVHTCSAAGGF STSDYDVGWESPHNGSGTWAYSAPDLLGAWSWGSCAVYDSGGYVQELGLSLEESRDRLRFLQLHNWLDNRSRAVFLE LTRYSPAVGLHAAVTLRLEFPAAGRALAALSVRPFALRRLSAGLSLPLLTSVCLLLFAVHFAVAEARTWHREGRWRV LRLGAWARWLLVALTAATALVRLAQLGAADRQWTRFVRGRPRRFTSFDQVAQLSSAARGLAASLLFLLLVKAAQQLR FVRQWSVFGKTLCRALPELLGVTLGLVVLGVAYAQLAILLVSSCVDSLWSVAQALLVLCPGTGLSTLCPAESWHLSP LLCVGLWALRLWGALRLGAVILRWRYHALRGELYRPAWEPQDYEMVELFLRRLRLWMGLSKVKEFRHKVRFEGMEPL PSRSSRGSKVSPDVPPPSAGSDASHPSTSSSQLDGLSVSLGRLGTRCEPEPSRLQAVFEALLTQFDRLNQATEDVYQ LEQQLHSLQGRRSSRAPAGSSRGPSPGLRPALPSRLARASRGVDLATGPSRTPLRAKNKVHPSST SEQ ID NO: 12, Polycystin-2 (PKD2), NCBI ref NP_000288.1, Homo sapiens, 968 aa MVN SSRVQPQQPGDAKRPPAPRAPDPGRLMAGCAAVGASLAAPGGLCEQRGLEIEMQRIRQAAARDPPAGAAASPSPPLS SCSRQAWSRDNPGFEAEEEEEEVEGEEGGMVVEMDVEWRPGSRRSAASSAVSSVGARSRGLGGYHGAGHPSGRRRRREDQGPPCPSPVGGGDPLHRHLPLEGQPPRVAWAERLVRGLRGLWGTRLMEESSTNREKYLKSVLRELVTYLLFLIVL CILTYGMMSSNVYYYTRMMSQLFLDTPVSKTEKTNFKTLSSMEDFWKFTEGSLLDGLYWKMQPSNQTEADNRSFIFY ENLLLGVPRIRQLRVRNGSCSIPQDLRDEIKECYDVYSVSSEDRAPFGPRNGTAWIYTSEKDLNGSSHWGIIATYSG AGYYLDLSRTREETAAQVASLKKNVWLDRGTRATFIDFSVYNANINLFCVVRLLVEFPATGGVIPSWQFQPLKLIRY VTTFDFFLAACEIIFCFFIFYYVVEEILEIRIHKLHYFRSFWNCLDVVIVVLSVVAIGINIYRTSNVEVLLQFLEDQ NTFPNFEHLAYWQIQFNNIAAVTVFFVWIKLFKFINFNRTMSQLSTTMSRCAKDLFGFAIMFFIIFLAYAQLAYLVF GTQVDDFSTFQECIFTQFRIILGDINFAEIEEANRVLGPIYFTTFVFFMFFILLNMFLAIINDTYSEVKSDLAQQKA EMELSDLIRKGYHKALVKLKLKNTVDDISESLRQGGGKLNFDELRQDLKGKGHTDAEIEAIFTKYDQDGDQELTEH EHQQMRDDLEKEREDLDLDHSSLPRPMSSRSFPRSLDDSEEDDDEDSGHSSRRRGSISSGVSYEEFQVLVRRVDRME HSIGSIVSKIDAVIVKLEIMERAKLKRREVLGRLLDGVAEDERLGRDSEIHREQMERLVREELERWESDDAASQISH In some embodiments, the transgene contains SEQ ID NO: 13 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 13 and remains similar when expressed as a protein.A nucleic acid sequence with the same function (e.g., a glucose residue cleaved by an immature glycoprotein), or a functional fragment thereof, or a codon-optimized version of a nucleic acid.

[0260] SEQ ID NO: 13, Glucosidase II α subunit (GANAB), also known as: G2AN, GIIA, PKD3, GLUII, GIIα; NCBI ref NM_001278192.2 position 15-2573, CCDS60818.1, human, 2559 nucleotides (nt) ATGGCGGCGGTAGCGGCAGTGGCGGCGCGTAGGAGGCGGCTTTCTGTCTCTCTGGTCGTGATGAGAACAGTGTGGAGTT AACCATGGCTGAGGGACCCTACAAGATCATCTTGACAGCACGGCCATTCCGCCTTGACCTACTAGAGGACCGAAGTC TTTTGCTTAGTGTCAATGCCCGAGGACTCTTGGAGTTTGAGCATCAGAGGGCCCCTAGGGTCTCTTTCTCGGATAAG GTTAATCTCACGCTTGGTAGCATATGGGATAAGATCAAGAACCTTTTCTCTAGGCAAGGATCAAAAGACCCAGCTGA GGGCGATGGGGCCCAGCCTGAGGAAACACCCAGGGATGGCGACAAGCCAGAGGAGACTCAGGGGAAGGCAGAGAAAG ATGAGCCAGGAGCCTGGGAGGAGACATTCAAAACTCACTCTGACAGCAAGCCGTATGGCCCCATGTCTGTGGGTTTG GACTTCTCTCTGCCAGGCATGGAGCATGTCTATGGGATCCCTGAGCATGCAGACAACCTGAGGCTGAAGGTCACTGA Instructions 83 / 145 Page 94 CN 121752299 A GGGTGGGGAGCCATATCGCCTCTACAATTTGGATGTGTTCCAGTATGAGCTGTACAACCCAATGGCCTTGTATGGGT CTGTGCCTGTGCTCCTGGCACACAACCCTCATCGCGACTTGGGCATCTTCTGGCTCAATGCTGCAGAGACCTGGGTT GATATATCTTCCAACACTGCCGGGAAGACCCTGTTTGGGAAGATGATGGACTACCTGCAGGGCTCTGGGGAGACCCCACAGACAGATGTTCGCTGGATGTCAGAGACTGGCATCATTGACGTCTTCCTGCTGCTGGGGCCCTCCATCTCTGATG TTTTCCGGCAATATGCTAGTCTCACAGGAACCCAGGCGTTGCCCCCACTCTTCTCCCTCGGCTACCACCAGAGCCGT TGGAACTACCGGGACGAGGCTGATGTGCTGGAAGTGGATCAGGGCTTTGATGATCACAACCTGCCCTGTGATGTCAT CTGGCTAGACATTGAACATGCTGATGGCAAGCGGTATTTCACCTGGGACCCCAGTCGCTTCCCTCAGCCCCGCACCA TGCTTGAGCGCTTGGCTTCTAAGAGGCGGAAGCTGGTGGCCATCGTAGACCCCCACATCAAGGTGGACTCCGGCTAC CGAGTTCACGAGGAGCTGCGGAACCTGGGGCTGTATGTTAAAACCCGGGATGGCTCTGACTATGAGGGCTGGTGCTG GCCAGGCTCAGCTGGTTACCCTGACTTCACTAATCCCACGATGAGGGCCTGGTGGGCTAACATGTTCAGCTATGACA ATTATGAGGGCTCAGCTCCCAACCTCTTTGTCTGGAATGACATGAACGAACCATCTGTGTTCAATGGTCCTGAGGTC ACCATGCTCAAGGATGCCCAGCATTATGGGGGCTGGGAGCACCGGGATGTGCATAACATCTATGGCCTTTATGTGCA CATGGCGACTGCTGATGGGCTGAGACAGCGCTCTGGGGGCATGGAACGCCCCTTTGTCCTGGCCAGGGCCTTCTTCG CTGGCTCCCAGCGCTTTGGAGCCGTGTGGACAGGGGACAACACTGCCGAGTGGGACCATTTGAAGATCTCTATTCCT ATGTGTCTCAGCTTGGGGCTGGTGGGACTTTCCTTCTGTGGGGCGGATGTGGGTGGCTTCTTCAAAAACCCAGAGCCAGAGCTGCTTGTGCGCTGGTACCAGATGGGTGCTTACCAGCCATTCTTCCGGGCACATGCCCACTTGGACACTGGGC GACGAGAGCCATGGCTGTTACCATCTCAGCACAATGATATAATCCGAGATGCCTTGGGCCAGCGATATTCTTTGCTG CCCTTCTGGTACACCCTCTTATATCAGGCCCATCGGGAAGGCATTCCTGTCATGAGGCCCCTGTGGGTGCAGTACCC TCAGGATGTGACTACCTTCAATATAGATGATCAGTACTTGCTTGGGGATGCGTTGCTGGTTCACCCTGTATCAGACT CTGGAGCCCATGGTGTCCAGGTCTATCTGCCTGGCCAAGGGGAGGTGTGGTATGACATTCAAAGCTACCAGAAGCAT CATGGTCCCCAGACCCTGTACCTGCCTGTAACTCTAAGCAGTATCCCTGTGTTCCAGCGTGGAGGGACAATCGTGCC In some embodiments, the transgene encodes a polypeptide comprising: SEQ ID NO: 14 or the same as SEQ ID NO: The sequence of 14 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 90%, at least 91%, at leastAn amino acid sequence or fragment thereof that is 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical and retains the same function (e.g., never cleaved glucose residues by an immature glycoprotein). SEQ ID NO: 14, Glucosidase II alpha subunit (GANAB), NCBI ref NP_001265121.1, human, 852 aa MAAVAAVAARRRRLSVSGRDENSVELTMAEGPYKIILTARPFRLDLLEDRSLLLSVNARGLLEFEHQRA PRVSFSDKVNLTLGSIWDKIKNLFSRQGSKDPAEGDGAQPEETPRDGDKPEETQGKAEKDEPGAWEETFKTHSDSKP YGPMSVGLDFSLPGMEHVYGIPEHADNLRLKVTEGGEPYRLYNLDVFQYELYNPMALYGSVPVLLAHNPHRDLGIFW LNAAETWVDISSNTAGKTLFGKMMDYLQGSGETPQTDVRWMSETGIIDVFLLLGPSISDVFRQYASLTGTQALPPLF SLGYHQSRWNYRDEADVLEVDQGFDDHNLPCDVIWLDIEHADGKRYFTWDPSRFPQPRTMLERLASKRRKLVAIVDP HIKVDSGYRVHEELRNLGLYVKTRDGSDYEGWCWPGSAGYPDFTNPTMRAWWANMFSYDNYEGSAPNLFVWNDMNEP SVFNGPEVTMLKDAQHYGGWEHRDVHNIYGLYVHMATADGLRQRSGGMERPFVLARAFFAGSQRFGAVWTGDNTAEW Instructions 84 / 145 Page 95 CN 121752299 A DHLKISIPMCLSLGLVGLSFCGADVGGFFKNPEPELLVRWYQMGAYQPFFRAHAHLDTGRREPWLLPSQHNDIIRDA LGQRYSLLPFWYTLLYQAHREGIPVMRPLWVQYPQDVTTFNIDDQYLLGDALLVHPVSDSGAHGVQVYLPGQGEVWY DIQSYQKHHGPQTLYLPVTLSSIPVFQRGGTIVPRWMRVRRSSECMKDDPITLFVALSPQGTAQGELFLDGHTFNYIn some embodiments, the transgene encodes a human protein. In some embodiments, the transgene encodes a mammalian protein. In some embodiments, the transgene encodes a protein from the same species as the subject. In some embodiments, the transgene encodes a protein from a different species than the subject.

