Method for renal administration of recombinant adeno-associated virus

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

Cross-references 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 includes a sequence list, which has been submitted in XML format via the Patent Center and 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 techniques described herein relate to methods for administering recombinant adeno-associated virus (rAAV) to the kidneys and pharmaceutical compositions containing rAAV for administration to the kidneys. Background Technology

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

[0005] The kidneys consist of over a million filtration units called nephrons. Nephrons, in order of filtrate passage, comprise the following regions: 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 salts; its thin descending limb reabsorbs water to concentrate urine, while 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 even more water, finely regulating the final urine products. Urine flows out of the collecting ducts and into the smaller and larger calyces of the kidney. Then, the urine 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 renal biopsies because the kidneys are highly vascularized, and punctures could lead to excessive bleeding. Since the kidneys are accessible via vascular tracts, another approach is renal artery cannulation, renal vein cannulation, and connecting a cannula to a pump to circulate the agent (e.g., rAAV) through the isolated and cannulated vascular system. However, this is a complex procedure that can lead to complications and discomfort. There is an urgent need for clinically meaningful administration methods to efficiently deliver rAAV to the kidneys. There is also a need for rAAVs that exhibit tropism and / or high efficiency in the kidneys. Summary of the Invention

[0007] Implementations of the technology described herein relate to a method of administering recombinant adeno-associated virus (rAAV) to the kidneys 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. This document also describes pharmaceutical compositions comprising recombinant adeno-associated virus (rAAV) for administration to the kidneys.

[0008] This article also describes specific rAAVs that exhibit high transduction in the kidneys after retrograde ureteral administration, including but not limited to those selected from Table 1 or Figure 4-5 The rAAV capsid. Furthermore, this article describes exemplary parameters for retrograde ureteral administration, including dosage, timing, and / or renal vascular occlusion.

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

[0010] In one aspect, this article describes a method for transducing nephrons in the kidneys 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 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 (kg being the subject's body weight), wherein the renal nephrons containing nephron cells are transduced by rAAV with high efficiency.

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

[0012] In some implementations of any aspect, a solution containing rAAV is administered to the kidney over approximately 1 to approximately 2 minutes.

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

[0014] In some embodiments in any aspect, 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 rAAV.

[0015] In some embodiments of any aspect, the rAAV transduction efficiency of a nephron 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 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 rAAV containing an AAV9 capsid to another kidney in the same manner.

[0017] In some implementations, 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 the proximal tubular cells of the nephron in the kidney is increased by 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 compared to 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 renal arteries, renal veins, and combinations thereof before administering a solution containing rAAV.

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

[0021] In some implementations of any aspect, the method also 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 renal arteries, renal veins, and combinations thereof during the conduct of the method.

[0023] In some embodiments of any aspect, a 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, a 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 implementations in any aspect, the subjects are humans, non-human primates, horses, dogs, or pigs.

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

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

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

[0028] In some implementations of any aspect, a balloon catheter is used to administer a solution containing rAAV.

[0029] In some implementations of any aspect, a balloon catheter is used to close the renal vessels.

[0030] In some implementations of any aspect, clamps are used to close the renal vessels, such as after laparoscopy.

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

[0032] In some implementations of any aspect, the renal vein of the kidney is not closed.

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

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

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

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

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

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

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

[0040] In some implementations of any aspect, the period of closure of at least one renal vessel is 15-45 minutes after closure.

[0041] In some implementations of any aspect, the period of closure of at least one renal vessel is 20-40 minutes after closure.

[0042] In some implementations of any aspect, the period of closure of at least one renal vessel is approximately 15-30 minutes after closure.

[0043] In some implementations of any aspect, the period of closure of at least one renal vessel is approximately 30 minutes after closure.

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

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

[0046] In some embodiments of any aspect, the amount of solution containing rAAV is from about 0.2 mL / kg to about 0.27 mL / kg, and the time period for blocking the renal vessels is about 30 minutes after blocking.

[0047] In some embodiments of any aspect, rAAV comprises an AAV capsid protein selected from the serotypes provided in Table 1. In some embodiments of any aspect, rAAV comprises an 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 implementations in any aspect, the capsid protein is AAV2G9.

[0050] In some implementations of any aspect, rAAV contains rational polyploid.

[0051] In some embodiments in any aspect, the solution contains a concentration of 10 8 One viral genome / mL (vg / mL) to 10 15 vg / mL rAAV.

[0052] In some embodiments in any aspect, the solution contains a concentration of 10 8 vg / mL to 10 13 vg / mL rAAV.

[0053] In some embodiments in any aspect, the solution contains a total of 1x10 13 Up to 2x10 13 One rAAV viral genome.

[0054] In some embodiments in any aspect, the solution contains a total of 5 x 10 13 Up to 6x10 13 One rAAV viral genome.

[0055] In some embodiments in any aspect, the solution contains a total of 1x10 10 One viral genome.

[0056] In some implementations in any aspect, rAAV contains genetic material.

[0057] In any embodiment of any aspect, the transgene is selected from: 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.

[0058] In any embodiment of any aspect, 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 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.

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

[0060] In some implementations in any aspect, rAAV is not neutralized in the subject's circulating serum after administration.

[0061] In some implementations of any aspect, the subject's circulating serum contains an antibody that neutralizes the rAAV to be administered and, after administration, the antibody does not neutralize rAAV in the kidneys.

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

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

[0064] In some embodiments of any aspect, the method uses rAAV to transduce the proximal tubules of the kidney.

[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 using rAAV.

[0066] In some implementations 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 implementations of any aspect, the kidney-specific promoter is a synthetic promoter.

[0069] In some implementations of any aspect, rAAV has a genome containing promoters specific to proximal convoluted tubules and / or collecting ducts.

[0070] In one aspect, this article describes a method for treating kidney-related conditions in subjects who require it, the method comprising administering recombinant adeno-associated virus (rAAV) to the subject by performing the method described herein.

[0071] In any of the implementation schemes, the 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 disease.

[0072] In any of the implementation schemes, the kidney-related conditions are selected from: episodic mineralocorticoid hyperthyroidism 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.

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

[0074] In some implementations in 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, this article describes a method 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 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 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 administration of the rAAV-containing solution, wherein the method results in the transduction of at least about 10% of nephrons in the kidney by rAAV.

[0076] In one aspect, this article 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 volume of rAAV-containing solution 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 (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 nephrons in the kidney being transduced by rAAV.

[0077] In one aspect, this article 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, wherein the method results in the transduction of at least about 25% of nephrons in the kidney by rAAV.

[0078] In one aspect, this article describes a method for transducing nephrons in a subject's kidney, 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 rAAV-containing solution, 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 implementations in any aspect, rAAV comprises capsid proteins selected from Table 1, but excluding AAV9.

[0080] In some embodiments of any aspect, 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.

[0081] In some embodiments of any aspect, the rAAV exhibits a 400-fold higher transduction efficiency in the kidney compared to AAV9. In some embodiments of any aspect, the rAAV exhibits 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, this article 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) 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 a rate 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 administration of the solution containing rAAV for a period of about 10 minutes to about 60 minutes, 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 for 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, wherein the first rAAV and the second rAAV are cross-reactive in serology, 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 a first and / or a 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 a first and / or a second rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 cm H2O.

[0085] In some implementations of any aspect, a solution containing a second rAAV is applied approximately one week later.

[0086] In some embodiments in 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 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).

[0087] In some embodiments in any aspect, the first and / or second rAAV is 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; (c) administering a solution containing rAAV into the kidney or the renal pelvis of a different kidney at a rate of about 0.13 mL / kg to about 0.33 mL / kg (kg being the subject's body weight) via the catheter; and (d) unoccluding the renal vessel after a period of about 10 minutes to about 60 minutes following occlusion and / or administration of the solution containing rAAV.

[0088] In some implementations of any aspect, the administration method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

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

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

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

[0092] In some implementations of any aspect, the time period for subsequent administration of the second rAAV is determined based on the efficacy or persistence of the first rAAV.

[0093] In some implementations in any aspect, the first rAAV is administered to the subject's first kidney, and the second rAAV is administered to the subject's second kidney.

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

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

[0096] In one aspect, this article describes a method for treating kidney disease in subjects who are 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, wherein the subject does not elicit a significant immune response to the rAAV therapeutic agent in the kidney.

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

[0098] In some embodiments in 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) occluding renal vessels of the kidney 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 into the renal pelvis of the kidney via 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) unoccluding the renal vessels after occlusion and / or after administration of the solution containing rAAV for a period of about 10 minutes to about 60 minutes, wherein said administration method results in at least about 25% of the nephrons in the kidney being transduced by rAAV.

[0100] In one aspect, this article 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 a rate 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 the transduction of at least about 25% of nephrons in the kidney by rAAV.

[0101] In one aspect, this article describes a method for treating kidney-related conditions in subjects with such needs, 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 in any aspect, the time period is 30-60 minutes after closure and / or after application of a solution containing rAAV.

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

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

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

[0108] In one aspect, this article describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid protein selected from Table 1; and (b) a transgene comprising: (i) a gene selected from: 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 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); 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 1 subunit α (SCNN1A), sodium channel epithelial 1 subunit β (SCNN1B), and uroregulatory hormone (UMOD); and (c) pharmaceutically acceptable carriers.

[0109] In one aspect, this document describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) comprising: (a) an AAV capsid protein selected from Table 1; and (b) a transgene comprising a gene 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); and 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 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 oculomotor-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); 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 implementations of any aspect, the pharmaceutically acceptable carrier comprises mannitol.

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

[0112] In some embodiments in any aspect, the solution containing rAAV is 10 8 One viral genome / mL (vg / mL) to 10 15 The concentration in vg / mL.

[0113] In some embodiments in any aspect, the solution containing rAAV is 10 8 One viral genome / mL (vg / mL) to 10 14 The concentration in vg / mL.

[0114] In some embodiments in any aspect, the solution containing rAAV is 10 8 vg / mL to 10 13 The concentration in vg / mL.

[0115] In some embodiments in any aspect, the pharmaceutical composition comprises a total of 1x10 13 Up to 2x10 13 One rAAV viral genome.

[0116] In some embodiments in any aspect, the pharmaceutical composition comprises a total of 5 x 10 13 Up to 6x10 13 One rAAV viral genome.

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

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

[0119] In some implementations of any aspect, the transgenic material contains a reporter protein.

[0120] In some implementations of any aspect, the rAAV genome contains 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 implementations of any aspect, the kidney-specific promoter is a synthetic promoter.

[0123] In some implementations of any aspect, the rAAV genome contains promoters 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 implementations of any aspect, rAAV is formulated for delivery in lipid nanoparticles (LNPs).

[0126] In one aspect, this article describes a method for transducing nephrons in the kidneys 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 rAAV to the renal pelvis of the kidney 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 AAV2G9, and wherein the nephrons in the kidney are transduced by rAAV with high efficiency. Attached Figure Description

[0127] Figure 1 Tissue sections of rat kidneys are shown following retrograde ureteral administration of the 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 Showing from Figure 1 These are magnified images. These images show that, compared to the negative control, the renal tubules were extensively transduced by the library.

[0129] Figure 3 A schematic diagram of the nephron and images illustrating various transduction pathways are shown.

[0130] Figure 4 The bar chart shows 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 according to Formula I as follows: (cDNA reads [%] / input [%]) / (cDNA AAV9 reads [%] / input AAV9 [%]).