[0262] In some embodiments, the transgene contains an inhibitor of a gene or protein such that when the expression of such a gene or protein is reduced, this reduction is effective in treating kidney-related conditions. In some embodiments, the transgene contains an inhibitor of a gene or protein selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD). In some embodiments, the inhibitor is a repressive nucleic acid. In some embodiments, the repressive nucleic acid is selected from: miRNA, siRNA, shRNA, RNAi, antisense oligonucleotides, crRNA, and gRNA. In some embodiments, the inhibitor is a protein that inhibits a gene or protein selected from the following: REN, SCNN1A, SCNN1B, and UMOD.

[0263] In some embodiments, the rAAV genome contains at least one transgene. In some embodiments, the rAAV genome contains 1, 2, 3, 4, 5, or more transgenes. Such multiple transgenes can be expressed using the same or different promoters. In some embodiments, the rAAV genome contains any combination of the following: (a) a reporter gene, (b) a gene that is effective in treating kidney-related conditions when expressed in a subject, and / or (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions. In some embodiments, the rAAV genome contains any combination of the following: (a) a reporter gene and (b) a gene that is effective in treating kidney-related conditions when expressed in a subject. In some embodiments, the rAAV genome contains any combination of the following: (b) a gene that is effective in treating kidney-related conditions when expressed in a subject and (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions. In some embodiments, the rAAV genome comprises any combination of the following: (a) a reporter gene and (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions. In some embodiments, the rAAV genome comprises any combination of the following: (a) a reporter gene, (b) a gene that is effective in treating kidney-related conditions when expressed in a subject, and (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions.

[0264] In some embodiments, the rAAV genome also contains at least one inverted terminal repeat (ITR) sequence. Typically, the ITR sequence is about 145 bp in length. Preferably, the rAAV genome uses a sequence that encodes essentially the entire ITR, but minor modifications to these sequences are permitted. The ability to modify these ITR sequences is within the scope of the art. (See, for example, texts such as Sambrook et al., "Molecular Cloning. A Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). One example of such a molecule used in this invention is a transgenic "cis-acting" plasmid in which the selected transgenic sequence and associated regulatory element are flanked by 5' and 3' AAV ITR sequences. The AAV ITR sequence can be obtained from any known AAV, including currently identified mammalian AAV types. In some embodiments, the isolated nucleic acid (e.g., an rAAV vector) comprises at least one ITR having a serotype selected from AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh8, AAVrh10, and variants thereof. In some embodiments, the isolated nucleic acid comprises a region encoding an AAV2 ITR (e.g., a first region). In some embodiments, the isolated nucleic acid comprises a region encoding an AAV9 ITR (e.g., a first region).

[0265] In some embodiments, the isolated nucleic acid further comprises a region containing a second AAV ITR (e.g., a second region, a third region, a fourth region, etc.). In some embodiments, the second AAV ITR has a serotype selected from AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh8, AAVrh10, and variants thereof. In some embodiments, the second ITR is a mutant ITR lacking a functional terminal cleavage site (TRS). The term "lacking a terminal cleavage site" can refer to an AAV ITR containing a mutation that negates the function of the terminal cleavage site (TRS) of the ITR (e.g., a sense mutation such as a nonsynonymous mutation or a missense mutation), or a truncated AAV ITR lacking a nucleic acid sequence encoding a functional TRS (e.g., ATRS).ITR). Not wishing to be bound by any particular theory, rAAV vectors containing ITRs lacking functional TRS are derived from complementary rAAV vectors, such as those described in McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0266] Kidney-related conditions In some embodiments, the methods described herein relate to treating subjects who have or are diagnosed with kidney-related conditions. In some aspects, methods of treating kidney-related conditions in subjects in need of this treatment include administering recombinant adeno-associated virus (rAAV) to the subject by performing a retrograde ureteral administration method as further described herein. Subjects with kidney-related conditions can be identified by a physician using existing methods for diagnosing kidney-related conditions. Symptoms and / or complications that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, nausea and vomiting, muscle cramps, loss of appetite, swelling of the ankles, dry and / or itchy skin, shortness of breath, difficulty sleeping, excessive or insufficient urination, metallic taste in the mouth, coldness, etc. Tests that may aid in the diagnosis of kidney-related conditions include, but are not limited to, blood tests (e.g., eGFR; serum creatinine; blood urea nitrogen (BUN); urine tests; renal ultrasound; renal biopsy; etc.). A family history of kidney-related conditions or risk factors for exposure to kidney-related conditions (e.g., diabetes; hypertension; cardiovascular disease; smoking; obesity; Black, Native American, or Asian American; renal structural abnormalities) may also help determine whether a subject is likely to have a kidney-related condition or to make a diagnosis of kidney-related condition.

[0267] In some embodiments, kidney-related conditions are selected from: autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome; autosomal dominant tubulointerstitial nephropathy (ADTKD); medullary cystic nephropathy; nephronial tuberculosis; Bart syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; thin basement membrane nephropathy; cystinuria; Liddle syndrome; papillary kidney syndrome; and cystin storage disorders; see, for example, Rubin et al., 2020, “Improving molecular therapy in the kidney,” Mol Diagn Ther. 24(4): 375–396, the contents of which are incorporated herein by reference in their entirety.

[0268] In some embodiments, the kidney-related conditions are selected from: episodic mineralocorticoid hyperplasia syndrome, autosomal dominant hypocalcemia, autosomal dominant hypomagnesemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia.Adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, type A cystinuria, type B cystinuria, type 1 Dent disease, type 2 Dent disease / Lowe syndrome, dicarboxyamino aciduria, distal RTA, EAST / SeSAME syndrome, Fanconi-Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2, Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Gitelman syndrome, glucocorticoid-suppressible aldosteronism. Hartnup syndrome, hereditary hypophosphatemic rickets with hypercalciuria, HNF1B-related nephropathy, BH4 deficiency hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 4 hypomagnesemia, type 5 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, hypomagnesemia, seizures and intellectual disability type 1, hypomagnesemia, seizures and intellectual disability type 2, iminoglycineuria, type 2 Kenny-Caffey syndrome, Liddle syndrome, lysineuria protein intolerance (instruction manual 86 / 145 pages 97 CN 121752299 A), type 2 neonatal inflammatory skin and intestinal diseases, nephrogenic diabetes insipidus, nephrogenic antidiuretic hormone secretion disorder syndrome, Type 1 pseudoketoalbuminemia, Type 1A pseudoketoalbuminemia, Type 2b pseudoketoalbuminemia, Type 2c pseudoketoalbuminemia, Type 2d pseudoketoalbuminemia, Type 2e pseudoketoalbuminemia, Type 3 renal tubular acidosis, and X-linked hypophosphatemic rickets, see, for example, Downie et al., 2020, “Inherited tubulopathies of the kidney,” Clin J Am Soc Nephrol. 16(4): 620-630, the contents of which are incorporated herein by reference in their entirety.

[0269] In some embodiments, the methods described herein include administering to a subject an effective amount of the composition described herein, such as a solution or pharmaceutical composition containing rAAV, to relieve symptoms of kidney-related conditions. As used herein, “relieving symptoms of kidney-related conditions” means improving any condition or symptom associated with kidney-related conditions. Compared to an equivalent untreated control, such reduction is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique.

[0270] For example, for cystinuria, the subject's symptoms of cystinuria can be evaluated (e.g., crystalluria (crystallization in urine); aminoaciduria (abnormally high levels of amino acids (e.g., cystine) in urine); flank or back pain (e.g.,The incidence of symptoms typically unilateral (such as pain), painful urination, hematuria, severe pain in the flank or back, pain in the groin, pelvis, or near the abdomen, nausea and vomiting, flank pain, back pain, recurrent abdominal pain, recurrent urinary tract infection, and / or fever may be reduced, any of which could indicate that the rAAV transduction efficiency in the kidneys is sufficient to treat cystinuria.

[0271] As another example, for polycystic kidney disease (e.g., ADPKD), the incidence of PKD symptoms (e.g., abdominal pain or tenderness, hematuria, nocturia, unilateral or bilateral flank pain, somnolence, arthralgia, nail abnormalities, hypertension, back or flank pain, abdominal distension, hepatomegaly, heart murmurs, and / or kidney or abdominal masses) may be evaluated as reduced, any of which could indicate that the rAAV transduction efficiency in the kidneys is sufficient to treat polycystic kidney disease (e.g., ADPKD).

[0272] As used herein, the term “effective amount” refers to the amount of a solution or pharmaceutical composition containing rAAV required to improve or alleviate at least one or more symptoms of kidney-related disease, and relates to an amount of solution or pharmaceutical composition sufficient to provide the desired effect. Therefore, the terms “therapeutic effective amount” or “pharmaceutical effective amount” refer to an amount of a solution or pharmaceutical composition containing rAAV sufficient to provide a specific anti-kidney-related disease effect when administered to a typical subject. As used herein, in various cases, an effective amount will also include an amount sufficient to delay the onset of disease symptoms, alter the course of symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. Therefore, specifying an exact “effective amount” is generally not feasible. However, for any given situation, a person skilled in the art can determine an appropriate “effective amount” using only routine experiments.

[0273] Effective amounts, toxicity, and efficacy can be determined by standard pharmaceutical procedures in cell culture or laboratory animals, for example, to determine the minimum effective dose and / or maximum tolerated dose. Dosages can vary depending on the dosage form and administration regimen employed. Therapeutic effective doses can be estimated first from cell culture assays. Alternatively, dosages can be formulated in animal models to achieve a dosage range between the minimum effective dose and the maximum tolerated dose. The effect of any specific dose can be monitored by appropriate bioassays. The dosage can be determined by a physician and adjusted as needed to accommodate observed therapeutic effects.

[0274] In some embodiments, the subject is a human. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a pig, which is a relevant animal model for human translational research due to its anatomical and physiological similarities to humans. In some embodiments, the subject is any animal requiring treatment for kidney-related conditions.

[0275] In some embodiments, when rAAV transduces cells in the kidney, resulting in kidney cells expressing an effective therapeutic effect...Kidney-related conditions are treated when there are inhibitors of transgenic and / or gene or protein in the kidney. To transduce cells, an rAAV vector enters a kidney cell and delivers its single-stranded DNA genome to the cell nucleus, whereby the genome is converted into a double strand, then transcribed and integrated into the subject's cellular genome. RAAV transduction in kidney cells can be detected by a variety of methods, including but not limited to sequencing of the rAAV genome (or barcodes therein) or expression of the reporter gene or transgene delivered by rAAV (e.g., RNA- or protein-based assays such as RT-qPCR, ELISA, histological staining, or flow cytometry). In some embodiments, rAAV transduction can be detected using anti-rAAV serology (e.g., in urine or blood) or the occurrence of symptoms associated with kidney-related conditions (to which rAAV has been administered for treatment).

[0276] In some embodiments, rAAV transduces nephrons in the kidney. In some embodiments, rAAV transduction is selected from the following nephron portions: the glomerulus, Bowman's capsule, proximal convoluted tubule, loop of Henry, or distal convoluted tubule of the kidney. In some embodiments, rAAV transduces the proximal convoluted tubule of the kidney. In some embodiments, rAAV transduces the collecting duct of the kidney. In some embodiments, rAAV transduces both the proximal convoluted tubule and the collecting duct of the kidney. In some embodiments, rAAV transduction is selected from the following renal tubules: the glomerulus (e.g., Bowman's capsule), proximal tubule (PT), thin descending limb of the loop of Henry (DL), thin ascending limb of the loop of Henry (AL), thick ascending limb of the loop of Henry (TALH), macula densa (MD), distal convoluted tubule (DCT), connecting tubule (CNT), or collecting duct (CT).

[0277] In some embodiments, rAAV transduction is selected from the following renal cells: vascular cells (e.g., glomerular endothelial cells); mesangial, smooth muscle cells (SMCs), or juxtaglomerular cells (JGs) (e.g., mesangial cells, SMCs, pericytes, and / or JGs); podocytes (e.g., adult podocytes and / or podocyte progenitor cells); proximal tubule (PT) cells (e.g., Pan-PT, proximal convoluted tubule, proximal straight tubule, PT progenitor cells, and / or damaged PT); loop of Henry (LOH), macula densa (MD) cells (e.g., the descending limb of the LOH, LOH...). The ascending limb of the lateral limb, the thick ascending limb of the LOH, and / or the plaque dense (MD)); distal convoluted tubule (DCT) or connecting tubule (CNT) cells (e.g., DCT1, DCT2, and / or CNT); and / or collecting duct (CD) cells (e.g., CD chief cells, Pan-CD intercalation cells, CD intercalation cells (type A), CD intercalation cells (type B), and / or CD transition cells).

[0278] In some embodiments, rAAV transduction is selected from the following renal cells: glomerular endothelial cells, mesangial cells, SMCs, periglomerular cells, and peripheral cells.Cells, JG, adult podocytes, podocyte progenitor cells, Pan-PT, proximal convoluted tubules, proximal straight tubules, PT progenitor cells, damaged PT, descending limb of LOH, ascending limb of LOH, thick ascending limb of LOH, dense plaque (MD), DCT1, DCT2, CNT, CD principal cells, Pan-CD intercalation cells, CD intercalation cells (type A), CD intercalation cells (type B) and / or CD transition cells.

[0279] In some embodiments, rAAV transduction is selected from the following kidney cells: vascular cells, including but not limited to glomerular endothelial cells (non-limiting examples of glomerular endothelial-specific marker genes include Plat, Emcn, Tsapn7, Mapt, Kdr, Smad6, Ehd3, Lpl, Flt1, Fbln2, Mgp, Trpv4, Bmx); mesangial / smooth muscle cells (SMC) / juxtaglomerular cells (JG), including but not limited to mesangial cells (non-limiting examples of mesangial cell-specific marker genes include Serpine2, Fhl2, Des, Prkca, Art3, Nt5e, Pdgfrb), SMC (Non-restricted examples of SMC-specific marker genes include Tagln, Myh11, Acta2, Gata3, Rergl, Map3k7c1), pericytes (non-restricted examples of pericyte-specific marker genes include Vim, Tagln, Myh11, Pdgfrb) and / or JG (non-restricted examples of JG-specific marker genes include Ren1, Akr1b7, Rgs5); podocytes, including but not limited to adult podocytes (non-restricted examples of adult podocyte-specific marker genes include Nphs1, Nphs2, Synpo, Cdkn1c, Wt1) and / or podocyte progenitor cells (non-restricted examples of podocyte progenitor-specific marker genes include Wt1, Foxc2, Mafb, Efnb2, Foxl1); proximal tubule (PT) cells, including but not limited to Pan-PT (non-restricted examples of Pan-PT-specific marker genes include Slc34a1, Lrp2, ... Hxyd2, Hrsp12, Acsm1, Acsm2, Cpt1a, Acox3, Slc26a6, Slc9a3, Glud1, Pck1, Aqp8, Hnf4a, Ppara), proximal convoluted tubules (non-restricted examples of proximal convoluted tubule-specific marker genes include Slc5a2, Slc5a12, Adra1a, Slc6a19, Slc7a8, Slc7a9), proximal straight tubules (non-restricted examples of proximal straight tubule-specific marker genes include Atp11a, Slc13a3, Slc16a9, Slc27a2, Slc7a13, S1c22a6 (S2 segment), Slc1a1), PT progenitor cells(Non-restricted examples of PT progenitor cell-specific marker genes include Notch2, Lgr4) and / or damaged PT (non-restricted examples of damaged PT-specific marker genes include Havcr1, Krt20, Hspa1a, Vcam1, Dcdc2a, Sema5a); Henry's specification 88 / 145 pages 99 CN 121752299 A Loop (LOH) / dense plaque (MD) cells, including but not limited to the descending limb of LOH (non-restricted examples of LOH descending limb-specific marker genes include Fst, Aqp1, Slc14a2, Bst1, Epha7, Cryab, Tshz2, Cald1, Bst1, Lypd2), and the ascending limb of LOH (non-restricted examples of LOH ascending limb-specific marker genes include Epha7, Mx2, Clcnka), the thick ascending limb of LOH (non-restricted examples of LOH thick ascending limb specific marker genes include Slc12a1, Umod, Tmem207, Foxq1, Cldn10, Ptger3, Kcnj1, Enox1, Thsd4, Mt2, Slc5a3) and / or plaques dense (MD) (non-restricted examples of MD specific marker genes include Enox1, Thsd4, Nos1, Avpr1a); distal convoluted tubule (DCT) / connecting tubule (CNT) cells, including but not limited to DCT1 (non-restricted examples of DCT1 specific marker genes include Pvalb, Slc12a3, Trpm7, Wnk1, Wnk4, Stk39, Calb1, Slc8a1, Egf, Trpm6, Cnnm2, Atp1a1, Atp1a2, Atp1a3, Atp1a4, Fxyd2), DCT2 (non-restricted examples of DCT2-specific marker genes include Slc12a3, Trpm7, Wnk1, Wnk4, Klhl3, Stk39, Calb1, Slc8a1, Egf, Trpm6, Cnnm2, Atp1a1, Atp1a2, Atp1a3, Atp1a4, Klk1, Trpv5, Trpm6, S100g, Atp2b1, Atp2b4, Scnn1b, Scnn1g, Kcne1, Fxyd2) and / or CNT (non-restricted examples of CNT-specific marker genes include Calb1, Slc8a1, Egf, Klk1, Trpv5, Trpm6, S100g, Atp2b1, Scann1b, Scann1g, Kcne7); and / or collecting duct (CD) cells, including but not limited to CD chief cells (non-restrictive examples of CD chief cell-specific marker genes include Scann1b, Scann1g, Aqp2, ...Avpr2, Hsd11b2, Rhbg, Elf5, Fxyd4, Aqp3, Apela, Kcne7, Npnt, Kcnj10), Pan-CD intercalation cells (non-restricted examples of pan-CD intercalation cell-specific marker genes include Tcfcp2l1, Foxi, Atp6v1g3, Atp6v0d2, Insr, Atp6v1b1), CD intercalation cells (type A) (non-restricted examples of CD intercalation cell (type A) specific marker genes include Atp4a, Slc4a1, Aqp6, Kit, Adgrf5, Mme), CD intercalation cells (type B) (CD intercalation cells (type B) Non-restricted examples of type-specific marker genes include Slc26a4, Hmx2, Spink8) and / or CD transition cells (non-restricted examples of CD transition cell-specific marker genes include Agp2, Hsd11b2, Rhbg, Atp6v1g3, Atp6v0d2, Insr, Atp6v1b1, Atp6v1b1, Parm1, Sec23b). See, for example, Balzer, “How Many Cell Types are in the Kidney and What Do they Do? Annu Rev Physiol. 10 Feb 2022; 84: 507–531; the contents of which are incorporated herein by reference in their entirety.

[0280] In some embodiments, the genome of rAAV contains a kidney-specific promoter that can be operatively linked to at least one transgene. As used herein, the term “kidney-specific promoter” refers to a promoter that preferentially activates in kidney cells and / or causes an increase in gene expression levels in kidney cells compared to cells and tissues outside the kidney. In some embodiments, kidney-specific promoter activation is increased by at least about 10%, at least about 20%, or at least about 30%, or at least about 40% in kidney cells compared to non-kidney cells (e.g., liver, heart, muscle, brain, lung, eye, joint cells, etc.). Or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or an increase of up to and including 100% or any increase between 10 and 100%, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times or at least about 10 times, or any increase between 2 and 10 times, or any increase between 10 and 100 times or greater. In some embodiments, the kidney-specific promoter is operatively linked to at least one transgene, such as (a) a reporter gene, (b) a gene effective in treating kidney-related conditions when expressed in a subject, and / or (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions.

[0281] In some embodiments, the rAAV genome contains a promoter (and / or other regulatory elements, such as enhancers) specific to the proximal convoluted tubule. In some embodiments, the rAAV genome contains a promoter specific to the collecting duct. In some embodiments, the rAAV genome contains promoters specific to both the proximal convoluted tubule and the collecting duct.