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

[0132] Figure 6A-6X A series of images and graphs illustrate the results of retrograde ureteral administration of AAV2G9 in pigs (see, for example, Example 2). Figure 6A Immunohistochemistry (IHC) of GFP after administration of AAV2G9 (1.41E+13VG) is shown. 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 This is a bar chart, showing... Figure 6A The quantitative results of the samples shown indicate the percentage of positive tubes. Figure 6C-6H Bar graphs and quantitative tables show renal AAV gDNA (gDNA) in pig subjects RU13 and RU14 after retrograde ureteral administration of AAV2G9 to the treated kidney (“2G9”) or the contralateral kidney (“con”). Figure 6C ), kidney AAV protein ( Figure 6D ), kidney AAVcDNA ( Figure 6E ), liver AAV gDNA ( Figure 6F ), liver AAV protein ( Figure 6G ) and liver AAV cDNA ( Figure 6H “NoRT” is a negative control for RT-PCR, indicating the absence of reverse transcriptase. Figure 6I-6L To show the contralateral control kidney of subject RU13 ( Figure 6I ), AAV2G9 administration to the kidneys of subject RU13 ( Figure 6J ), the contralateral control kidney of subject RU14 ( Figure 6K ) and subject RU14's AAV2G9 administration to the kidney ( Figure 6L Images of the puncture sites in kidney samples 1-26 are shown in the image. 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... Figure 6I-6L . Figure 6M-6R The bar chart shows the viral load results in porcine subject RU13 after retrograde administration of AAV2G9 to the treated kidney (“2G9”) or the contralateral kidney (“con”): renal 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 ). Figure 6S-6X The bar chart shows the viral load results in porcine subject RU14 after retrograde administration of AAV2G9 to the treated kidney (“2G9”) or the contralateral kidney (“con”): renal 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] Figure 7A-7MA series of images and graphs illustrate the results of retrograde ureteral administration of AAV2G9 in nonhuman primates (see, for example, Example 3). Figures 7A-7F In this study, kidney sections from NHP-RU1 were stained with periodic acid Schiff (PAS) and subjected to immunohistochemistry for green fluorescent protein GFP (brown staining; indicating successful transduction of rAAV in renal cells). Figure 7A Histological images of the contralateral kidney from NHP-RU1 are shown. Figure 7B Histological images of the kidney after rAAV administration from NHP-RU1 are shown. Figure 7C Showing from Figure 7A High-resolution images of designated locations in the anterior inferior section of the kidney cortex. Figure 7D Showing from Figure 7B High-resolution images of designated locations in the anterior inferior section of the kidney cortex. Figure 7E Showing from Figure 7B Multiple high-resolution images of designated locations in the renal medulla in the anterior superior slice. Figure 7F Showing from Figure 7B Multiple high-resolution images of designated locations in the renal cortex of anterior superior slices; in the high-resolution images shown, transduction is nearly 100% visible in the proximal convoluted tubules. Figures 7A-7B The scale in the text is 2 mm. Figure 7C-7F The scale bar in the magnified image is 200 µm. Figure 7G The bar chart shows the vector copy number (VCN) of each diploid genome in the specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 8. Figure 7H The bar chart shows the eGFP cDNA levels (relative fold change (log10) 2^ddCT) in the specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 9. Figure 7I RNA analysis of NHP_RU2 is shown, comparing the level of eGFP cDNA in the specified samples (left bar in each sample) with the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) control (right bar in each sample); "NoRT" indicates no reverse transcriptase and "NTC" indicates no template control. Figure 7J The bar chart shows the relative light units (RLU) of protein per mg in the specified samples from NHP-RU1 and NHP-RU2; the results are quantified in Table 10. Figure 7K-7M A series of tables show the location of renal biopsies from NHP-RU1 and NHP-RU2 after AAV2G9 administration; biopsies 1, 2, 3 and 10 are anterosuperior; biopsies 7, 8, 9 and 11 are anteroinferior; biopsies 4, 5 and 6 are renal midline. Figure 7KThe table shows viral DNA (vg / dg), eGFP RNA (relative fold change in cDNA), luciferase protein (RLU / mg), and %GFP+ in proximal convoluted tubules (PCTs). Each column (DNA / RNA / protein) in the table was analyzed independently. Consistency was maintained between molecular assays and histological staining for each biopsy. Figure 7L The ranking of biopsies based on transduction (viral DNA vg / dg) is shown. Figure 7M The biopsy rankings are shown based on functional transduction (luciferase protein RLU / mg).

[0134] Figures 8A-8E A series of images showing co-staining of non-human primate kidneys after administration of AAV2G9 via the retrograde ureteral route (see, for example, Example 3). Figure 8A The analysis process of VISIOPHARM® is shown. Figures 8B-8E Counterstaining was performed on AAV2G9 (GFP - purple staining), proximal convoluted tubules (CD13 - yellow staining), and distal convoluted tubules and collecting tubules (CK19 - blue staining). Figure 8B The image shows a histological co-stained image of the contralateral kidney from NHP-RU1. Figure 8C Histological co-stained images of NHP-RU1 kidneys treated with AAV-2G9 are shown. Figure 8D The image shows a histological co-stained image of the contralateral kidney from NHP-RU2. Figure 8E Histological co-stained images of NHP-RU2 kidneys treated with AAV-2G9 are shown. Figures 8B-8E The results are quantified in Table 11-12.

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

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

[0137] The implementation of the technology described herein relates to a method of administering recombinant adeno-associated virus (rAAV) to the kidneys of a subject using the retrograde ureteral route. As used herein, the terms "retrograde ureteral route," "retrograde ureter (RU)," "retrograde route," "retrograde route to the ureter," "retrograde injection via the ureter," "retrograde administration," or "retrograde administration" are interchangeable and refer to administering the 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 an administration method can be used to treat kidney-related conditions in subjects who require it. This document also describes pharmaceutical compositions containing recombinant adeno-associated virus (rAAV) for administration to the kidneys.

[0138] This article also describes specific rAAVs that exhibit high transduction in the kidneys after retrograde ureteral administration, including but not limited to those selected from Table 1 and Figure 4-5 The rAAVs with capsids described herein. Compared to AAV9, these specific rAAVs (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. Compared to other routes of administration (e.g., intravenous), retrograde ureteral administration results in improved renal transduction levels.

[0139] Application and treatment methods In several respects, this article describes methods for administering recombinant adeno-associated virus (rAAV) to the kidneys of subjects and methods for treating kidney-related conditions in subjects with such need. In one respect, this article describes a method 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, the method comprising administering a measured amount of rAAV-containing solution into the ureter of the kidney via a retrograde route, wherein the rAAV-containing solution is administered to the kidneys for a sufficient time and / or under intrarenal pressure sufficient to result in a pharmaceutically effective level of rAAV transduction in the kidney nephrons.

[0140] In one aspect, this article describes a method for transducing nephrons in the kidneys 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 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), 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 an rAAV-containing solution 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. In some embodiments, the administration of the rAAV-containing solution lasts for a period of about 0.5 minutes to about 2 minutes. In some embodiments, the administration of the rAAV-containing solution lasts for a period of about 1 minute to 60 minutes. In some embodiments, the method further includes the step of occluding renal vessels selected from renal arteries, renal veins, and combinations thereof prior to the administration of rAAV. In some embodiments, the method further includes unoccluding the renal vessels after occlusion and / or after administration of the rAAV-containing solution for a period of about 10 minutes to about 60 minutes.

[0142] In one aspect, this article 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 paper describes a method for transducing at least about 20% of the nephrons in a subject's kidney using rAAV.

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

[0145] In one aspect, this article 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 retrogradely into the ureter of the kidney; 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 the transduction of at least about 25% of the nephrons in the kidney 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 renal vessels 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 vessels 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 2-fold higher transduction efficiency than a corresponding administration using rAAV9 instead of the rAAV. In some embodiments, the rAAV has a transduction efficiency at least 400-fold or at least 3500-fold higher than that of rAAV9.

[0147] In one aspect, this article describes a method for transducing at least about 30% of nephrons in a subject's kidney using rAAV. In another aspect, this article describes a method for administering recombinant adeno-associated virus (rAAV) to the kidney of a subject and / or treating kidney-related conditions in subjects in need of it, the method comprising: (a) occluding at least one renal vessel selected from the renal artery, renal vein, and combinations thereof; (b) administering a quantity of an rAAV-containing solution into the ureter of the kidney via a retrograde route, wherein the quantity 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 rAAV-containing solution.

[0148] In one aspect, this article 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 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 article 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 doing so, the method comprising: (a) blocking at least one renal vessel of the kidney selected from the renal artery, renal vein, and combinations thereof; (b) administering a solution containing rAAV in an amount of about 0.13 mL / kg to about 0.33 mL / kg via a retrograde route into the ureter of the kidney, wherein the rAAV contains a capsid protein derived from serotype AAV2G9; and (c) unblocking said at least one renal vessel after a certain period of time following the blocking and / or the administration of the solution containing rAAV.

[0150] In some implementations of any aspect, the steps of “closing at least one renal vessel of the kidney selected from the renal artery, renal vein and combination thereof” or “closing the renal vessel of the kidney selected from the renal artery, renal vein and combination thereof” or “closing the renal vessel of the kidney selected from the renal artery, renal vein and combination thereof” are replaced by the step of “isolating the kidney from the systemic circulation”.

[0151] In some implementations of any aspect, the steps of "unsealing at least one of the renal vessels after a certain period of time" or "unsealing the renal vessels after a certain period of time" or "unsealing at least one renal vessel after a certain period of time" are replaced by the step of "reintegrating 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 nephrons in a subject's kidney with recombinant adeno-associated virus (rAAV), the method comprising: (a) isolating the kidney from systemic circulation; (b) administering a volume of rAAV-containing solution retrogradely into the ureter of the kidney, 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) re-introducing the kidney into 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 with 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 involves 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 approximately 0.13 mL / kg to approximately 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 this administration, the nephrons of the kidney are transduced with rAAV with high efficiency.

[0154] In some aspects, this document describes a method for transducing nephrons of a subject's kidney using rAAV. The method involves 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 approximately 0.13 mL / kg to approximately 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 this administration, the nephrons of the kidney are transduced with rAAV with high efficiency.

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

[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 "therapeuticly effective" or "pharmaceutically effective" transduction level refer to a transduction level sufficient to provide transgenic expression (e.g., a transgene encoded by the rAAV genome) in the transduced kidney cells, such expression being sufficient to treat or improve at least one symptom caused or resulting from a kidney-related condition in a subject, or to inhibit, slow, minimize, or reverse the progression of a kidney-related condition in a subject. In various embodiments, a therapeutically and / or pharmaceutically effective level corresponds to a nephron transduction efficiency or a 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 implementations, 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., removing the clamp) or re-introducing the kidney into the systemic circulation, the rAAV transduction of the nephrons will be at least 30%, which has not been previously reported.

[0158] As used herein, the terms “transducing a nephron” or “transducing nephrons” may 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 known as 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 known as 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 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 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 a total of 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 out of those 1,000,000 proximal tubules are transduced. In other words, in this 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 in each of the 100,000 individual proximal tubules out of the total 1,000,000 individual proximal tubules is transduced.

[0160] Because a transduced nephron component can have more than one transduced cell, this aspect can be discussed from the perspective of "cell transduction efficiency." For example, a single nephron can 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 can 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 the transduction of other components of the same nephron. For example, cells can 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 ducts.

[0161] Therefore, in some respects, the prior art provides both nephron transduction efficiency and cell transduction efficiency. As an example, the term "at least 20% proximal tubule transduction efficiency and at least 30% proximal tubule cell transduction efficiency" means 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 of a certain percentage of nephron transduction in the kidney in this article may refer to nephron transduction efficiency, transduction efficiency of nephron components, and / or cell transduction efficiency.

[0163] In some embodiments, the method results in a proximal tubule transduction efficiency 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%, 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.

[0164] In some implementations, 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 approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more of the nephrons in the kidney being transduced by rAAV, or in other words, approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, or more of the total number of "nephron cells" (cells of the nephron) in the kidney being transduced by rAAV. 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 are transduced by rAAV. In some embodiments, 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 approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more of the nephrons in the kidney being transduced by rAAV, or in other words, approximately 5%, 10%, 15%, 20%, or 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. 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 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 tubules (see, for example, Example 3). In some embodiments, the transduced cells are epithelial cells from the loop of Henry. 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 both the distal convoluted tubule and the 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 of the proximal tubules of the kidney or epithelial cells of the proximal tubules of the kidney (see, for example, Table 11). 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. 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 renaldiseases,” Am J Physiol Renal Physiol. Dec 1, 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 Henry. 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 epithelial cells transduced from the loop of Henry. It should be understood that each of the individual percentages described in this article can be used to define the lower and upper limits of a 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 epithelial cells from the distal convoluted tubule of the kidney. It should be understood that each of the individual percentages described in this article can be used to define the lower and upper limits of a 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 the 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 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 epithelial cells from the collecting ducts of the kidney. 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 derived 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 epithelial cells from the distal convoluted tubule and / or collecting duct of the kidney (see, for example, Table 12). It should be understood that each of the individual percentages described in this article can be used to define the lower and upper limits of a percentage range.

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

[0177] In some embodiments, the nephron transduction percentage may represent the transduction percentage of all proximal tubules in the kidney. As a non-limiting example, 30% nephron transduction means the transduction of 30% of the proximal tubules or proximal tubular cells in each treated kidney. In some embodiments, at least 30% of the nephrons or nephron cells in each treated kidney are transduced, and other nephrons may be transduced at sites other than the proximal tubules. In some embodiments, the nephron transduction percentage in the kidney may be higher than the proximal tubule transduction percentage, for example, when considering transduction at other sites in the proximal tubules and nephrons.

[0178] In some embodiments, the method results in the transduction of cells and / or nephrons in at least one renal pyramid and / or associated cortical region of the kidney. A renal pyramid is cone-shaped renal tissue within 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 that 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 cells and / or nephrons in at least one renal pyramid and / or associated 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% of the kidneys. Transduction of at least one cell or at least one nephron in the renal pyramidal and / or related cortical regions at 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 the lower and upper limits of the percentage range.

[0179] In some embodiments, the amount of solution containing rAAV is from about 0.13 mL / kg of subject to about 0.33 mL / kg of subject. “kg” (kilograms) represents the subject’s weight. 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 can be adjusted according to the subject’s weight. For example, a maximum amount of 30 mL can be administered to a 90 kg subject instead of the maximum amount of 25 mL for a 75 kg subject. In some embodiments, the subject’s weight is 60 kg to 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 solution containing rAAV is 0.13 mL / kg, 0.14 mL / kg, 0.15 mL / kg, 0.16 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. 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.

[0181] In some embodiments, the amount of the 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, 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, 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 amounts described herein can be used to define a lower and upper limit for the range of amounts.

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

[0183] In some implementations, a solution containing rAAV is administered to the kidney under intrarenal pressure that causes tubular reflux and venous return without causing fornix rupture. The term "tubular reflux" (also 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 appear as a blush of the renal pyramids. The term "venous return" (also called "renal pelvic reflux") refers to the drainage of fluid from the renal pelvis into the renal venous system; venous return can occur when an abnormal amount of intrarenal pressure occurs in the opposite direction to normal. The term "renal fornix" (or "fornix") refers to a small, dot-like projection extending 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 caused by increased pressure in the renal pelvis; rupture of one or more renal fornixes may lead to 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 tubular reflux. IRP between 41 cm H2O and 68 cm H2O can cause venous reflux. IRP below 27 cm H2O does not cause tubular reflux or venous reflux and is therefore not effective for the method described herein. IRP between 81 cm H2O and 95 cm H2O may cause fornix rupture. Therefore, the method described herein uses IRP of 80 cm H2O or less. IRP above normal (e.g., for extended periods, such as several days) may be associated with infectious and hemorrhagic complications as well as 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. June 2022; 11(12): 3429; the contents of which are incorporated herein by reference in their entirety.

[0185] In some implementations, the dosage and the resulting intrarenal pressure are determined in subjects such as humans, non-human primates, or pigs, which serve as relevant animal models for human translational studies due to their anatomical and physiological similarities to humans. Intrarenal pressure can be measured using methods known in the art, such as sensor leads (e.g., leads containing pressure sensors; e.g., placed within 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 implementations, 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 smaller doses and / or smaller body size subjects, the administration time of rAAV can be shorter (e.g., for subjects weighing 8–12 kg, 2.5 mL is administered at 5 mL / min over approximately 0.5–1.0 minutes); for larger doses and / or larger body size subjects, the administration time of rAAV can be longer (e.g., for subjects weighing 60–80 kg, 18 mL is administered at 9 mL / min over approximately 1–2 minutes). Shorter rAAV administration times 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., about 25 cm H2O to about 55 cm H2O or about 27 cm H2O to about 80 cm H2O). In some embodiments, the rAAV solution is administered 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, a 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, a solution containing rAAV is administered to the kidney at an intrarenal pressure of about 46 cm H2O. In some embodiments, a solution containing rAAV 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, a solution containing rAAV is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 41 cm H2O. In some embodiments, a solution containing rAAV is administered to the kidney at an intrarenal pressure of about 41 cm H2O to about 68 cm H2O. In some embodiments, a solution containing rAAV is administered to the kidney at an intrarenal pressure of about 68 cm H2O to about 80 cm H2O. In some embodiments, a solution containing 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 solution containing rAAV is administered to the kidney at an intrarenal pressure of at most 25 cm H2O, at most 30 cm H2O, at most 35 cm H2O, at most 40 cm H2O, at most 45 cm H2O, at most 50 cm H2O, at most 55 cm H2O, at most 60 cm H2O, at most 65 cm H2O, at most 70 cm H2O, at most 75 cm H2O, or at most 80 cm H2O. In some implementations, 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 in this article 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) and / or flexible. 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) can 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) can 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 kidneys. The catheter may be inserted transurethral, ​​transbladder, and ascend through the ureter into 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 towards the kidney). In some embodiments, 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 administering 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 administration of the amount of rAAV-containing solution into the ureter of the kidney via a retrograde route is performed by injecting rAAV (e.g., a composition containing rAAV) into the ureter. The injection can be performed using a syringe that is connected to a catheter and is 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 s, 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. Injection time intervals of approximately 1.75 min, 2 min, 2.25 min, 2.5 min, 2.75 min, 3 min, 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 interval is less than or equal to approximately 5 min, less than or equal to approximately 4 min, less than or equal to approximately 3 min, less than or equal to approximately 2 min, or less than or equal to approximately 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 stated amount of rAAV-containing solution does not include continuous infusion 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 to and in fluid communication with a catheter in the ureter) for a period of up to 5 minutes, and the isolated kidney is reintegrated into the systemic circulation after a period of about 10 to about 60 minutes following isolation. In some embodiments, the isolation period is replaced by a closure period of about 10 to about 60 minutes following closure (e.g., using a balloon catheter or clamp), and the rAAV-containing solution is administered to the closed kidney using 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. The up to 5-minute administration of rAAV can be performed at any time during the 10-minute to 60-minute isolation or closure period of the kidney. For example, rAAV is administered at approximately 0-5 minutes, 5-10 minutes, 10-15 minutes, 15-20 minutes, 20-25 minutes, 25-30 minutes, 30-35 minutes, 35-40 minutes, 40-45 minutes, 45-50 minutes, 50-55 minutes, or 55-60 minutes during a 10- to 60-minute renal isolation or closure period. In some embodiments, the rAAV-containing solution is administered in discontinuous increments, for example, injection for approximately 1 minute, followed by isolation or closure for approximately 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 a solution containing rAAV. 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. With each heartbeat, oxygenated blood is carried through arteries to organs and tissues (where arteries convert to arterioles) and then to capillaries where gas exchange occurs. Deoxygenated blood is then transferred from the capillaries to venules, which convert to 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 remove deoxygenated blood from the kidneys). "Isolating the kidneys from the systemic circulation" means slowing, minimizing, substantially stopping, or stopping blood flow through a particular kidney. Isolation of the kidneys from the systemic circulation can be achieved by closing at least one renal vessel of the kidney (i.e., the renal artery and / or renal vein) and / or by diverting circulation away 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 kidneys are “isolated,” any agents delivered to the systemic circulation will not enter the kidneys and / or circulate through them. Isolating the kidneys from the systemic circulation results in reduced or absent urine production in the nephrons of the kidneys (and thus in the kidneys themselves). Thus, urine production is minimized or reduced compared to kidneys that are not isolated from or have 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 occluding at least one renal vessel of the kidney. By occluding at least one renal vessel, the kidney is isolated from the systemic circulation. In some embodiments, only the renal artery is closed and not the renal vein. In other embodiments, only the renal vein is closed and not the renal artery. In still other embodiments, both the renal artery and the renal vein are closed. Therefore, only one renal vessel may be closed, or both renal vessels may be closed. Closure of the renal vessel can be performed by methods known in the art, including by occlusion or clamping. Closure of the 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 the need for an external or auxiliary circuit. In some embodiments, neither the renal artery nor the renal vein is closed.

[0194] Occlusion of at least one renal vessel involves introducing an occluder into at least one vessel to slow, minimize, substantially stop, or stop blood flow through said at least one renal vessel. Such an occluder can be introduced using a catheter, such as a dilating catheter, balloon catheter, or perfusion catheter, as non-limiting examples. 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. Occlusion of at least one renal vessel is performed prior to the administration of a solution containing rAAV. As discussed herein, at least one renal vessel is unblocked approximately 10 to 60 minutes after closure and / or administration of a solution containing rAAV. 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 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] Closing at least one renal vessel involves 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, arterial clamps, vascular clamps, arterial clips, vascular clips, renal vein clamps, renal vein clips, Dieffenbach clamps, and hemostatic forceps. 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. Closure of at least one renal vessel is performed before the administration of a solution containing rAAV. As discussed herein, said 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 solution containing rAAV. Unblocking at least one renal vessel is done, for example, by releasing the clamp or releasing the clamp, which will restore blood flow to the kidney and reintroduce 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 a perfusion catheter in the renal artery of the kidney; (b) positioning a recovery catheter in the renal vein of the kidney, wherein the perfusion catheter and the recovery catheter, together with a membrane oxygenation device, form a closed external perfusion circuit through the kidney; and (c) allowing perfusion fluid to flow through the external circuit, wherein the external circuit isolates the perfusion through the kidney from the subject's systemic circulation. Positioning the perfusion catheter and the recovery 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 the method. Such isolation of the kidney from the systemic circulation using an external circuit 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 implementations, to avoid ischemia and / or hypoperfusion, the flow rate of the perfusion fluid circulating through the closed loop does not deviate significantly from the patient's own blood flow rate. In some implementations, blood from the recovery catheter in the renal vein is recirculated back into the perfusion catheter in the renal artery to form an external loop. While isolating the kidney from systemic circulation through the external loop, rAAV can be administered retrogradely via the ureter.

[0197] In some implementations, methods for reintegrating the kidney into the systemic circulation include 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 the systemic circulation to flow into the renal artery and out through the renal vein back into the systemic circulation. Because the external circuit allows for a continuous supply of oxygen to the kidney, reintegration of the kidney into the systemic circulation can be performed over a longer period than renal 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 encompassing the kidney. In some embodiments, the method does not include a basic closed system encompassing the kidney. In some embodiments, the method does not include shunting circulation away from the kidney. In some embodiments, the method does not involve 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 implementations, during the period in which the kidney is isolated from the systemic circulation (e.g., by occluding at least one renal vessel), the isolated kidney is maintained at a normal temperature "warm" condition (i.e., body temperature). Non-limiting examples of normal temperature conditions for the isolated kidney include about 36°C, about 36.1°C, about 36.2°C, about 36.3°C, about 36.4°C, about 36.5°C, about 36.6°C, about 36.7°C, about 36.8°C, about 36.9°C, about 37°C, about 37.1°C, about 37.2°C, about 37.3°C, about 37.4°C, about 37.5°C, about 37.6°C, about 37.7°C, about 37.8°C, about 37.9°C, or about 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 is approximately 15 minutes after isolation. In some embodiments, the period of isolation is 10-60 minutes after isolation. In some embodiments, the period of isolation is 30-60 minutes after isolation. In some embodiments, the period of isolation is 30-45 minutes after isolation. In some embodiments, the period of isolation is 15-45 minutes after isolation. In some embodiments, the period of isolation is 20-40 minutes after isolation. In some embodiments, the period of isolation is approximately 15-30 minutes after isolation. In some implementations, the kidney is isolated from the systemic circulation for approximately 30 minutes after isolation. In other implementations, 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, 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 after isolation. 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 in this document can be used to define the lower and upper limits of the time range.

[0202] In some implementations, the period of isolation of the kidney from the systemic circulation is at most 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, 36 min, 37 min, 38 min, 39 min, 40 min, 41 min, 42 min, 43 min, 44 min, 45 min, 46 min, and 47 min after isolation. The time range is defined as 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 implementations, the kidneys are isolated from the systemic circulation for approximately 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55 minutes after isolation. min, approximately 56 min, approximately 57 min, approximately 58 min, approximately 59 min, or approximately 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.

[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 isolation of 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 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 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 and / or 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 kidneys of the subject.

[0206] In some embodiments, subjects have been previously 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 the level in samples with similar cell types, sample types, sample treatments, and / or in samples obtained from subjects with similar age, sex, and other demographic parameters. 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" refers to a sample collected or isolated from a living organism, such as 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 a kidney), blood, serum, plasma, or urine. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (or pre-processed) 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 population 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 abnormal levels 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 abnormal levels 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 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 receiving the results of a determination / measurement of the analyte level in the subject on the 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 a report, result, or other means identifying the subject as having reduced analyte levels.