[0282] In some embodiments, a kidney-specific promoter is used for genes that are naturally and specifically expressed in kidney cells or tissues. In some embodiments, the kidney-specific promoter is selected from: the kidney-specific cadherin (KSPC) gene promoter; the Na+ / glucose cotransporter (SGLT2) gene promoter; the sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and the E-cadherin (ECAD) gene promoter. The KSPC gene promoter specifically activates expression throughout the nephron. The SGLT2 gene promoter specifically activates expression in the S1 and S2 segments of the proximal tubule. The NKCC2 gene promoter specifically activates expression in the thick ascending limb of the loop of Henry (TALH). The ECAD gene promoter specifically activates expression in the collecting duct (CD). See, for example, Asico et al., “Nephron segment-specific gene expression using AAV vectors,” Biochem Biophys Res Commun. 26 Feb 2018, 497(1): 19–24; the contents of which are incorporated herein by reference in their entirety.

[0283] In some embodiments, the kidney-specific promoter is a synthetic promoter. In some embodiments, the synthetic promoter comprises a portion of at least one natural kidney-specific promoter (e.g., the KSPC, SGLT2, NKCC2, or ECAD gene promoter). In some embodiments, the synthetic promoter comprises a synthetic sequence. In some embodiments, the synthetic promoter comprises a portion of at least one natural kidney-specific promoter (e.g., the KSPC, SGLT2, NKCC2, or ECAD gene promoter) and a synthetic sequence. Such synthetic promoter sequences can be designed to be selectively active in the proximal tubules, distal convoluted tubules, and / or collecting ducts of the kidney. Promoter sequences can be tested in vitro for specific activation (e.g., reporter protein expression) in kidney cells, for example, using kidney cells (e.g., 293 cells, proximal tubular epithelial cells (PTEC cells), Madin-Darby canine kidney (MDCK) cells, primary kidney cells, etc.) versus non-kidney cells (e.g., liver, heart, muscle, brain, lung, eye, joint cells, etc.). Promoter sequences that show increased activation in kidney cells compared to non-kidney cells can be tested in vitro.Further screening is conducted in animal models such as mice, pigs, or non-human primates. Specific activation of the promoter sequence in vivo can be evaluated using co-staining of tissue markers and reporter genes (e.g., histology, flow cytometry, etc.).

[0284] In some embodiments, the rAAV genome contains a ubiquitous promoter that can be operatively linked to at least one transgene. As used herein, the term "ubiquitous promoter" refers to a promoter that is activated in all cells of the subject, including kidney cells and non-kidney cells. In some embodiments, the ubiquitous promoter is selected from: cytomegalovirus (CMV), β-actin (β-Act), chicken β-actin promoter (CAG), elongation factor-1α (EF1), early growth response 1 (EGR1), eukaryotic initiation factor 4A1 (eIF4A1), ferritin heavy chain (FerH), ferritin light chain (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose regulatory protein 78 (GRP78), glucose regulatory protein 94 (GRP94), heat shock protein 70 (HSP70), β-kinin (β-Kin), phosphoglycerate kinase 1 (PGK-1), Rosa26, or ubiquitin B promoter, or combinations thereof.

[0285] As used herein, when a coding sequence (e.g., at least one transgenic) and a regulatory sequence (e.g., a kidney-specific promoter) are covalently linked in a manner that brings the expression or transcription of the coding sequence under the influence or control of the regulatory sequence, they are described as “operably” or “effectively” linked or conjugated. If it is desired to translate a coding sequence into a functional protein, then the two DNA sequences are described as operatively conjugable if inducing a promoter in the 5′ regulatory sequence results in transcription of the coding sequence and if the nature of the connection between the two DNA sequences does not (1) lead to the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, if a promoter region is capable of influencing the transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide, then the promoter region is operatively conjugable to the coding sequence.

[0286] When a nucleic acid molecule encoding any polypeptide described herein is expressed in a cell, a variety of transcriptional control sequences (e.g., promoter / enhancer sequences) can be used to direct its expression. A promoter can be a natural promoter, i.e., a promoter of a gene in its endogenous environment that provides normal regulation of gene expression. In some embodiments, a promoter can be constitutive, i.e., an unregulated promoter that allows for continuous transcription of its associated gene. A variety of conditional promoters, such as promoters controlled by the presence or absence of a molecule, can also be used. Instruction manual, pages 90 / 145, 101 CN 121752299 A

[0287] The precise nature of the regulatory sequences required for gene expression can vary by species or cell type, but generally may include, as needed, initiation 5′ non-transcriptional sequences and 5′ non-translational sequences, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5′ non-transcriptional regulatory sequences will include promoter regions that include promoter sequences for transcriptional control of the gene for operatively binding. Regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. Vectors of the present invention may optionally include 5′ leader or signal sequences. The selection and design of suitable vectors is within the competence and judgment of those skilled in the art.

[0288] In some embodiments, one or more recombinantly expressed transgenes may be integrated into the genome of a cell. Such genomic integration may allow for stable expression of nucleic acids or proteins effective for the treatment of kidney-related conditions.

[0289] Subsequent administration methods described herein are methods for subsequent administration or re-administration of rAAV (containing the same or different serotype capsids) after a period of time. Such re-administration is possible because at least the first administration does not elicit an immune response against rAAV in the kidneys.

[0290] In some embodiments, the subject's circulating serum does not neutralize rAAV after administration or re-administration. In some embodiments, antibodies neutralizing the administered rAAV are present in the subject's circulating serum. In some embodiments, circulating serum antibodies do not neutralize rAAV in the kidneys after administration. In some embodiments, the subject is seropositive for rAAV prior to administration of the solution containing rAAV. In some embodiments, neutralizing antibodies are not present in renal fluid and / or urine even if they circulate in the serum. In some embodiments, subsequent administration of rAAV as described herein can be performed without causing a substantial inflammatory response in the kidneys. In some embodiments, subsequent administration is performed at least one day later. In some embodiments, subsequent administration is performed at least one month later.

[0291] In one aspect, this document describes a method of treating kidney disease in a subject with such need, the method comprising: administering a first recombinant adeno-associated virus (rAAV) encoding a transgene that has a therapeutic effect on kidney disease to the kidney of the subject; and, following the administration of the first rAAV, administering a second rAAV encoding the transgene or a different transgene that has a therapeutic effect on kidney disease to the kidney of the subject or a different kidney. In some embodiments, the first rAAV and the second rAAV are cross-serologically reactive. As used herein, the term "cross-serologically reactive" refers to rAAV that binds to the same antibody. In some embodiments, the subject does not elicit a significant immune response to the second rAAV in the kidney. As used herein, the term "significant immune response" refers to an antibody that causes a reduction in the efficacy or transduction of the first and / or second rAAV.Immune response to the second rAAV.

[0292] In one aspect, this document describes a method of treating kidney disease in a subject who is seropositive for a recombinant adeno-associated virus (rAAV) therapeutic agent, the method comprising: administering to the kidney of the subject a transgenic rAAV therapeutic agent encoding a therapeutic effect on kidney disease. In some embodiments, the subject does not elicit a significantly adverse immune response to the rAAV therapeutic agent in the kidney.

[0293] In some embodiments, the first and second rAAVs are administered by a method comprising: guiding a catheter through the subject's urethra, bladder, and ureter; and administering a solution containing the first or second rAAV to the renal pelvis of the kidney in an amount of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight).

[0294] In some embodiments, the first and second rAAVs are administered by a method comprising: (a) occluding a renal vessel selected from the renal artery, renal vein, and combinations thereof; (b) administering a volume of a solution containing the first or second rAAV into the ureter of the said kidney or a second ureter of a different kidney via a retrograde route, wherein the volume is from about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject’s weight; and (c) unoccluding the renal vessel after a period of about 10 minutes to about 60 minutes following occlusion and / or administration of the solution containing the first and / or second rAAVs. In some embodiments, the first and second rAAVs are administered by a method comprising: (a) isolating the kidney from the systemic circulation; (b) administering a volume of a solution containing the first or second rAAV into the ureter of said kidney or a second ureter of a different kidney via a retrograde route, wherein said volume is from about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject’s weight; and (c) reintroducing the kidney into the systemic circulation after a period of about 10 minutes to about 60 minutes following isolation.

[0296] In some embodiments, at least one solution containing the first rAAV and / or the second rAAV (and / or the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more rAAVs) is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O. In some embodiments, at least one rAAV comprising a first rAAV and / or a second rAAV (and / or a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth or more rAAVs) is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.Solution. In some embodiments, at least one solution containing a first rAAV and / or a second rAAV (and / or a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more rAAV) is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 41 cm H2O. In some embodiments, at least one solution containing a first rAAV and / or a second rAAV (and / or a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more rAAV) is administered to the kidney at an intrarenal pressure of about 41 cm H2O to about 68 cm H2O. In some embodiments, at an intrarenal pressure of about 68 cm H2O to about 80 cm H2O, at least one solution containing a first rAAV and / or a second rAAV (and / or a third rAAV, a fourth rAAV, a fifth rAAV, a sixth rAAV, a seventh rAAV, an eighth rAAV, a ninth rAAV, a tenth rAAV or more) is administered to the kidney.

[0297] In some embodiments, a solution containing at least one rAAV is administered to the kidney at an intrarenal pressure of at least 20 cm H2O, at least 25 cm H2O, at least 30 cm H2O, at least 35 cm H2O, at least 40 cm H2O, at least 45 cm H2O, at least 50 cm H2O, at least 55 cm H2O, at least 60 cm H2O, at least 65 cm H2O, at least 70 cm H2O, or at least 75 cm H2O. In some embodiments, a first rAAV and / or a second rAAV (and / or a third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more) is administered to the kidney at intrarenal pressures of approximately 25 cm H2O, 30 cm H2O, 35 cm H2O, 40 cm H2O, 45 cm H2O, 50 cm H2O, 55 cm H2O, 60 cm H2O, 65 cm H2O, 70 cm H2O, 75 cm H2O, or 80 cm H2O. In some embodiments, the first rAAV and / or a second rAAV (and / / a third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more) is administered to the kidney at intrarenal pressures of approximately 20–30 cm H2O, 25–35 cm H2O, 30–40 cm H2O, 35–45 cm H2O, 40–50 cm H2O, or 45–55 cm H2O.The solution containing at least one rAAV is administered to the kidney under intrarenal pressure ranging from 50-60 cm H2O, 55-65 cm H2O, 60-70 cm H2O, 65-75 cm H2O, 70-80 cm H2O, 20-40 cm H2O, 20-50 cm H2O, 20-60 cm H2O, 20-70 cm H2O, 20-80 cm H2O, 25-85 cm H2O, 30-80 cm H2O, 40-80 cm H2O, 50-80 cm H2O, or 60-80 cm H2O. It should be understood that each of the individual intrarenal pressures described herein can be used to define the lower and upper limits of the intrarenal pressure range.