[0210] In one aspect of any embodiment, this document describes a method for treating kidney-related conditions in a subject with such need, the method comprising: a) determining whether the subject has an abnormal level of the analyte described herein; and b) if the analyte level 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 in 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 implementations of any aspect, the methods 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 the second dose and / or treatment may include: treatment 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 conditions (e.g., statins such as atorvastatin or simvastatin); potassium-lowering medications (e.g., sodium zirconium cyclosilicate); reduction of fluid intake; 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., quitting smoking; 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, that subject may also be given a second dose and / or treatment known to be beneficial to subjects experiencing pain or inflammation. Examples of such doses 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., cortisol, 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 further 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, said at least one immunosuppressant is selected from: prednisone, cyclosporine, tacrolimus, azathioprine, morphifencaltate, sirolimus, everolimus, alemtuzumab, and is DEPO-MEDROL® (methylprednisolone acetate). In some embodiments, the at least one immunosuppressant is administered approximately 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, on the day of rAAV administration via retrograde ureteral administration, 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. 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 immunogenicity markers, therefore at least one immunosuppressant is not administered.

[0215] Recombinant adeno-associated virus (rAAV) This article describes a method for administering recombinant adeno-associated virus (rAAV) to the kidneys of a subject. The recombinant AAV (rAAV) vector typically consists of at least a transgene and its regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). As further described herein, the transgene may contain one or more regions encoding one or more repressive RNAs (e.g., miRNAs) containing nucleic acids that target 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. The isolated nucleic acid (e.g., the recombinant AAV vector) may be packaged into a capsid protein and administered to the 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 coat selected from the coats described in Table 1. In other embodiments, rAAV includes a coat selected from the coats described in Table 1, but does not include AAV9. The exemplary coats 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.

[0217] In some implementations, 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 implementations, 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.

[0219] In some implementations, 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 One or more AAV capsid proteins, including 3xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5, AAV4E, and AAV4A.

[0220] In some embodiments, rAAV comprises an AAV capsid protein selected from AAV2, AAV6, AAVLK03, AAVDJ, AAV9A2i, AAV9A6i, 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 renal tropism, i.e., it has the ability to preferentially and efficiently infect renal cells and / or tissues.

[0222] In some implementations, 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 implementations, rAAV does not contain an AAV9 capsid.

[0224] In some embodiments, rAAV includes a garment selected from the following: AAV2G9, AAV2.5, AAVDJ, AAV2, AAVKP1, AAVKP2, AAVKP3, and AAV2.7m8. In some embodiments, rAAV includes a garment selected from the following: AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, and AAV2.7m8. In some embodiments, rAAV includes a garment selected from the following: AAVKP1, AAVKP2, and AAVKP3. In some embodiments, rAAV includes a garment of AAV2.7m8. In some embodiments, rAAV includes a garment of AAV-DJ.

[0225] In some implementations, 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 implementations, rAAV administered via the retrograde ureteral route has a transduction efficiency in the kidneys (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct) that is 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, or at least [a higher efficiency] compared to rAAV administered via other routes (e.g., IV). 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, or at least 3500 times the transduction efficiency.

[0226] In some embodiments, the rAAV exhibits at least 3500-fold higher transduction efficiency in the kidney (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct) compared to AAV9 (e.g., administered via the retrograde route as described herein). In some embodiments, the rAAV exhibits at least 100-fold higher transduction efficiency in the kidney (e.g., cells of the nephron, proximal tubule) compared to AAV9. In some embodiments, the rAAV exhibits at least 800-fold higher transduction efficiency in the kidney (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct) compared to AAV9. In some embodiments, compared to AAV9, the rAAV exhibits 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, and at least 13-fold higher activity in the kidneys (e.g., cells of the nephron, proximal tubule, loop of Henry, distal convoluted tubule, and / or collecting duct). 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, or at least 3500 times the transduction efficiency.

[0227] In some implementations, rAAVs comprise rational polyploids. As used herein, the term "rational polyploid" refers to an AAV vector composed of capsids from two or more AAV serotypes, which 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 "rational polyploid" virions to describe the fact that the capsid proteins VP1, VP2, and VP3 originate 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 originate 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 originate from three different serotypes. In particular, such reasonable polyploid (e.g., reasonable 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 contains 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 directly involve or are adjacent to Gal recognition sites 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 its variants contain the A266S mutation. In some embodiments, the AAV2G9 VP3 capsid protein or its variants do not contain the A266S mutation. In some embodiments, the AAV2G9 VP3 capsid protein or its variants contain the A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A mutations. In some embodiments, the AAV2G9 VP3 capsid protein or its variants contain the Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A mutations. Regardless of the presence or absence of the A266S mutation, the differences between 2G9 capsids are negligible. 2G9 may also include other variations that do not affect its general properties (e.g., tropism, transduction efficacy in the kidney (e.g., PCT)).

[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), at the following insertion sites: 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) MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 2, AAV9 VP3 capsid protein (see, for example, SEQ ID NO: 75 of WO2014144229), 736 aa MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL 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 highlighted in bold and underlined text: A266S, Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F and S501A (AAV2 VP3 numbers). MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLV EEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSG S SNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPP FPADVFFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS V AG P S NMAV Q G RNWLPGPCYRQQRVSKTSADNNNSE FAWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL SEQ ID NO: 4, exemplary AAV2G9 VP3 capsid protein, with the following mutations indicated in bold, underlined text: Q464V, A467P, D469N, I470M, R471A, D472V, S474G, Y500F, and S501A (AAV2 VP3 numbering); see, e.g., SEQ ID NO: 24 of US20220354969. MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFS V AG P S NMAV Q G RNWLPGPCYRQQRVSKTSADNNNSE FAWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIW AKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL 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 implementations, rAAV comprises 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 receptor). 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 implementations, rAAV contains a capsid protein derived from serotype AAVDJ. AAV-DJ is a highly recombinant hybridization vector created through experiments involving DNA shuffling of eight AAV serotypes. AAV-DJ is a chimera of AAV types 2 / 8 / 9. AAV2 and AAV8 are the closest parent vectors of AAV-DJ. Mutations at ubiquitination or phosphorylation sites 137 / 251 / 503 of the AAV2 or AAV8 capsid have been reported to result in 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,” JVirol. June 2008; 82(12): 5887–5911; Mao et al., “Single point mutation in adeno-associated viral vectors -DJ capsid leads to improvement for gene delivery invivo,” 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 isolated from human DNA in 2004. See, for example, Samulski et al., “Rescue of adeno-associated virus 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 in treating kidney-related conditions. In some embodiments, the solution contains a concentration of 10... 8 One viral genome / mL (vg / mL) to 10 15 vg / mL, 10 9 vg / mL to 10 15 vg / mL, 10 10 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 15 vg / mL, 10 12 vg / mL to 10 15 vg / mL, 10 13 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 12 vg / mL, 10 12 vg / mL to 10 13 vg / mL, 10 13 vg / mL to 10 14 vg / mL, 10 14 vg / mL to 10 15 vg / mL, 10 8 vg / mL to 10 14 vg / mL, 10 8 vg / mL to 10 13 vg / mL, 10 8 vg / mL to 10 12 vg / mL, 10 8 vg / mL to 10 11 vg / mL, 10 8 vg / mL to 10 10 vg / mL or 10 8 vg / mL to 10 9 vg / mL rAAV. In some embodiments, the solution contains a concentration of 10 8 vg / mL to 10 13 vg / mL of rAAV. In some embodiments, the solution contains at least 10 vg / mL of rAAV. 8 vg / mL, at least 10 9 vg / mL, at least 10 10 vg / mL, at least 10 11 vg / mL, at least 10 12 vg / mL, at least 10 13 vg / mL, at least 10 14vg / mL, at least 10 15 vg / mL or higher rAAV. In some embodiments, the solution contains a concentration of up to 10 9 vg / mL, up to 10 10 vg / mL, up to 10 11 vg / mL, up to 10 12 vg / mL, up to 10 13 vg / mL, up to 10 14 vg / mL or up to 10 15 rAAV in vg / mL. In some implementations, rAAV is approximately 5.2 x 10⁻⁶. 10 A concentration of vg / kg, for example, in a 75 kg subject. In some embodiments, the solution contains a concentration of about 1 x 10⁻⁶. 10 vg / kg, approximately 2 x 10 10 vg / kg, approximately 3 x 10 10 vg / kg, approximately 4 x 10 10 vg / kg, approximately 5 x 10 10 vg / kg, approximately 6 x 10 10 vg / kg, approximately 7 x 10 10 vg / kg, approximately 8 x 10 10 vg / kg, approximately 9 x 10 10 rAAV concentrations of 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 the lower and upper limits of the rAAV concentration range.

[0237] In some implementations, the solution contains a total of 1x10 for every 75 kg of subject. 11 Up to 1x10 14 vg, Total 1x10 11 Up to 1x10 13 vg, Total 1x10 11 Up to 1x10 12 vg, Total 1x10 12 Up to 1x10 13 vg, Total 1x10 12 Up to 1x10 14 vg, Total 1x10 13 Up to 1x10 14 vg, Total 1x10 13 Up to 6x10 13 vg, Total 2x10 13 Up to 5x10 13 vg or a total of 1x10 13 Up to 2x10 13Each rAAV viral genome. In some embodiments, the solution contains a total of 5 x 10n 13 Up to 6x10 13 One rAAV viral genome. In some embodiments, the solution contains a total of at least 1 x 10n 13 At least 2x10 13 At least 3x10 13 At least 4x10 13 At least 5x10 13 At least 6x10 13 At least 7x10 13 At least 8x10 13 At least 9x10 13 One or more rAAV viral genomes. In some embodiments, the solution contains a total of up to 1x102 13 At most 2x10 13 At most 3x10 13 At most 4x10 13 At most 5x10 13 At most 6x10 13 At most 7x10 13 At most 8x10 13 At most 9x10 13 One or more rAAV viral genomes. In some embodiments, the solution contains a total of approximately 1 x 102 11 1 vg, approximately 2 x 10 in total 11 1 vg, approximately 3 x 10 in total 11 1 vg, approximately 4 x 10 in total 11 5 vg, approximately 5 x 10 11 1 vg, approximately 6x10 in total 11 1 vg, approximately 7 x 10 in total 11 8 vg, approximately 8x10 in total 11 1 vg, approximately 9x10 in total 11 10 vg, approximately 10x10 in total 11 One vg, approximately 1x10 in total 12 1 vg, approximately 2 x 10 in total 12 1 vg, approximately 3 x 10 in total 12 1 vg, approximately 4 x 10 in total 12 5 vg, approximately 5 x 10 12 1 vg, approximately 6x10 in total 12 1 vg, approximately 7 x 10 in total 12 8 vg, approximately 8x10 in total 12 1 vg, approximately 9x10 in total 12 10 vg, approximately 10x10 in total 12 One vg, approximately 1x10 in total 131 vg, approximately 2 x 10 in total 13 1 vg, approximately 3 x 10 in total 13 1 vg, approximately 4 x 10 in total 13 5 vg, approximately 5 x 10 13 1 vg, approximately 6x10 in total 13 1 vg, approximately 7 x 10 in total 13 8 vg, approximately 8x10 in total 13 1 vg, approximately 9x10 in total 13 10 vg, approximately 10x10 in total 13 One vg or approximately 1x10 in total 14 Each vg. It should be understood that each of the individual rAAV quantities described in this article can be used to define the lower and upper limits of the range of the rAAV quantity.

[0238] In some implementations, the solution contains a total of 1x10 10 One viral genome. In some implementations, the solution contains a total of 1x10^6 viral genomes. 10 4x10 viral genomes (e.g., 4x10) 8 vg / mL to 1x10 9 (vg / mL).

[0239] In some implementations, the rAAV genome 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 into cells, the rAAV vector enters the cell and delivers its single-stranded DNA genome to the cell nucleus, where 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 implementations, the transgenic gene has a therapeutic effect on kidney-related conditions. In some implementations, the kidney-related conditions for which the transgenic gene 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).

[0243] In some implementations, the transgene contains a gene that is effective in treating kidney-related conditions when expressed in subjects (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 implementation schemes, the kidney-related conditions for which transgenics 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, 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, iminoglycinuria, 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 are listed in Table 2B.

[0245] Table 2B: Exemplary Kidney-Related Diseases (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). Exemplary tubular diseases are listed, grouped by affected nephron segment, underlying 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.

[0246] In some implementations, the kidney-related conditions for which the transgene has a therapeutic effect are associated with the proximal tubule and selected from: A-type cystinuria, B-type 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, iminoglycinuria, lysineuria protein intolerance, type 3 renal tubular acidosis, and X-linked hypophosphatemic rickets.