[0298] In some embodiments, the administration method results in at least about 15% of the nephrons in the kidney being transduced by the first and / or second rAAV. In some embodiments, the method results in about 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more of the nephrons in the kidney being transduced by the first and / or second rAAV.

[0299] In one aspect, this document describes a method for transducing at least about 15% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: (a) occluding renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) administering a first solution containing a first rAAV into the ureter of the kidney via a retrograde route, wherein the amount of the first solution is about 0.13 mL / kg to about 0.33 mL / kg, where kg is the weight of the subject; (c) unoccluding the renal vessels after a first occlusion period of about 10 minutes to about 60 minutes following the first occlusion; and (d) administering a second solution containing a first rAAV or a second rAAV by repeating steps (a)-(c), wherein the first occlusion period or the second occlusion period of about 10 minutes to about 60 minutes following the second occlusion is used after a subsequent administration period, wherein the amount of the second solution is about 0.13 mL / kg to about 0.33 mL / kg. In some embodiments, neither the first nor the second solution triggers an immune response in the kidneys.

[0300] In one aspect, this document describes a method for administering recombinant adeno-associated virus (rAAV) to the kidneys of a subject and / or for treating kidney-related conditions in subjects in need of such administration, comprising: (a) blocking at least one renal vessel selected from the renal artery, renal vein, and combinations thereof; (b) administering a first solution containing a first rAAV into the ureter of the kidney via a retrograde route; (c) unblocking the at least one renal vessel after a first blocking period following the first blocking; and (d) subsequently administering a second solution containing either the first or second rAAV by repeating steps (a)–(c) after a certain period. In some embodiments, neither the first nor the second solution elicits an immune response in the kidney.

[0301] In one aspect, this document describes a method for transducing at least about 25% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: (a) blocking renal vessels of the kidney selected from renal arteries, renal veins, and combinations thereof; (b) administering a first solution containing a first rAAV into the ureter of the kidney via a retrograde route, wherein the amount of the first solution is about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; (c) unblocking the renal vessels after a first blocking period of about 10 minutes to about 60 minutes following the first blocking; and (d) administering a second solution containing a first rAAV or a second rAAV by repeating steps (a)-(c), wherein the first blocking period or the second blocking period of about 10 minutes to about 60 minutes following the second blocking is used after a subsequent administration period, wherein the amount of the second solution is about 0.13 mL / kg to about 0.33 mL / kg. In some embodiments, neither the first solution nor the second solution induces an immune response in the kidney.

[0302] In some embodiments of any aspect, the steps of “closing at least one renal vessel selected from the renal artery, renal vein, and combinations thereof” or “closing the renal vessel selected from the renal artery, renal vein, and combinations thereof” or “closing the renal vessel selected from the renal artery, renal vein, and combinations thereof” are replaced by the step of “isolating the kidney from the systemic circulation.”

[0303] In some embodiments of any aspect, the steps of “unclosing the renal vessel after a first closure period” or “unclosing the at least one renal vessel after a first closure period” are replaced by the step of “reintegrating the kidney into the systemic circulation after a first isolation period” after the first isolation.

[0304] In some embodiments of any aspect, “first closure period” is replaced by “first isolation period”. Both the first closure period and the first isolation period (whichever is implied) are measured from the moment of closure or isolation. In some embodiments of any aspect, “second closure period” is replaced by “second isolation period”. In some embodiments of any aspect, the firstrAAV administration includes occluding at least one renal vessel, while the second rAAV administration includes isolating the kidney from the systemic circulation. In some embodiments of any aspect, the second rAAV administration includes occluding at least one renal vessel, while the first rAAV administration includes isolating the kidney from the systemic circulation. In some embodiments of any aspect, the first and second rAAV administrations include occluding at least one renal vessel. In some embodiments of any aspect, the first and second rAAV administrations include isolating the kidney from the systemic circulation.

[0305] In some embodiments, steps (a)–(c) are repeated using a first rAAV, a second rAAV, a third rAAV, a fourth rAAV, a fifth rAAV, a sixth rAAV, a seventh rAAV, an eighth rAAV, a ninth rAAV, a tenth rAAV, or more. In some embodiments, steps (a)–(c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more, for example, using the same or different rAAVs as described in CN 121752299 A, page 93 / 145, or combinations thereof.

[0306] In some embodiments, the capsid of the first rAAV has the same serotype as the capsid of the second rAAV. In some embodiments, the capsid of the first rAAV has a different serotype than the capsid of the second rAAV. In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from the capsids described in Table 1. In other implementations, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAVrh74, AAVrh10, po1, AAV9-PHP.B, AAV9-PHP.eB, AAVLK03, AAVAnc80L65, AAVDJ, AAV1A6ii, AAV1P5ii, AAV4A1ii, AAV7P4i, AAV9A1i, AAV9A2i, AAV9A6i, AAV9P1i, AAV9P2i, AAV9P5i, AAVrh10A1i, AAVrh10A2i, AAVrh10P1i, AAV12P2ii, AAVS10P1i, AAV JEA, AAV2 3xA P2i, AAVDJ P2i, AAV2i8, AAV2G9, AAV2.5, AAV4E, and AAV4A. It should be understood that the capsids may all be the same, all be different, or some may be the same and some may be different.

[0307] In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from the capsids described in Table 1. In other implementations, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from: AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV2.5G9, AAV2.5, AAVrh8, AAVrh10, AAVrh74, AAV10, AAV11, and AAVDJ. It should be understood that the capsids may all be the same, all be different, or some may be the same while some are different.

[0308] In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from the capsids described in Table 1. In other embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from: AAV2, AAV6, AAVLK03, AAVDJ, AAV9A2i, AAV9A6i, AAVrh10A2i, AAV2g9, and AAV2.5 (see, for example, Figure 4). In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are independently selected from: AAV2, AAVDJ, AAVJEA (minimal), AAV2g9, or AAV2.5 (see, for example, Figure 5). It should be understood that the capsids may all be the same, all be different, or some may be the same and some may be different.

[0309] In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are reasonable polyploids.

[0310] In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, sixth rAAV, and more capsids are reasonable polyploids.The first rAAV, the second rAAV, the third rAAV, the fourth rAAV, the fifth rAAV, the sixth rAAV, the seventh rAAV, the eighth rAAV, the ninth rAAV, the tenth rAAV, or more capsids are independently selected from: AAV2G9, AAV2.5, AAVDJ, and AAV2.

[0311] In some embodiments, the first rAAV, the second rAAV, the third rAAV, the fourth rAAV, the fifth rAAV, the sixth rAAV, the seventh rAAV, the eighth rAAV, the ninth rAAV, the tenth rAAV, or more capsids are AAV2G9. In some embodiments, the first rAAV, the second rAAV, the third rAAV, the fourth rAAV, the fifth rAAV, the sixth rAAV, the seventh rAAV, the eighth rAAV, the ninth rAAV, the tenth rAAV, or more capsids are AAV2.5. In some implementations, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are AAVDJ. In some implementations, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more capsids are AAV2.

[0312] In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth, CN 121752299 A, specification 94 / 145 pages, 105, and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV is 108 viral genomes / mL (vg / mL) to 1015vg / mL, 109vg / mL to 1015vg / mL, 1010vg / mL to 1015vg / mL, 1011vg / mL to 1015vg / mL, 1012vg / mL to 1015vg / mL, 1013vg / mL to 1015vg / mL, 1011vg / mL to 1012vg / mL, 1012vg / mL to 1013 ... Concentrations ranging from 10¹⁴ vg / mL, 10¹⁴ vg / mL to 10¹⁵ vg / mL, 10⁸ vg / mL to 10¹⁴ vg / mL, 10⁸ vg / mL to 10¹³ vg / mL, 10⁸ vg / mL to 10¹² vg / mL, 10⁸ vg / mL to 10¹¹ vg / mL, 10⁸ vg / mL to 10¹⁰ vg / mL, or 10⁹ vg / mL. In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV concentrations are from 10⁸ vg / mL to 10¹³ vg / mL. In some embodiments...In the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV is at least 108 vg / mL, at least 109 vg / mL, at least 1010 vg / mL, at least 1011 vg / mL, at least 1012 vg / mL, at least 1013 vg / mL, at least 1014 vg / mL, at least 1015 vg / mL or higher concentrations. In some implementations, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV concentrations are at most 109 vg / mL, at most 1010 vg / mL, at most 1011 vg / mL, at most 1012 vg / mL, at most 1013 vg / mL, at most 1014 vg / mL, or at most 1015 vg / mL. It should be understood that each of the individual rAAV concentrations described herein can be used to define a lower and upper limit for the range of rAAV concentrations.

[0313] In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) solutions contain a total of 1x10¹³ to 2x10¹³ first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV viral genomes. In some embodiments, the first and / or second solutions (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) contain a total of 5x10¹³ to 6x10¹³ first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV viral genomes. In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) solutions contain a total of at least 1x10¹³, at least 2x10¹³, at least 3x10¹³, at least 4x10¹³, at least 5x10¹³, at least 6x10¹³, at least 7x10¹³, at least 8x10¹³, at least 9x10¹³ or more of the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV viral genomes. In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) solutions contain a total of up to 1x10¹³, up to 2x10¹³, up to 3x10¹³, up to 4x10¹³, up to 5x10¹³, etc.6x10¹³, up to 7x10¹³, up to 8x10¹³, up to 9x10¹³, a total of 1x10¹¹ to 1x10¹⁴ vg, a total of 1x10¹¹ to 1x10¹³ vg, a total of 1x10¹¹ to 1x10¹² vg, a total of 1x10¹² to 1x10¹³ vg, a total of 1x10¹² to 1x10¹⁴ vg, a total of 1x10¹³ to 1x10¹⁴ vg, a total of 1x10¹³ to 6x10¹³ vg, a total of 2x10¹³ to 5x10¹³ vg, or a total of 1x10¹³ to 2x10¹³ or more first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV viral genomes. It should be understood that each of the individual rAAV quantities described herein can be used to define a lower and upper limit for the range of rAAV quantities.