[0247] In some implementations, 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 implementations, 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 pseudoketoaldosteronism, type 2c pseudoketoaldosteronism, type 2d pseudoketoaldosteronism, and type 2e pseudoketoaldosteronism.

[0249] In some implementations, the kidney-related conditions for which the transgene has a therapeutic effect are associated with collecting ducts and selected from: episodic mineralocorticoid hyperplasia, 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 implementations, the transgene contains a gene that is effective in treating kidney-related conditions when expressed in subjects (e.g., at approximately physiological levels). In some implementations, the transgene includes genes 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 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 receptor 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 any combination thereof.

[0251] In some implementations, the transgene contains 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(0)AT1, b(0,+)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, protein containing WD repeat 72, WNK1, WNK4, y(+)LAT1 and any combination thereof.

[0252] In some implementations, 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); painful urination; 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 an amino acid transporter (b0,+ transporter system) whose function is to reabsorb cystine from urine into the renal tubules. In some embodiments, the kidney-related condition is A-type cystinuria, and the transgene is SLC3A1. In some embodiments, the kidney-related 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] In 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, Solute Carrier Family 3 Member 1 (SLC3A1), NCBI ref NP_000332.2, Human, 685 amino acids (aa) MAEDKSKRDSIEMSMKGCQTNNGFVHNEDILEQTPDPGSSTDNLKHSTRGILGSQEPDFKGVQPYAGMPKEVLFQFSGQARYRIPREILFWLTVASVLVLIAATIAIIALSPKCLDWWQEGPMYQIYPRSFKDSNKDGNGDLKGIQDKLDYITALNIKTVWITSFYKSSLK DFRYGVEDFREVDPIFGTMEDFENLVAAIHDKGLKLIIDFIPNHTSDKHIWFQLSRTRTGKYTDYYIWHDCTHENGKTIPPNNWLSVYGNSSWHFDEVRNQCYFHQFMKEQPDLNFRNPDVQEEIKEILRFWLTKGVDGFSLDAVKFLLEAKHLRDEIQVNKTQIPDTVTQ YSELYHDFTTTQVGMHDIVRSFRQTMDQYSTEPGRYRFMGTEAYAESIDRTVMYYGLPFIQEADFPFNNYLSMLDTVSGNSVYEVITSWMENMPEGKWPNWMIGGPDSRLTSRLGNQYVNVMNMLLFTLPGTPITYYGEEIGMGNIVAANLNESYDINTLRSKSPMQWDN SSNAGFSEASNTWLPTNSDYHTVNVDVQKTQPRSALKLYQDLSLLHANELLLNRGWFCHLRNDSHYVVYTRELDGIDRIFIVVLNFGESTLLNLHNMISGLPAKMRIRLSTNSADKGSKVDTSGIFLDKGEGLIFEHNTKNLLHRQTAFRDRCFVSNRACYSSVLNILYTSC SEQ ID NO: 8, Solute Carrier Family 7 Member 9 (SLC7A9), NCBI ref NP_001119807.1, Human, 487 aa MGDTGLRKRREDEKSIQSQEPKTTSLQKELGLISGISIIVGTIIGSGIFVSPKSVLSNTEAVGPCLIIWAACGVLATLGALCFAELGTMITKSGGEYPYLMEAYGPIPAYLFSWASLIVIKPTSFAIICLSFSEYVCAPFYVGCKPPQIVVKCLAAAAILFISTVNSLSVRLGSYVQNIFTAAKLVIVAIIIISGLVLLAQGNTKNFDNSFEGAQLSVGAISLAFYNGLWAYDGWNQLNYITEELRNPYRNLPLAIIIGIPLVTACYILMNVSYFTVMTATELLQSQAVAVTFGDRVLYPASWIVPLFVAFSTIGAANGTCFTAGRLIYVAGREGHMLKVLSYISVRRLTPAPAIIFYGIIATIYIIPGDINSLVNYFSFAAWLFYGLTILGLIVMRFTRKELERPIKVPVVIPVLMTLISVFLVLAPIISKPTWEYLYCVLFILSGLLFYFLFVHYKFGWAQKISKPITMHLQMLMEVVPPEEDPE 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 (e.g., distal convoluted tubules) and collecting ducts (CDs). Symptoms of polycystic kidney disease include, but are not limited to, 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 a mass in the kidneys or abdomen. In some implementations, the transgenes are 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 tubular formation, and affect growth and fluid secretion function. Mutations in the PKD1 or PKD2 genes produce dysfunctional cells and ultimately lead to cystic growths 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, 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 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) when expressed as a protein.

[0258] In some embodiments, the transgene encodes a polypeptide comprising one of SEQ ID NO: 11-12 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 one of SEQ ID NO: 11-12 and maintaining the same function (e.g., regulating renal cells, influencing the formation of renal tubular structures, and influencing renal fluid secretion function), or a functional fragment thereof.

[0259] SEQ ID NO: 12, Polycystin-2 (PKD2), NCBI ref NP_000288.1, Human, 968 aa MVNSSRVQPQQPGDAKRPPAPRAPDPGRLMAGCAAVGASLAAPGGLCEQRGLEIEMQRIRQAAARDPPAGAAASPSPPLSSCSRQAWSRDNPGFEAEEEEEEVEGEEGGMVVEMDVEWRPGSRRSAASSAVSSVGARSRGLGGYHGAGHPSGRRRRREDQGPPCPSPVGGGDPLHRHLPLEGQPPRVAWAERLVRGLRGLWGTRLMEESSTNREKYLKSVLRELVTYLLFLIVLCILTYGMMSSNVYYYTRMMSQLFLDTPVSKTEKTNFKTLSSMEDFWKFTEGSLLDGLYWKMQPSNQTEADNRSFIFYENLLLGVPRIRQLRVRNGSCSIPQDLRDEIKECYDVYSVSSEDRAPFGPRNGTAWIYTSEKDLNGSSHWGIIATYSGAGYYLDLSRTREETAAQVASLKKNVWLDRGTRATFIDFSVYNANINLFCVVRLLVEFPATGGVIPSWQFQPLKLIRYVTTFDFFLAACEIIFCFFIFYYVVEEILEIRIHKLHYFRSFWNCLDVVIVVLSVVAIGINIYRTSNVEVLLQFLEDQNTFPNFEHLAYWQIQFNNIAAVTVFFVWIKLFKFINFNRTMSQLSTTMSRCAKDLFGFAIMFFIIFLAYAQLAYLVFGTQVDDFSTFQECIFTQFRIILGDINFAEIEEANRVLGPIYFTTFVFFMFFILLNMFLAIINDTYSEVKSDLAQQKAEMELSDLIRKGYHKALVKLKLKKNTVDDISESLRQGGGKLNFDELRQDLKGKGHTDAEIEAIFTKYDQDGDQELTEHEHQQMRDDLEKEREDLDLDHSSLPRPMSSRSFPRSLDDSEEDDDEDSGHSSRRRGSISSGVSYEEFQVLVRRVDRMEHSIGSIVSKIDAVIVKLEIMERAKLKRREVLGRLLDGVAEDERLGRDSEIHREQMERLVREELERWESDDAASQISHGLGTPVGLNGQPRPRSSRPSSSQSTEGMEGAGGNGSSNVHV. In some embodiments, the transgene comprises a nucleic 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: 13 and retaining the same function when expressed as a protein (e.g., cleaving glucose residues from immature glycoproteins), or a functional fragment thereof, or a codon-optimized version of the nucleic acid.

[0260] In some embodiments, the transgene encodes a polypeptide comprising: SEQ ID NO: 14 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: 14 and retaining the same function (e.g., cleaving glucose residues from an immature glycoprotein), or a functional fragment thereof.

[0261] SEQ ID NO: 14, Glucosidase II alpha subunit (GANAB), NCBI ref NP_001265121.1, Homo sapiens, 852 aa MAAVAAVAARRRRLSVSGRDENSVELTMAEGPYKIILTARPFRLDLLEDRSLLLSVNARGLLEFEHQRAPRVSFSDKVNLTLGSIWDKIKNLFSRQGSKDPAEGDGAQPEETPRDGDKPEETQGKAEKDEPGAWEETFKTHSDSKPYGPMSVGLDFSLPGMEHVYGIPEHADNLRLKVTEGGEPYRLYNLDVFQYELYNPMALYGSVPVLLAHNPHRDLGIFWLNAAETWVDISSNTAGKTLFGKMMDYLQGSGETPQTDVRWMSETGIIDVFLLLGPSISDVFRQYASLTGTQALPPLFSLGYHQSRWNYRDEADVLEVDQGFDDHNLPCDVIWLDIEHADGKRYFTWDPSRFPQPRTMLERLASKRRKLVAIVDPHIKVDSGYRVHEELRNLGLYVKTRDGSDYEGWCWPGSAGYPDFTNPTMRAWWANMFSYDNYEGSAPNLFVWNDMNEPSVFNGPEVTMLKDAQHYGGWEHRDVHNIYGLYVHMATADGLRQRSGGMERPFVLARAFFAGSQRFGAVWTGDNTAEWDHLKISIPMCLSLGLVGLSFCGADVGGFFKNPEPELLVRWYQMGAYQPFFRAHAHLDTGRREPWLLPSQHNDIIRDALGQRYSLLPFWYTLLYQAHREGIPVMRPLWVQYPQDVTTFNIDDQYLLGDALLVHPVSDSGAHGVQVYLPGQGEVWYDIQSYQKHHGPQTLYLPVTLSSIPVFQRGGTIVPRWMRVRRSSECMKDDPITLFVALSPQGTAQGELFLDDGHTFNYQTRQEFLLRRFSFSGNTLVSSSADPEGHFETPIWIERVVIIGAGKPAAVVLQTKGSPESRLSFQHDPETSVLVLRKPGINVASDWSIHLR In 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: 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: (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: (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: (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 contains any combination of: (a) a reporter gene and (c) an inhibitor of a gene or protein that is effective in treating kidney-related conditions. In some implementations, 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 (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", 2ded., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., JVirol., 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 also includes 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., an ATRS ITR). Without being bound by any particular theory, rAAV vectors containing ITRs lacking functional TRS generate self-complementary rAAV vectors, as described, for example, in McCarthy (2008) Molecular Therapy 16(10): 1648-1656.

[0266] Kidney-related diseases In some implementations, the methods described herein involve treating subjects who have or are diagnosed with kidney-related conditions. In some aspects, methods for treating kidney-related conditions in subjects with this need include administering recombinant adeno-associated virus (rAAV) to the subject via retrograde ureteral administration 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 and feet, dry and / or itchy skin, shortness of breath, difficulty sleeping, excessive or insufficient urination, metallic taste in the mouth, coldness, etc. Tests that can 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; kidney ultrasound; kidney 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; kidney structural abnormalities) can also help determine whether a subject is likely to have kidney-related conditions or to make a diagnosis of kidney-related conditions.

[0267] In some implementations, 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 cystine storage disease; 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 implementation schemes, kidney-related conditions are selected from: episodic mineralocorticoid hyperthyroidism 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 Hypoplasmosis, 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 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, 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. This reduction, compared to an equivalent untreated control, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or more, as measured by any standard technique.

[0270] For example, in the case of cystinuria, the incidence of cystinuria symptoms (e.g., crystalluria (crystallization in urine); aminoaciduria (abnormally high levels of amino acids (e.g., cystine) in 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 infection; and / or fever) can be evaluated to determine if any of these symptoms are reduced.

[0271] As another example, for polycystic kidney disease (e.g., ADPKD), it is possible to evaluate whether 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) in a subject is reduced, any of which may 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 doses, toxicity, and efficacy can be determined using standard pharmaceutical procedures in cell culture or laboratory animals, for example, to determine the minimum effective dose and / or maximum tolerated dose. Dosage can vary depending on the dosage form and administration regimen used. Therapeuticly effective doses can be estimated initially using cell culture assays. Alternatively, doses can be formulated in animal models to achieve a dose range between the minimum effective dose and the maximum tolerated dose. The effect of any specific dose can be monitored using appropriate bioassays. Dosage can be determined by a physician and adjusted as needed to accommodate observed therapeutic effects.