[0314] In some embodiments, the first and / or second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) solutions contain a total of 1 x 10¹⁰ viral genomes (e.g., 4 x 10⁸ vg / mL to 1 x 10⁹ vg / mL).

[0315] In some embodiments, the transgene of the first rAAV is the same as that of the second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV. In some embodiments, the first rAAV is a transgenic gene different from the second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh, eighth and / or ninth and / or tenth, etc.) rAAV. In some embodiments, the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV or more transgenic genes are selected from: (a) reporter genes, (b) genes that are effective in treating kidney-related conditions when expressed in subjects, (c) inhibitors of genes or proteins that are effective in treating kidney-related conditions, or any combination thereof.

[0316] In some embodiments, the transgene of the first rAAV is activated by at least one kidney-specific promoter, said at least one kidney-specific promoter being the same as at least one kidney-specific promoter that activates the transgene of the second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV. In some embodiments, the transgene of the first rAAV is activated by at least one kidney-specific promoter, said at least oneThe kidney-specific promoter is different from at least one kidney-specific promoter that activates the second (and / or third and / or fourth and / or fifth and / or sixth and / or seventh and / or eighth and / or ninth and / or tenth, etc.) rAAV transgene.

[0317] In some embodiments, the subsequent administration of the second solution is carried out over a period of at least one day. In some embodiments, the time period for subsequent applications of the second solution between the application of the first solution and the second solution (or between the second solution and the third solution, or between the third solution and the fourth solution, or between the fourth solution and the fifth solution, or between the fifth solution and the sixth solution, or between the sixth solution and the seventh solution, or between the seventh solution and the eighth solution, or between the eighth solution and the ninth solution, or between the ninth solution and the tenth solution) is at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 1 year, or longer.

[0318] In some embodiments, the time period for subsequent applications of the second solution is determined based on the efficacy or persistence of the application of the first solution containing rAAV. In some embodiments, the time period of subsequent administration between the administration of the first and second solutions (or between the second and third solutions, or between the third and fourth solutions, or between the fourth and fifth solutions, or between the fifth and sixth solutions, or between the sixth and seventh solutions, or between the seventh and eighth solutions, or between the eighth and ninth solutions, or between the ninth and tenth solutions) is determined based on the efficacy or durability of administration of at least one of the previous solutions containing rAAV.

[0319] Efficacy or durability can be determined, for example, by examining the rAAV transduction of the kidney (e.g., sequencing of the rAAV genome (or barcodes therein); expression of the reporter gene or transgene delivered by rAAV (e.g., RNA- or protein-based assays such as RT-qPCR, ELISA, histological staining, or flow cytometry)), anti-rAAV serology (e.g., in urine or blood), or the occurrence of symptoms associated with kidney-related conditions (to which rAAV has been administered). If efficacy or persistence falls below a certain level, the subject is given a solution containing a subsequent rAAV (containing the same or different capsid as the previous rAAV; containing the same or different transgene as the previous rAAV).

[0320] In various embodiments, the first solution is administered to the subject's first kidney, and the second (i.e., contralateral) kidney is not treated simultaneously. Instead, retrograde administration of the solution to the contralateral kidney is delayed relative to retrograde rAAV administration to the first kidney.The administration of retrograde rAAV to the first kidney is delayed to confirm its effectiveness and does not result in substantial side effects that would prompt a medical professional to treat the contralateral kidney in the same or similar manner. Therefore, retrograde rAAV administration to the contralateral kidney may be delayed relative to retrograde rAAV administration to the first kidney by at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, or at least 5 weeks. In other embodiments, the contralateral kidney is treated on the same day as the first kidney by retrograde rAAV administration.

[0321] In some embodiments, a first solution is administered to the subject's first kidney, while a second solution is administered to the subject's second kidney. (Page 96 / 145, CN 121752299 A) In some embodiments, a first solution (or a second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more) is administered to the subject's first kidney, while a second solution (or a first, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more) is administered to the subject's second kidney.

[0322] In some embodiments, a solution containing a first rAAV is administered to the subject's first kidney, and a solution containing a second rAAV is administered to the subject's first kidney. In some embodiments, at least one solution containing a first rAAV, a second rAAV, a third rAAV, a fourth rAAV, a fifth rAAV, a sixth rAAV, a seventh rAAV, an eighth rAAV, a ninth rAAV, a tenth rAAV, or more rAAVs is each administered to the same kidney of the subject. In some embodiments, at least one solution comprising a first rAAV, a second rAAV, a third rAAV, a fourth rAAV, a fifth rAAV, a sixth rAAV, a seventh rAAV, an eighth rAAV, a ninth rAAV, a tenth rAAV, or more rAAVs is each administered to the same or different kidneys of the subject, or a combination thereof.

[0323] In some embodiments, a solution comprising a first rAAV is administered to both kidneys of the subject, and a solution comprising a second rAAV is administered to both kidneys of the subject. In some embodiments, at least one solution comprising...

Claims

1. A method for transducing nephrons in the kidneys of a subject using recombinant adeno-associated virus (rAAV), the method comprising: The catheter is guided through the subject's urethra, bladder, and ureter; and A solution containing the rAAV is administered to the renal pelvis of the kidney via the catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight, wherein the nephrons of the kidney are transduced with the rAAV at high efficiency.

2. The method of claim 1, wherein the solution containing the rAAV is administered to the kidney for about 0.5 minutes to about 60 minutes.

3. The method of claim 1, wherein the solution containing the rAAV is administered to the kidney for about 1 minute to about 2 minutes.

4. The method of claim 1, wherein the solution containing the rAAV is administered at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O.

5. The method of claim 1, wherein the method results in 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 being transduced by the rAAV.

6. The method of claim 1, wherein the rAAV transduction efficiency of the nephron in the kidney is increased by at least 2, at least 5, at least 10, at least 50, at least 100, at least 400, at least 1000, or at least 3500 times compared to the transduction efficiency of the corresponding nephron in another kidney treated by intravenous administration of the solution containing the rAAV.

7. The method of claim 1, wherein the rAAV does not contain an AAV9 capsid, and the rAAV transduction efficiency of the nephron in the kidney is increased by at least 2, at least 5, at least 10, at least 50, at least 100, at least 400, at least 1000, or at least 3500 times compared to the corresponding transduction efficiency obtained by administering an AAV containing an AAV9 capsid to another kidney in the same manner.

8. The method of claim 1, wherein the rAAV does not contain an AAV9 capsid, and the rAAV transduction efficiency in the proximal tubular cells of the nephron in the kidney is increased by at least 2, at least 5, at least 10, at least 50, at least 100, at least 400, at least 1000, or at least 3500 times compared to the corresponding transduction efficiency in the proximal tubular cells obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

9. The method of claim 1, further comprising the step of blocking renal vessels selected from renal arteries, renal veins, and combinations thereof in the kidney prior to administering the solution containing the rAAV.

10. The method of claim 9, further comprising the step of unblocking the renal vessels after a period of about 10 minutes to about 60 minutes following the administration of the solution containing the rAAV.

11. The method of claim 1, wherein the kidney is not isolated from the systemic circulation.

12. The method of claim 1, wherein the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof are not blocked during the execution of the method.

13. The method of claim 1, wherein the solution containing the rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

14. The method of claim 1, wherein the subject is a human, a non-human primate, a horse, a dog, or a pig.

15. The method of claim 1, wherein at least about 30% of the nephrons in the kidney are transduced by the rAAV.

16. The method of claim 1, wherein the amount of the solution containing the rAAV administered to the subject is from about 0.13 mL / kg to about 0.33 mL / kg.

17. The method of claim 1, wherein the amount of the solution containing the rAAV administered to the subject is from about 0.27 mL / kg to about 0.33 mL / mg.

18. The method of claim 1, wherein the solution containing the rAAV is administered using a balloon catheter.

19. The method of claim 9, wherein a balloon catheter is used to close the renal vessels.

20. The method of claim 9, wherein clamps are used to close the renal vessels.

21. The method of claim 9, wherein only one of the renal artery or renal vein is blocked.

22. The method of claim 1, wherein the renal vein of the kidney is not blocked.

23. The method of claim 1, wherein the method does not include continuous perfusion of the isolated kidney.

24. The method of claim 1, wherein the method does not include a closed loop comprising the kidney.

25. The method of claim 1, wherein the method does not include a basic closure system comprising the kidney.

26. The method of claim 1, wherein the method does not include shunting circulation from the kidney.

27. The method of claim 1, wherein the method does not include bypassing the kidney.

28. The method of claim 1, wherein the method is performed in vivo.

29. The method according to claim 1, wherein the method is performed without leaving the body.

30. The method of claim 10, wherein the time period for occluding the at least one renal vessel is 15-45 minutes after the occlusion.

31. The method of claim 10, wherein the time period for occluding the at least one renal vessel is 20-40 minutes after the occlusion.

32. The method of claim 10, wherein the time period for occluding the at least one renal vessel is approximately 15-30 minutes after the occlusion.

33. The method of claim 10, wherein the amount of the solution containing the rAAV is from about 0.13 mL / kg to about 0.33 mL / kg, and wherein the time period for blocking the renal vessels is about 15-30 minutes after the blocking.

34. The method of claim 1, wherein the rAAV comprises AAV capsid proteins selected from Table 1.

35. The method of claim 1, wherein the rAAV comprises a capsid protein selected from AAV2G9, AAV2.5, AAVDJ, and AAV2.

36. The method of claim 35, wherein the capsid protein is AAV2G9.

37. The method of claim 1, wherein the rAAV comprises a reasonable polyploid.

38. The method of claim 1, wherein the solution contains a concentration of 10 8 One viral genome / mL (vg / mL) to 10 15 The rAAV in vg / mL.

39. The method of claim 1, wherein the solution contains a concentration of 10 8 vg / mL to 10 13 The rAAV in vg / mL.

40. The method of claim 1, wherein the solution comprises a total of 1x10 13 Up to 2x10 13 One rAAV viral genome.

41. The method of claim 1, wherein the solution comprises a total of 5 x 10 13 Up to 6x10 13 One rAAV viral genome.

42. The method of claim 1, wherein the solution comprises a total of 1x10 10 One viral genome.

43. The method of claim 1, wherein the rAAV comprises a genetically modified organism.

44. The method according to claim 43, wherein the transgene is selected from: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome with sensorineural deafness (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); potassium inward rectifier channel subfamily J member 1 (KCNJ1); MAGED2 (type V); mucin 1 (MUC1); renin 1 (NPHP1); Nephrotic protein (NPHS1); Nephrotic protein 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL); Polycystic protein 1 (PKD1); Polycystic protein 2 (PKD2); Polycystic kidney and liver disease 1 (PKHD1); Protein transporter Sec61 subunit α isoform 1 (SEC61A1); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 7 member 9 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and combinations thereof.

45. The method according to claim 43, wherein the transgene is selected from: aquaporin 2 (AQP2); ATPase Na+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport V0 subunit A4 (ATP6V0A4); ATPase H+ transport V1 subunit B1 (ATP6V1B1); arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); carbonic anhydrase 2 (CA2); calcium-sensitive receptor (CaSR); chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); chloride voltage-gated channel Kb (CLCNKB); tight junction protein 16 (CLDN16); tight junction protein 19 (CLDN19); cyclin and CBS domain divalent metal cation transporter mediator 2 (CNNM2); Cullin 3 (CUL3); Cytochrome P450 family 11 subfamily B member 1 (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; Alpha stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; Pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); sodium channel epithelial subunit 1 α (SCNN1A); sodium channel epithelial subunit 1 β (SCNN1B); sodium channel epithelial subunit 1 γ (SCNN1G);Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 1 member 1 (SLC1A1); Solute carrier family 2 member 2 (SLC2A2); Solute carrier family 34 member 1 (SLC34A1); Solute carrier family 34 member 3 (SLC34A3); Solute carrier family 36 member 2 (SLC36A2); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 4 member 1 (SLC4A1); Solute carrier family 6 member 19 (SLC6A19); Solute carrier family 6 member 20 (SLC6A20); Solute carrier family 7 member 7 (SLC7A7); Solute carrier family 7 member 9 (SLC7A9); Transient acceptor potential cation channel subfamily M member 6 (TRPM6); WD repeat domain 72 (WDR72); lysine-deficient (WNK) protein kinase 1 (WNK1); lysine-deficient (WNK) protein kinase 4 (WNK4); and combinations thereof.

46. ​​The method of claim 43, wherein the transgene comprises an inhibitor of a gene or protein selected from: renin (REN), sodium channel epithelial 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD).

47. The method of claim 1, wherein the circulating serum of the subject does not neutralize the rAAV after administration.

48. The method of claim 1, wherein the subject's circulating serum contains an antibody that neutralizes the rAAV to be administered and the antibody does not neutralize the rAAV in the kidney after administration.

49. The method of claim 1, wherein subsequent administration of the rAAV of claim 1 does not result in an inflammatory response in the parenchyma of the kidney.

50. The method of claim 49, wherein the subsequent application is performed at least one day later.

51. The method of claim 49, wherein the subsequent application is performed at least one month later.

52. The method of claim 1, wherein the method transduces the proximal tubule of the kidney using the rAAV.

53. The method of claim 1, wherein the method uses the rAAV to transduce at least one cell population of the glomerulus, capsule of the glomerulus, proximal convoluted tubule, loop of Henry, distal convoluted tubule, or collecting duct of the kidney.

54. The method of claim 1, wherein the rAAV comprises a kidney-specific promoter.

55. The method of claim 54, wherein the kidney-specific promoter is selected from: the kidney-specific cadherin (KSPC) gene promoter; the Na+ / glucose cotransporter (SGLT2) gene promoter; the sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and the E-cadherin (ECAD) gene promoter.

56. The method of claim 54, wherein the kidney-specific promoter is a synthetic promoter.

57. The method of claim 1, wherein the rAAV has a genome comprising a promoter specific to the proximal convoluted tubule and / or collecting duct.

58. A method of treating kidney-related conditions in a subject with such need, the method comprising administering recombinant adeno-associated virus (rAAV) to the subject by performing the method according to claim 1.

59. The method of claim 58, wherein the kidney-related condition is selected from: autosomal dominant polycystic kidney disease (ADPKD); Alport syndrome; autosomal dominant tubulointerstitial nephropathy (ADTKD); medullary cystic nephropathy; nephron tuberculosis; Bart syndrome; Von Hippel-Lindau syndrome; Gitelman syndrome; congenital nephrotic syndrome; primary hyperoxaluria; Dent disease; thin basement membrane nephropathy; cystinuria; Liddle syndrome; papillary kidney syndrome; and cystin storage disease.

60. The method of claim 58, wherein the kidney-related condition is selected from: episodic mineralocorticoid hyperplasia syndrome, autosomal dominant hypocalcemia, autosomal dominant hypomagnesemia, type 1 Bart syndrome, type 2 Bart syndrome, type 3 Bart syndrome, type 4a Bart syndrome, type 4b Bart syndrome, type 5 Bart syndrome, type 1 congenital adrenal hyperplasia, type 2 congenital adrenal hyperplasia, type 4 congenital adrenal hyperplasia, type 5 congenital adrenal hyperplasia, type A cystinuria, type B cystinuria, type 1 Dent disease, type 2 Dent disease / Lowe syndrome, dicarboxyaminoaciduria, distal RTA, EAST / SeSAME syndrome, Fanconi Bickel syndrome, Fanconi tubular syndrome 1, Fanconi tubular syndrome 2, Fanconi tubular syndrome 3, Fanconi tubular syndrome 4, Gitelman syndrome, glucocorticoid-suppressible aldosteronism, Hartnup syndrome, hereditary hypophosphatemic rickets with hypercalciuria, HNF1B-related nephropathy, BH4 deficiency with hyperphenylalaninemia, type 1 hypomagnesemia / hypomagnesemia with secondary hypocalcemia, type 2 hypomagnesemia, type 3 hypomagnesemia / familial hypomagnesemia with hypercalciuria and nephrocalcinosis, type 4 hypomagnesemia, type 5 hypomagnesemia / familial hypomagnesemia with... Hypercalciuria and nephrocalcinosis, hypomagnesemia, seizures and intellectual disability type 1, hypomagnesemia, seizures and intellectual disability type 2, iminoglycinuria, type 2 Kenny-Caffey syndrome, Liddle syndrome, lysineuria protein intolerance, type 2 neonatal inflammatory skin and intestinal diseases, nephrogenic diabetes insipidus, nephrogenic syndrome of abnormal antidiuretic hormone secretion, type 1 pseudoketoalbuminemia, type 1A pseudoketoalbuminemia, type 2b pseudoketoalbuminemia, type 2c pseudoketoalbuminemia, type 2d pseudoketoalbuminemia, type 2e pseudoketoalbuminemia, type 3 renal tubular acidosis and X-linked hypophosphatemic rickets.

61. The method of claim 58, wherein the kidney-related condition is cystinuria, and the transgene is SLC3A1 and / or SLC7A9.

62. The method of claim 58, wherein the kidney-related condition is autosomal dominant polycystic kidney disease (ADPKD), and the transgene is PKD1, PKD2, and / or GANAB.

63. A method for transducing at least about 10% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a solution containing the rAAV into the renal pelvis of the kidney via the catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in at least about 10% of the nephrons in the kidney being transduced by the rAAV.

64. A method for transducing at least about 25% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a solution containing the rAAV into the renal pelvis of the kidney via the catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

65. A method for transducing at least about 25% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) Close the renal artery of the kidney without closing the renal vein of the kidney; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a given amount of solution containing the rAAV into the renal pelvis of the kidney via the catheter; and d) Unblock the renal artery approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

66. A method for transducing nephrons in the kidney of a subject, the method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a given amount of a solution containing rAAV, not rAAV9, into the renal pelvis of the kidney via the catheter; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in a transduction efficiency that is at least twice as high as that obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

67. The method of claim 66, wherein the rAAV comprises capsid proteins selected from Table 1.

68. The method of claim 66, wherein the rAAV has at least twice the transduction efficiency in the kidney compared to the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

69. The method of claim 66, wherein the rAAV has a transduction efficiency 400 times higher in the kidney than the corresponding transduction efficiency obtained by administering rAAV containing an AAV9 capsid to another kidney in the same manner.

70. A method for transducing at least about 25% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) Isolate the kidney from the systemic circulation; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administer a solution containing the rAAV to the renal pelvis of the kidney via the catheter at a rate of about 0.13 mL / kg to about 0.33 mL / kg, where kg is the subject's body weight; as well as d) The kidney is reintroduced into systemic circulation approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

71. A method for treating kidney disease in a subject with this need, the method comprising: The subject was given a first recombinant adeno-associated virus (rAAV) encoded with a therapeutic effect on the kidney condition; as well as Following administration of the first rAAV, a second rAAV encoding a therapeutic effect on the kidney or a different kidney of the subject is administered. The first rAAV and the second rAAV are cross-reactive with serum, and The subject in the kidney did not elicit a significant immune response to the second rAAV.

72. The method of claim 71, wherein at least one solution comprising the first and / or the second rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O.

73. The method of claim 71, wherein the solution containing the second rAAV is applied approximately one week later.

74. The method of claim 71, wherein the first and / or second rAAV is applied by an application method comprising: The catheter is guided through the subject's urethra, bladder, and ureter; and A solution containing the first or second rAAV is administered to the renal pelvis of the kidney via the catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight.

75. The method of claim 71, wherein the first and / or second rAAV is applied by an application method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a solution containing the first or second rAAV to the kidney or the renal pelvis of a different kidney at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg via the catheter, wherein the kg is the subject's body weight; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the first or second rAAV.

76. The method of claim 71, wherein the administration method results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

77. The method of claim 71, wherein the subject has a neutralizing antibody against the first rAAV therapeutic agent prior to administration.

78. The method of claim 71, wherein the capsid protein of the first rAAV has the same serotype as the capsid protein of the second rAAV.

79. The method of claim 71, wherein the capsid protein of the first rAAV has a different serotype from the capsid protein of the second rAAV.

80. The method of claim 71, wherein the time period for subsequent application of the second rAAV is determined based on the efficacy or persistence of the application of the first rAAV.

81. The method of claim 71, wherein the first rAAV is applied to the subject's first kidney, and the second rAAV is applied to the subject's second kidney.

82. The method of claim 71, wherein the first rAAV is applied to the first kidney of the subject, and the second rAAV is applied to the first kidney of the subject.

83. The method of claim 71, wherein the first rAAV is applied to both kidneys of the subject, and the second rAAV is applied to both kidneys of the subject.

84. A method of treating kidney disease in a subject with this need, said subject being seropositive for a recombinant adeno-associated virus (rAAV) therapeutic agent, said method comprising: The rAAV therapeutic agent, which encodes a genetically modified form that has a therapeutic effect on the kidney condition, was administered to the kidneys of the subject. The subjects in the subjects did not elicit a significant immune response in the kidneys from the rAAV therapeutic agent.