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

[0275] In some embodiments, kidney-related conditions are treated when rAAV transduces cells in the kidney, resulting in the expression of transgenic and / or gene or protein inhibitors effective in treating kidney-related conditions. To transduce cells, an rAAV vector enters a kidney cell and delivers its single-stranded DNA genome to the cell nucleus, where the genome becomes double-stranded, 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 transgenic gene 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 the nephron of 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 transduces the following renal tubules: the glomerulus of the kidney (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 kidney cells: vascular cells (e.g., glomerular endothelial cells); mesangial, smooth muscle (SMC) or juxtaglomerular (JG) cells (e.g., mesangial cells, SMC, pericytes and / or JG); 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 (LO). H), plaque-dense (MD) cells (e.g., the descending limb of the LOH, the ascending limb of the LOH, the thick ascending limb of the LOH, and / or 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 principal cells, Pan-CD intercalation cells, CD intercalation cells (type A), CD intercalation cells (type B), and / or CD transition cells).

[0278] In some implementations, rAAV transduction is selected from the following kidney cells: glomerular endothelial cells, mesangial cells, SMCs, pericytes, JGs, adult podocytes, podocyte progenitor cells, Pan-PTs, proximal convoluted tubules, proximal straight tubules, PT progenitor cells, damaged PTs, the descending limb of the LOH, the ascending limb of the LOH, the thick ascending limb of the LOH, plaque dense (MD), DCT1, DCT2, CNTs, CD chief cells, Pan-CD intercalation cells, CD intercalation cells (type A), CD intercalation cells (type B), and / or CD transition cells.

[0279] In some implementations, rAAV transduction is selected from renal cells including: 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 (SMCs) / juxtaglomerular cells (JGs), 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-restrictive examples of JG-specific marker genes include Ren1, Akr1b7, Rgs5 ); podocytes, including but not limited to adult podocytes (non-limiting examples of adult podocyte-specific marker genes include Nphs1, Nphs2, Synpo, Cdkn1c, Wt1 ) and / or podocyte progenitor cells (non-limiting examples of podocyte progenitor cell-specific marker genes include Wt1, Foxc2, Mafb, Efnb2, Foxl1 ); proximal tubule (PT) cells, including but not limited to Pan-PT (non-limiting 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 tubule (non-restrictive examples of proximal convoluted tubule-specific marker genes include Slc5a2, Slc5a12 Adra1a, Slc6a19, Slc7a8, Slc7a9 ), proximal tubules (non-restrictive examples of proximal 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 impaired PT (non-limiting examples of impaired PT-specific marker genes include Havcr1, Krt20, Hspa1a, Vcam1, Dcdc2a, Sema5a ); Loop Henry (LOH) / dense plaque (MD) cells, including but not limited to the descending segment of the LOH (non-limiting examples of LOH descending segment-specific marker genes include Fst, Aqp1, Slc14a2, Bst1, Epha7, Cryab, Tshz2, Cald1, Bst1, Lypd2 ), ascending limb segments of LOH (non-restricted examples of LOH ascending limb segment-specific marker genes include Epha7 Mx2, Clcnka The thick ascending limb of LOH (non-restricted examples of LOH-specific marker genes include...) Slc12a1, Umod, Tmem207, Foxq1, Cldn10, Ptger3, Kcnj1, Enox1, Thsd4, Mt2, Slc5a3 ) and / or dense spots (MD) (non-restrictive 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-limiting 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 CNTs (non-restrictive examples of CNT-specific marker genes include Calb1, Slc8a1, Egf, Klk1, Trpv5, Trpm6, S100g, Atp2b1, Scnn1b, Scnn1g, Kcne7 ); and / or collecting duct (CD) cells, including but not limited to CD chief cells (non-limiting examples of CD chief cell-specific marker genes include Scnn1b, Scnn1g, 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) (non-restricted examples of CD intercalation cell (type B) 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, Sec23bSee, 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 rAAV genome 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 in kidney cells is increased by at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, 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 up to and including 100% or any increase between 10 and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold, or any increase between 10-fold and 100-fold, or greater. In some implementations, a kidney-specific promoter is operatively linked to at least one transgene, such as (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.

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

[0282] In some embodiments, kidney-specific promoters are used for genes that are naturally and specifically expressed in kidney cells or tissues. In some embodiments, kidney-specific promoters are 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 AAVvectors,” 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 programmed to be selectively active in the proximal tubules, distal convoluted tubules, and / or collecting ducts of the kidney. The promoter sequence can be tested in vitro for specific activation in kidney cells (e.g., reporter protein expression), 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.) and 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 further screened in animal models such as mice, pigs, or non-human primates. Specific activation of promoter sequences in vivo can be evaluated using co-staining with 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, 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 the translation of the coding sequence into a functional protein is desired, then the two DNA sequences are described as operablely conjugated if inducing the 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 the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Therefore, a promoter region is operablely conjugated to a coding sequence if it can influence the transcription of the DNA sequence, thereby allowing the resulting transcript to be translated into the desired protein or polypeptide.

[0286] When a nucleic acid molecule encoding any of the polypeptides described herein is expressed in a cell, its expression can be directed using a variety of transcriptional control sequences (e.g., promoter / enhancer sequences). A promoter can be a natural promoter, i.e., a promoter of the gene in its endogenous environment that provides normal regulation of gene expression. In some embodiments, the promoter can be constitutive, i.e., the promoter is unregulated, thereby allowing continuous transcription of its associated gene. Various conditional promoters can also be used, such as promoters controlled by the presence or absence of the molecule.

[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 involved in transcription and translation respectively, such as TATA boxes, capping sequences, CAAT sequences, etc. In particular, such 5′ non-transcriptional regulatory sequences will include a promoter region comprising a promoter sequence for transcriptional control of the gene for operatively binding. The regulatory sequences may also include enhancer sequences or upstream activator sequences as needed. The vectors of the present invention may optionally include a 5′ leader or signal sequence. The selection and design of suitable vectors is within the competence and judgment of those skilled in the art.

[0288] In some implementations, one or more recombinantly expressed transgenes can be integrated into the cell's genome. Such genomic integration can allow for the stable expression of nucleic acids or proteins that are effective in treating kidney-related conditions.

[0289] Subsequent application methods This article describes a method for subsequent or re-administered rAAV (containing the same or different serotypes of the capsid) 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 for treating kidney disease in a subject with this 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 same 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 immune response to the second rAAV that results in a reduction in the efficacy or transduction of the first and / or second rAAV.

[0292] In one aspect, this document describes a method for treating kidney disease in subjects who are seropositive for a recombinant adeno-associated virus (rAAV) therapeutic agent, the method comprising administering to the subject's kidney 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 at a rate 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 body 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.

[0295] 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 the 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 body 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 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 25 cm H2O to about 55 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 rAAVs) is administered to the kidney at an intrarenal pressure of about 27 cm H2O to about 80 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 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 implementations, 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) are 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, at least one rAAV comprising a first rAAV and / or a second rAAV is administered to the kidney under 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. (and / or solutions of the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more rAAV). 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 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 first and / or second rAAV.

[0299] 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 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 volume 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 volume 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 elicits an immune response in the kidney.

[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 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; (b) administering a first solution containing a first rAAV into the ureter of the kidney via a retrograde route, wherein the volume 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 volume 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 elicits an immune response in the kidney.

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

[0303] In some implementations of any aspect, the steps such as "unblocking the renal vessels after the first closure period" or "unblocking the at least one renal vessel after the first closure period" are replaced by the step of "reintegrating the kidney into the systemic circulation after the first isolation period".

[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 first rAAV administration includes closure of at least one renal vessel, while the second rAAV administration includes isolation of the kidney from the systemic circulation. In some embodiments of any aspect, the second rAAV administration includes closure of at least one renal vessel, while the first rAAV administration includes isolation of the kidney from the systemic circulation. In some embodiments of any aspect, both the first and second rAAV administrations include closure of at least one renal vessel. In some embodiments of any aspect, both the first and second rAAV administrations include isolation of the kidney from the systemic circulation.

[0305] In some implementations, 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 implementations, steps (a)–(c) are repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, for example, using the same or different rAAVs 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, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, 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: 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. It should be understood that the captions can be all the same, all different, or some the same and some 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 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: 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 be the same and some be 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 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: AAV2, AAVDJ, AAVJEA (minimal), AAV2g9, or AAV2.5 (see example). Figure 5 It should be understood that the outer shells can all be the same, all be different, or some be the same and some be different.

[0309] In some implementations, the capsids of the first rAAV, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more are reasonably polyploid.

[0310] 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 independently selected from: AAV2G9, AAV2.5, AAVDJ, and AAV2.

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

[0312] 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 is 10. 8 One viral genome / mL (vg / mL) to 10 15 vg / mL, 10 9 vg / mL to 10 15 vg / mL, 10 10 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 15 vg / mL, 10 12 vg / mL to 10 15 vg / mL, 10 13 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 12 vg / mL, 10 12 vg / mL to 10 13 vg / mL, 10 13 vg / mL to 10 14 vg / mL, 10 14vg / mL to 10 15 vg / mL, 10 8 vg / mL to 10 14 vg / mL, 10 8 vg / mL to 10 13 vg / mL, 10 8 vg / mL to 10 12 vg / mL, 10 8 vg / mL to 10 11 vg / mL, 10 8 vg / mL to 10 10 vg / mL or 10 8 vg / mL to 10 9 The concentration is 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 is 10. 8 vg / mL to 10 13 The concentration is 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 is at least 10. 8 vg / mL, at least 10 9 vg / mL, at least 10 10 vg / mL, at least 10 11 vg / mL, at least 10 12 vg / mL, at least 10 13 vg / mL, at least 10 14 vg / mL, at least 10 15 Vg / mL or higher concentrations. 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 is up to 10 9 vg / mL, up to 10 10 vg / mL, up to 10 11 vg / mL, up to 10 12 vg / mL, up to 10 13 vg / mL, up to 10 14 vg / mL or up to 10 15 The concentration is expressed in vg / mL. It should be understood that each of the individual rAAV concentrations described herein can be used to define the lower and upper limits of the rAAV concentration range.

[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 1x10 13 Up to 2x10 13 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 5 x 10 13 Up to 6x10 13 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 1 x 102 13 At least 2x10 13 At least 3x10 13 At least 4x10 13 At least 5x10 13 At least 6x10 13 At least 7x10 13 At least 8x10 13 At least 9x10 13 One 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. 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 1x10 13 At most 2x10 13 At most 3x10 13 At most 4x10 13 At most 5x10 13 At most 6x10 13 At most 7x10 13 At most 8x10 13 At most 9x10 13 Total 1x10 11 Up to 1x10 14 vg, Total 1x10 11 Up to 1x10 13 vg, Total 1x10 11 Up to 1x10 12vg, Total 1x10 12 Up to 1x10 13 vg, Total 1x10 12 Up to 1x10 14 vg, Total 1x10 13 Up to 1x10 14 vg, Total 1x10 13 Up to 6x10 13 vg, Total 2x10 13 Up to 5x10 13 vg or a total of 1x10 13 Up to 2x10 13 One 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 the lower and upper limits of 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 1x10 10 4x10 viral genomes (e.g., 4x10) 8 vg / mL to 1x10 9 (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 transgene of the first rAAV is different from 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, second rAAV, third rAAV, fourth rAAV, fifth rAAV, sixth rAAV, seventh rAAV, eighth rAAV, ninth rAAV, tenth rAAV, or more transgenes are selected from: (a) a reporter gene, (b) a gene that is effective in treating kidney-related conditions when expressed in a subject, (c) an inhibitor of a gene or protein that is 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, which is 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, which is different from 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.

[0317] In some embodiments, the subsequent application of the second solution is for at least one day. In some embodiments, the subsequent application period 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 application 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 for subsequent application 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 determined based on the efficacy or persistence of the application of at least one of the previous solutions containing rAAV.

[0319] Efficacy or durability can be determined, for example, by examining renal rAAV transduction (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 renal-related conditions (treated with rAAV). If efficacy or durability falls below a certain level, the subject is given a solution containing 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 to confirm that retrograde rAAV administration to the first kidney is effective 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. Thus, retrograde rAAV administration to the contralateral kidney may be delayed relative to retrograde rAAV administration to the first kidney for 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, the first solution is administered to the subject's first kidney, while the second solution is administered to the subject's second kidney. In some embodiments, the first solution (or the second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more solutions) is administered to the subject's first kidney, while the second solution (or the first, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more solutions) 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, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more rAAVs is each administered to the same kidney of the subject. In some embodiments, at least one solution containing a first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, 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 containing a first rAAV is administered to both kidneys of the subject, and a solution containing a second rAAV is administered to both kidneys of the subject. 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 both kidneys of the subject. 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 one kidney, both kidneys, or a combination thereof of the subject.