85. The method of claim 84, wherein the subject has a neutralizing antibody against the rAAV therapeutic agent prior to administration.

86. The method of claim 84, wherein the rAAV is applied by an application method comprising: The catheter is guided through the subject's urethra, bladder, and ureter; and A solution containing the first or second rAAV is administered to the renal pelvis of the kidney via the catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight.

87. The method of claim 84, wherein the rAAV is applied by an application method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administer a solution containing the rAAV to the renal pelvis of the kidney at an amount of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The administration method described therein results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

88. A method for transducing at least about 25% of nephrons in the kidney of a subject using recombinant adeno-associated virus (rAAV), the method comprising: a) Close the renal vessels of the kidney selected from the renal artery, renal vein, and combinations thereof; b) Guide the catheter through the subject's urethra, bladder, and ureter; c) Administering a solution containing the rAAV into the renal pelvis of the kidney via the catheter at an amount of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight, wherein the rAAV contains capsid proteins selected from Table 1; and d) Unblock the renal vessels approximately 10 minutes to approximately 60 minutes after administration of the solution containing the rAAV. The method described therein results in at least about 25% of the nephrons in the kidney being transduced by the rAAV.

89. A method of treating a kidney-related condition in a subject with such need, the method comprising administering the rAAV to the subject by performing the method according to claim 88.

90. The method of claim 89, wherein the rAAV is administered to the kidney at an intrarenal pressure of about 25 cm H2O to about 55 cm H2O.

91. The method of claim 89, wherein the amount of the solution is from 0.27 mL / kg to 0.33 mL / kg.

92. The method of claim 89, wherein the time period is 30-60 minutes after the application of the solution containing the rAAV.

93. The method of claim 89, wherein the subject is serologically positive for the rAAV prior to administration of the solution containing the rAAV.

94. The method according to any one of claims 1, 58, 63-66, 70, 71, 84, 88 or 89, wherein the rAAV is administered in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles or vesicles.

95. The method according to any one of claims 1, 58, 63-66, 70, 71, 84, 88 or 89, wherein the rAAV is applied in lipid nanoparticles (LNP).

96. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV), said pharmaceutical composition comprising: a) AAV capsid proteins selected from Table 1; b) Genetically modified organisms, wherein the genetically modified organisms comprise: i) Genes selected from the following: alanine-glyoxylate aminotransferase (AGXT); Bart syndrome with sensorineural deafness (BSND); chloride voltage-gated channel 5 (CLCN5); chloride voltage-gated channel Ka (CLCNKA); chloride voltage-gated channel Kb (CLCNKB); type IV collagen α3 chain (COL4A3); type IV collagen α4 chain (COL4A4); type IV collagen α5 chain (COL4A5); glucosidase II α subunit (GANAB); glyoxylate and hydroxypyruvate reductase (GRHPR); hepatic nuclear factor 1 (HNF1) homeobox B (HNF1B); 4-hydroxy-2-ketoglutarate aldolase 1 (HOGA1); potassium inward rectifier channel subfamily J member 1 (KCNJ1); MAGED2 (type V); mucin 1 (MUC1); renin 1 (NPHP1); Nephrotic protein (NPHS1); Nephrotic protein 2 (NPHS2; Podocin); Inositol polyphosphate-5-phosphatase (OCRL); Polycystic protein 1 (PKD1); Polycystic protein 2 (PKD2); Polycystic kidney and liver disease 1 (PKHD1); Protein transporter Sec61 subunit α isoform 1 (SEC61A1); Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 7 member 9 (SLC7A9); Von Hippel-Lindau tumor suppressor (VHL); and combinations thereof; or ii) Inhibitors of the following genes or proteins: renin (REN), sodium channel epithelial subunit 1 α (SCNN1A), sodium channel epithelial subunit 1 β (SCNN1B), and uroregulatory hormone (UMOD); and c) Pharmaceutically acceptable carriers.

97. A pharmaceutical composition comprising recombinant adeno-associated virus (rAAV), said pharmaceutical composition comprising: a) AAV capsid proteins selected from Table 1; b) A transgene comprising a gene selected from the following: aquaporin 2 (AQP2); ATPase Na+ / K+ transport subunit α1 (ATP1A1); ATPase H+ transport V0 subunit A4 (ATP6V0A4); ATPase H+ transport V1 subunit B1 (ATP6V1B1); arginine angiotensin receptor 2 (AVPR2); Barttin CLCNK (chloride channel K) type helper subunit β (BSND); carbonic anhydrase 2 (CA2); calcium-sensitive receptor (CaSR); chloride voltage-gated channel 5 (CLCN5); CLCNKA (chloride voltage-gated channel Ka); chloride voltage-gated channel Kb (CLCNKB); tight junction protein 16 (CLDN16); tight junction protein 19 (CLDN19); cyclin and CBS domain divalent metal cation transporter 2 (CNNM2); Cullin 3 (CUL3); member of cytochrome P450 family 11 subfamily B. (CYP11B1); Cytochrome P450 family 11 subfamily B member 2 (CYP11B2); Cytochrome P450 family 17 subfamily A member 1 (CYP17A1); Cytochrome P450 family 21 subfamily A member 2 (CYP21A2); Epidermal growth factor (EGF); Epidermal growth factor receptor (EGFR); Enyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase (EHHADH); FAM111 (family 111) trypsin-like peptidase A (FAM111A); Forkhead box I1 (FOXI1); Ion transport regulator 2 containing FXYD domain / motif (FXYD2); Glycine amidotransferase (GATM); Guanine nucleotide-binding protein; Alpha stimulation (GNAS); Hepatocyte nuclear factor 1 (HNF1) homeobox B (HNF1B); Hepatocyte nuclear factor 4α (HNF4A); Hydroxysteroid 11-β dehydrogenase 2 (HSD11B2); Hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2 (HSD3B2); Potassium voltage-gated channel subfamily A member 1 (KCNA1); Potassium inward rectifier channel subfamily J member 1 (KCNJ1); Potassium inward rectifier channel subfamily J member 10 (KCNJ10); Kelch-like family member 3 (KLHL3); Melanoma antigen gene family member D2 (MAGED2); Nuclear receptor subfamily 3 group C member 2 (NR3C2); Lowe's oculoencephalopathy-renal syndrome (OCRL) inositol polyphosphate-5-phosphatase; Pterin-4 α-methanolamine dehydratase 1 (PCBD1); X-linked phosphate-regulated endopeptidase (PHEX); Sodium channel epithelial subunit 1 α (SCNN1A); Sodium channel epithelial subunit 1 β (SCNN1B); Sodium channel epithelial subunit 1 γ (SCNN1G);Solute carrier family 12 member 1 (SLC12A1); Solute carrier family 12 member 3 (SLC12A3); Solute carrier family 1 member 1 (SLC1A1); Solute carrier family 2 member 2 (SLC2A2); Solute carrier family 34 member 1 (SLC34A1); Solute carrier family 34 member 3 (SLC34A3); Solute carrier family 36 member 2 (SLC36A2); Solute carrier family 3 member 1 (SLC3A1); Solute carrier family 4 member 1 (SLC4A1); Solute carrier family 6 member 19 (SLC6A19); Solute carrier family 6 member 20 (SLC6A20); Solute carrier family 7 member 7 (SLC7A7); Solute carrier family 7 member 9 (SLC7A9); Transient acceptor potential cation channel subfamily M member 6 (TRPM6); WD repeat domain 72 (WDR72); lysine-deficient (WNK) protein kinase 1 (WNK1); lysine-deficient (WNK) protein kinase 4 (WNK4); and combinations thereof; and; c) Pharmaceutically acceptable carriers.

98. The pharmaceutical composition according to claim 96 or 97, wherein the pharmaceutically acceptable carrier comprises mannitol.

99. The pharmaceutical composition according to claim 96 or 97, wherein the AAV comprises the capsid protein of AAV2G9.

100. The pharmaceutical composition according to claim 96 or 97, wherein the solution containing the rAAV is 10 8 One viral genome / mL (vg / mL) to 10 15 The concentration in vg / mL.

101. The pharmaceutical composition according to claim 96 or 97, wherein the solution containing the rAAV is 10 8 vg / mL to 10 13 The concentration in vg / mL.

102. The pharmaceutical composition according to claim 96 or 97, wherein the pharmaceutical composition comprises a total of 1x10 13 Up to 2x10 13 One rAAV viral genome.

103. The pharmaceutical composition according to claim 96 or 97, wherein the pharmaceutical composition comprises a total of 5 x 10 13 Up to 6x10 13 One rAAV viral genome.

104. The pharmaceutical composition according to claim 96 or 97, wherein the pharmaceutical composition is a unit dose of about 0.13 mL / kg to about 0.33 mL / kg, wherein kg is the weight of the subject.

105. The pharmaceutical composition according to claim 96 or 97, wherein the pharmaceutical composition is a unit dose of about 0.27 mL / kg to about 0.33 mL / kg.

106. The pharmaceutical composition according to claim 96 or 97, wherein the transgene comprises a reporter protein.

107. The pharmaceutical composition of claim 96 or 97, wherein the genome of said rAAV contains a kidney-specific promoter.

108. The pharmaceutical composition according to claim 107, wherein the kidney-specific promoter is selected from: the kidney-specific cadherin (KSPC) gene promoter; the Na+ / glucose cotransporter (SGLT2) gene promoter; the sodium-potassium-chloride cotransporter (NKCC2) gene promoter; and the E-cadherin (ECAD) gene promoter.

109. The pharmaceutical composition according to claim 107, wherein the kidney-specific promoter is a synthetic promoter.

110. The pharmaceutical composition of claim 96 or 97, wherein the genome of said rAAV contains a promoter specific to the proximal convoluted tubule and / or collecting duct.

111. The pharmaceutical composition according to claim 96 or 97, wherein the rAAV is formulated for delivery in liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, or vesicles.

112. The pharmaceutical composition according to claim 96 or 97, wherein the rAAV is formulated for delivery in lipid nanoparticles (LNPs).

113. A method for transducing nephrons in the kidneys of a subject using recombinant adeno-associated virus (rAAV), the method comprising: The catheter is guided through the subject's urethra, bladder, and ureter; The solution containing the rAAV was administered to the renal pelvis of the kidney at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg, where kg is the subject's body weight, wherein the rAAV contains AAV2G9, and wherein the nephrons of the kidney were efficiently transduced by the rAAV.

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