[0324] In some embodiments, at least one solution comprising 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, including 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, is approximately 0.13 mL / kg to approximately 0.33 mL / kg, approximately 0.20 mL / kg to approximately 0.27 mL / kg, approximately 0.27 mL / kg to approximately 0.33 mL / kg, approximately 0.13 mL / kg to approximately 0.35 mL / kg, approximately 0.15 mL / kg to approximately 0.35 mL / kg, approximately 0.2 mL / kg to approximately 0.35 mL / kg, approximately 0.25 mL / kg to approximately 0.35 mL / kg, approximately 0.3 mL / kg to approximately 0.35 mL / kg, approximately 0.13 mL / kg to approximately 0.30 mL / kg. 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 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 solution is at least 0.13 mL / kg, at least 0.20 mL / kg, or at least 0.27 mL / kg. In some embodiments, the amount 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 solution is up to 0.33 mL / kg.

[0325] In some embodiments, the amounts 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 solutions are 0.13 mL / kg, 0.14 mL / kg, 0.15 mL / kg, 0.16 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, etc. mL / kg, 0.33 mL / kg, 0.34mL / kg, 0.35 mL / kg, 0.05-0.15 mL / kg, 0.10-0.20 mL / kg, 0.15-0.25 mL / kg, 0.20-0.30mL / 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.

[0326] In some embodiments, the amount 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 solution is selected from 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, and at least 0.27 mL / kg. 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, at least 0.33 mL / kg, at least 0.34 mL / kg, at least 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, 0.10-0.35 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 The concentrations are 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. In some embodiments, the amount 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 solution is selected from up to 0.30 mL / kg of the subject, up to 0.31 mL / kg of the subject, up to 0.32 mL / kg of the subject, up to 0.33 mL / kg of the subject, up to 0.34 mL / kg of the subject, or up to 0.35 mL / kg of the subject.The amount 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 solution is approximately 0.24 mL / kg. It should be understood that each of the individual amounts described herein can be used to define the lower and upper limits of the range of amounts.

[0327] In some embodiments, the amounts 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.) solutions are the same as the amounts 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.) solutions. In some embodiments, the amounts 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.) solutions are different from the amounts 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.) solutions. In some implementations, the amount 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.) solution can be adjusted by those skilled in the art (such as physicians or other medical professionals).

[0328] In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is such that ischemic injury to the kidneys does not occur. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is approximately 15 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is 10-60 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is 15-45 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is 20-40 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is 30-60 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is approximately 15-30 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is approximately 30 minutes after isolation or closure. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is no more than 45 minutes after isolation or closure.

[0329] In some implementations, the isolation or closure period for 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.) kidneys of at least one renal vessel 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, at least 43 min, at least 44 min, at least 45 min, at least 46 min, etc., after isolation or closure. The time ranges are: 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.

[0330] In some implementations, the isolation or closure period for 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.) kidneys of at least one renal vessel is at most 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, 36 min, 37 min, 38 min, 39 min, or 40 min after isolation or closure. The time range is defined as follows: min, up to 41 min, up to 42 min, up to 43 min, up to 44 min, up to 45 min, up to 46 min, up to 47 min, up to 48 min, up to 49 min, up to 50 min, up to 51 min, up to 52 min, up to 53 min, up to 54 min, up to 55 min, up to 56 min, up to 57 min, up to 58 min, up to 59 min, or up to 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.

[0331] In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is the same as the isolation or closure period for 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.) kidneys. In some embodiments, the isolation or closure period for 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.) kidneys of at least one renal vessel is different from the isolation or closure period for 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.) kidneys. 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.) isolation or closure periods can be adjusted by those skilled in the art (such as physicians or other medical professionals).

[0332] In aspects involving at least one subsequent rAAV administration, each rAAV administration may be performed as described herein. In some embodiments, at least one solution containing 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, including 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, is administered to the ureter via a catheter in a retrograde manner. In some embodiments, the catheter used for administering 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 solutions is a balloon catheter. In some embodiments, the balloon catheter is inflated to close the ureter after administration of a 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.) solution containing 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 solutions. In some embodiments, the balloon catheter is deflated to release the ureter after the first 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.) closure period.

[0333] In some embodiments, a catheter is used to isolate or block at least one renal vessel, for example, during at least one of subsequent rAAV administrations via a retrograde ureteral route. In some embodiments, the catheter used to isolate or block at least one renal vessel is a balloon catheter. In some embodiments, the balloon catheter is inflated to isolate or block at least one renal vessel and occlude blood supply to the kidney before administration of a solution containing 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. In some embodiments, a clamp is used to isolate or block at least one renal vessel, for example, during at least one of subsequent rAAV administrations via a retrograde ureteral route.

[0334] Pharmaceutical Composition This document describes pharmaceutical compositions comprising at least one rAAV as described herein and optionally a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprising at least one rAAV comprises one or more AAV capsid proteins selected from Table 1. In other embodiments, the pharmaceutical composition comprising at least one rAAV includes a serum type 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 One or more AAV capsid proteins, namely JEA, AAV2 3xA P2i, AAVDJ P2i, AAV 2i8, AAV2G9, AAV2.5, AAV4E, and AAV4A.

[0335] In some embodiments, a pharmaceutical composition comprising at least one rAAV contains one or more AAV capsid proteins selected from AAV2, AAV6, AAVLK03, AAVDJ, AAV9A2i, AAV9A6i, AAVrh10A2i, AAV2g9, or AAV2.5 (see, for example...). Figure 4 In some embodiments, the pharmaceutical composition comprising at least one rAAV contains one or more AAV capsid proteins selected from AAV2, AAVDJ, AAVJEA (minimal), AAV2g9, or AAV2.5 (see, for example...). Figure 5 In some embodiments, the pharmaceutical composition comprising at least one rAAV is a rational polyploid.

[0336] In some embodiments, a pharmaceutical composition comprising at least one rAAV contains one or more AAV capsid proteins selected from serum types AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV2G9, AAV2.5G9, AAV2.5, AAVrh8, AAVrh10, AAVrh74, AAV10, AAV11, and AAVDJ.

[0337] In some embodiments, the pharmaceutical composition comprising at least one rAAV includes a capsid selected from AAV2G9, AAV2.5, AAVDJ, and AAV2. In some embodiments, the pharmaceutical composition comprising at least one rAAV includes a capsid from AAV2G9. In some embodiments, the pharmaceutical composition comprising at least one rAAV includes a capsid from AAV2.5. In some embodiments, the pharmaceutical composition comprising at least one rAAV includes a capsid from AAVDJ. In some embodiments, the pharmaceutical composition comprising at least one rAAV includes a capsid from AAV2.

[0338] In some embodiments, the pharmaceutical composition comprising at least one rAAV does not contain a capsid protein derived from serum-type AAV9. In some embodiments, the rAAV is not rAAV9.

[0339] In one aspect, this article describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) comprising: (a) AAV2G9 capsid protein; (b) a transgene comprising: a gene selected from AGXT (type I), BSND (type IV), CLCN5 (type I), CLCNKA (type IV), CLCNKB, CLCNKB (types III and IV), COL4A3, COL4A4, COL4A5, GANAB, GRHPR (type II), HNF1B, HOGA1 (type III), KCNJ1 (type II), MAGED2 (type V), MUC1 (type I), NPHP1, NPHS1, NPHS2, OCRL (type II), PKD1, PKD2, PKHD1, SEC61A1, SLC12A1, SLC12A3, SLC3A1, SLC7A9, VHL, and combinations thereof; and (c) a pharmaceutically acceptable carrier.

[0340] In one aspect, this article describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) comprising: (a) AAV2G9 capsid protein; (b) 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 transport 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 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; and (c) pharmaceutically acceptable carriers.

[0341] In one aspect, this article describes a pharmaceutical composition comprising recombinant adeno-associated virus (rAAV) comprising: (a) AAV2G9 capsid protein; (b) a transgene comprising: an inhibitor of a gene or protein selected from REN, SCNN1A, SCNN1B and UMOD; and (c) a pharmaceutically acceptable carrier.

[0342] In some embodiments, the active ingredient of the pharmaceutical composition comprises rAAV as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists substantially of rAAV as described herein. In some embodiments, the active ingredient of the pharmaceutical composition consists of rAAV as described herein.

[0343] In some embodiments, the pharmaceutical composition comprises a total of 1x10 13 Up to 2x10 13 Each rAAV viral genome. In some embodiments, the pharmaceutical composition comprises a total of 5 x 10rAAV 13 Up to 6x10 13 One rAAV viral genome. In some embodiments, the pharmaceutical composition comprises a total of at least 1x10 rAAV viral genomes. 13 At least 2x10 13 At least 3x10 13 At least 4x10 13 At least 5x10 13 At least 6x1013 At least 7x10 13 At least 8x10 13 At least 9x10 13 One or more rAAV viral genomes. In some embodiments, the pharmaceutical composition contains a total of up to 1x10 13 At most 2x10 13 At most 3x10 13 At most 4x10 13 At most 5x10 13 At most 6x10 13 At most 7x10 13 At most 8x10 13 At most 9x10 13 One or more rAAV viral genomes. In some embodiments, the pharmaceutical composition comprises 10 8 One viral genome / mL (vg / mL) to 10 15 vg / mL, 10 9 vg / mL to 10 15 vg / mL, 10 10 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 15 vg / mL, 10 12 vg / mL to 10 15 vg / mL, 10 13 vg / mL to 10 15 vg / mL, 10 11 vg / mL to 10 12 vg / mL, 10 12 vg / mL to 10 13 vg / mL, 10 13 vg / mL to 10 14 vg / mL, 10 14 vg / mL to 10 15 vg / mL, 10 8 vg / mL to 10 14 vg / mL, 10 8 vg / mL to 10 13 vg / mL, 10 8 vg / mL to 10 12 vg / mL, 10 8 vg / mL to 10 11 vg / mL, 10 8 vg / mL to 10 10 vg / mL or 10 8 vg / mL to 10 9rAAV concentrations in vg / mL. It should be understood that each of the individual rAAV concentrations described herein can be used to define the lower and upper limits of the rAAV concentration range.

[0344] In some embodiments, the pharmaceutical composition comprises a total of 1x10 10 rAAV viral genomes (e.g., 4x10) 8 vg / mL to 1x10 9 (vg / mL).

[0345] In some embodiments, the pharmaceutical composition (e.g., a pharmaceutically acceptable carrier, such as a solution as described herein) is in a unit dose of about 0.13 mL / kg to 0.33 mL / kg, about 0.20 mL / kg to about 0.27 mL / kg, about 0.27 mL / kg to about 0.33 mL / kg, 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 (kg being the subject's body weight). In some embodiments, the pharmaceutical composition is in a unit dose of at least 0.13 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of at least 0.20 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of at least 0.27 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of about 0.24 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of up to 0.33 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of 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, at least 0.33 mL / kg, at least 0.34 mL / kg, or at least 0.35 mL / kg. In some embodiments, the pharmaceutical composition is a unit dose of up to 0.30 mL / kg, up to 0.31 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.

[0346] In some embodiments, the pharmaceutical composition is selected from the group consisting of 0.13 mL / kg, 0.14 mL / kg, 0.15 mL / kg, 0.16 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, 0.35 mL / kg, 0.05-0.15 mL / kg, 0.10-0.20 mL / kg, 0.15-0.25 mL / kg, 0.20-0.30 Unit doses of 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.

[0347] According to the methods described herein, the dosage range of a pharmaceutical composition containing rAAV depends on, for example, the form of the pharmaceutical composition, its potency (e.g., the transduction efficiency of rAAV, such as that measured by an efficiency index, see, for example...). Figure 4-5 The dosage should be adjusted to reduce the severity of symptoms, markers, or indicators of the kidney-related conditions described herein. The dosage should not be excessive to avoid adverse side effects such as immunogenicity (e.g., immune responses to AAV capsid antigens or genetically modified products). Dosage may vary depending on the patient's age, condition, and sex, and can be determined by someone skilled in the art. Individual physicians may also adjust the dosage if any complications occur.

[0348] The efficacy of the pharmaceutical composition in, for example, the treatment of kidney-related conditions as described herein can be determined by a skilled clinician. However, a treatment is considered “effective” as used herein if one or more signs or symptoms of the kidney-related condition described herein change in a beneficial manner, other clinically recognized symptoms improve or even resolve, or induce a desired response, such as by at least 10% change, improvement, or induction following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring biomarkers, indicators, symptoms, and / or incidence of kidney-related conditions treated according to the methods described herein. Efficacy can also be measured by the absence of individual deterioration, such as by hospitalization or assessment requiring medical intervention (i.e., cessation of progression of kidney-related conditions). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of the condition in an individual or animal and includes: (1) suppressing kidney-related conditions, for example, preventing the worsening of symptoms; or (2) reducing the severity of kidney-related conditions, for example, causing symptom resolution. An effective dose for treating a condition refers to an amount sufficient, when administered to a subject in need, to result in an effective treatment of the condition (as defined herein). The efficacy of an agent can be determined by assessing physical indicators of the condition or desired response. Those skilled in the art are fully capable of monitoring the efficacy of administration and / or treatment by measuring any one or any combination of such parameters. Efficacy can be assessed in animal models of the conditions described herein, such as the treatment of specific kidney-related conditions.

[0349] For example, Livrozet et al., “An animal model of type A cystinuria due to spontaneous mutation in 129S2 / SvPasCrl mice.” PLoS One. July 21, 2014, 9(7):e102700, describe a mouse model of type A cystinuria, the contents of which are incorporated herein by reference in their entirety. This mouse model of type A cystinuria was identified by comparing the Slc3a1 gene sequences of 129S2 / SvPasCrl (high incidence of cystinuria-related symptoms, including crystalluria and aminoaciduria) and C57BL / 6J (low incidence of these symptoms). A 1232G>A missense mutation was identified in the 129S2 / SvPasCrl Slc3a1 gene; this mutation corresponds to the E383K mutation in the expressed rBAT peptide. In some embodiments, the mouse model of A-type cystinuria contains a 1232G>A missense mutation in the Slc3a1 gene, which corresponds to the E383K mutation in the expressed rBAT peptide. The 1232G>A mutation in mouse Slc3a1 / rBAT (see, for example, SEQ ID NO: 17) corresponds to the 1150G>A mutation in human Slc3a1 / rBAT CDS (see, for example, SEQ ID NO: 5). The E383K mutation in mouse Slc3a1 / rBAT (see, for example, SEQ ID NO: 18) corresponds to the E384K mutation in human Slc3a1 / rBAT (see, for example, SEQ ID NO: 7). Other animal models of A-type or B-type cystinuria known to those skilled in the art may also be used.

[0350] SEQ ID NO: 17, House mouseG SEQ ID NO: 18, House mouse Solute carrier family 3 member 1 (Slc3a1), NCBI ref NP_033231.2, 685 aa; the E384 residue (bold, double underline) in SEQ ID NO: 18 may be mutated to K384 in a mouse model of A-type cystinuria. MDEDKGKRDPIQMSLKGCRTNNGFVQNEDIPEQDPDPGSRDTPQPNAVSIPAPEEPHLKAVRPYAGMPKEVLFQFSGQARYRVPREILFWLTVVSVFLLIGATIAIIVISPKCLDWWQAGPIYQIYPRSFKDSDKDGNGDLKGIQEKLDYITALNIKTLWITSFYKSLKDFRYAVEDFKEIDPIFGTMKD FENLVAAIHDKGLKLIIDFIPNHTSDKHPWFQSSRTRSGKYTDYYIWHNCTHVNGVTTPPNNWLSVYGNSSWHFDEVRKQCYFHQFLKEQPDLNF RNPAVQEEIKEIITFWLSKGVDGFSFDAVKFLLEAKDLRNEIQVNTSQIPDTVTHYSELYHDFTTTQVGMHDIVRDFRQTMNQYSREPGRYRFMGA E ASAESIERTMMYYGLPFIQEADFPFNKYFTTIGTLSGHTVYEVITSWMENMPEGKWPNWMTGGPETPRLTSRVGSEYVNAMHMLLFTLPGTPITYYGEEIGMGDISVTNFNESYDSTTLVSKSPMQWDNSSNAGFTEANHTWLPTNSDYHT VNVDVQKTQPSSALRLYQDLSLLHATELVLSRGWFCLLRDDSHSVVYTRELDGIDNVFLVVLNFGESSTVLNLQGIISDLPPELRIRLSTNSASKGSAVDTRAISLEKGEGLVLEHSTKAPLHQQAAFRDRCFVSSRACYSSALDILYSSC In experimental animal models, therapeutic efficacy is confirmed when a statistically significant change in biomarkers is observed. For example, in an animal model of cystinuria, biomarkers demonstrating effective treatment after rAAV administration could be a reduction in crystalluria and / or aminoaciduria levels. in vitro Animal model assays allow for the evaluation of drug compositions at given doses.

[0351] As described herein, solutions or pharmaceutical compositions may contain at least one pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, microcrystalline cellulose, and cellulose acetate; (4) tragacanth gum powder; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) Buffers, such as magnesium hydroxide and aluminum hydroxide; (15) Alginate; (16) pyrogen-free water; (17) Isotonic saline; (18) Ringer's solution; (19) Ethanol; (20) pH buffer solutions; (21) Polyesters, polycarbonates and / or polyanhydrides; (22) Fillers, such as peptides and amino acids; (23) Serum components, such as serum albumin, HDL and LDL; (24) C2-C 12 Alcohols; and (25) other non-toxic and compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as “excipient,” “carrier,” and “pharmaceutically acceptable carrier” are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent (e.g., rAAV as described herein).

[0352] Other non-limiting examples of pharmaceutically acceptable carriers include: sterile water; water for injection (USP); saline solution; glucose solution; aqueous media such as, but not limited to, sodium chloride injection, Ringer's solution, dextran injection, dextran and sodium chloride injection, and lactated Ringer's solution; water-miscible media such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous media such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0353] In some embodiments, the pharmaceutically acceptable carrier comprises mannitol. In some embodiments, the pharmaceutical composition comprises 25% mannitol. In some embodiments, the pharmaceutical composition comprises at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% mannitol. In some embodiments, the pharmaceutical composition comprises up to 5%, up to 10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, or up to 40% mannitol. In some embodiments, the effect of a specific pharmaceutically acceptable carrier (such as mannitol) can be tested in animal models (e.g., rats, pigs). In some embodiments, the pharmaceutically acceptable carrier does not significantly reduce the efficacy of rAAV. In some embodiments, the pharmaceutically acceptable carrier significantly enhances the efficacy of rAAV.

[0354] The rAAV of this disclosure can be introduced into suitable host cells (e.g., renal tubular cells) using delivery media such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, lipid nanoparticles, vesicles, etc. In particular, the transgene delivered by the rAAV vector can be formulated and encapsulated in lipid particles, liposomes, vesicles, nanospheres, or nanoparticles for delivery.

[0355] Such formulations may be preferred for use in pharmaceutically acceptable formulations that introduce the nucleic acid or rAAV constructs disclosed herein. The formation and use of liposomes are well known to those skilled in the art. Liposomes with improved serum stability and circulating half-life have been developed (US Patent No. 5,741,516). Furthermore, various methods describing liposomes and liposome-like formulations as drug carriers have been seen (US Patent Nos. 5,567,434; 5,552,157; ​​5,565,213; 5,738,868 and 5,795,587).

[0356] Liposomes have been successfully used in many cell types that are typically resistant to transfection by other procedures. Furthermore, liposomes do not have the DNA length limitations typical of virus-based delivery systems. Liposomes have been effectively used to introduce genes, drugs, radiotherapeutic agents, viruses, transcription factors, and allosteric effectors into a wide variety of cultured cell lines and animals. In addition, several successful clinical trials have been completed to examine the effectiveness of liposome-mediated drug delivery.

[0357] Liposomes are formed from phospholipids dispersed in an aqueous medium and spontaneously form multilayered concentric bilayered vesicles (also known as multilayered vesicles (MLVs)). MLVs typically have a diameter of 25 nm to 4 μm. Sonication of MLVs results in the formation of small monolayered vesicles (SUVs) with diameters ranging from 200 to 500 Å, which contain an aqueous solution in their core.

[0358] Alternatively, nanocapsule formulations of rAAV can be used. Nanocapsules typically capture substances in a stable and reproducible manner. To avoid side effects from intracellular polymer overload, such ultrafine particles (approximately 0.1 µm in size) should be designed using polymers that are biodegradable in vivo. Consider using biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements.

[0359] In one embodiment, nanoparticles (e.g., lipid nanoparticles (LPN)) may be used instead of the AAV capsid to deliver the viral AAV payload. The term "nanoparticle" also encompasses liposomes and lipid particles having a nanoparticle size. Exemplary liposomes may include, for example, DSPC, DPPC, DSPG, cholesterol, hydrogenated soybean phosphatidylcholine, soybean phosphatidylcholine, methoxy polyethylene glycol (mPEG-DSPE) phosphatidylcholine (PC), phosphatidylglycerol (PG), distearate phosphatidylcholine, and combinations thereof. In some embodiments in any aspect, the load is lipid nanoparticles (LNP), contains LNP, or is composed of LNP. Lipid nanoparticles may contain a variety of components, including, for example, ionizable lipids (such as MC3, DLin-MC3-DMA, ALC-0315, or SM-102), polyethylene glycol-modified lipids (such as PEG2000-C-DMG, PEG2000-DMG, ALC-0159), phospholipids (such as DSPC), and cholesterol.

[0360] In some embodiments, the LNP comprises (4-hydroxybutyl)azanediylbis(hexane-6,1-diyl)bis(2-hexyldecanoate), an ionizable cationic lipid (ALC-0315, CAS Registry No. 2036272-55-4); (2-hexyldecanoate), 2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide, a PEG lipid (ALC-0159, CAS 1849616-42-7); 1,2-distearyl-sn-glycerol-3-phosphocholine, an accessory lipid (DSPC, CAS 816-94-4); and / or cholesterol, an accessory lipid (Chol, CAS 57-88-5). In some embodiments, the LNP comprises 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid heptadecan-9-yl ester, an ionizable cationic lipid (SM-102, CAS 2089251-47-6); 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000, a PEG lipid (PEG2000-DMG, CAS 160743-62-4); 1,2-distearate-sn-glycerol-3-phosphocholine, an accessory lipid (DSPC, CAS 816-94-4); and / or cholesterol, an accessory lipid (Chol, CAS 57-88-5). In some embodiments, the LNP comprises ALC-0315, ALC-0159, SM-102, PEG2000-DMG, DSPC, cholesterol, or any combination thereof. See, for example, Alshrari et al., Journal of Infection and Public Health 15 (2022) 123–131; Tenchov et al., ACS Nano 2021, 15, 16982-17015; Schoenmaker et al., International Journal of Pharmaceutics 601 (2021) 120586; Suzuki et al., Drug Metabolism and Pharmacokinetics 41 (2021) 100424; the contents of which are incorporated herein by reference in their entirety.

[0361] Typically, lipid nanoparticles have an average diameter selected to provide the desired therapeutic effect. Thus, in some aspects, lipid nanoparticles have an average diameter of about 30 nm to about 150 nm, more typically about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 85 nm to about 105 nm, and preferably about 100 nm. In some aspects, this disclosure provides lipid particles that are relatively large compared to common nanoparticle sizes and have a size of about 150 nm to 250 nm. The size of the lipid nanoparticles can be determined by quasi-elastic light scattering, for example using a MALVERNZETASIZER NANO ZS (Malvern, UK) system.

[0362] Depending on the intended use of the lipid particles, the proportions of the components can vary, and the delivery efficiency of a particular formulation can be measured using, for example, endosomal release parameter (ERP) assays.

[0363] Nucleic acids can be complexed with or encapsulated within the lipid portion of lipid nanoparticles. In some embodiments, the nucleic acid can be completely encapsulated within the lipid portion of the lipid nanoparticles, thereby protecting it from degradation by nucleases, for example, in aqueous solution. In some embodiments, the nucleic acid in the lipid nanoparticles does not undergo substantial degradation after exposure to nucleases at 37°C for, for example, at least about 20, 30, 45, or 60 minutes. In some embodiments, the nucleic acid in the lipid nanoparticles does not undergo substantial degradation after incubation in serum at 37°C for, for example, at least about 30, 45, or 60 minutes, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours.

[0364] In some implementations, lipid nanoparticles are substantially non-toxic to mammals such as humans.

[0365] In some embodiments, the lipid nanoparticles are solid core particles having at least one lipid bilayer. In other embodiments, the lipid nanoparticles have a non-bilayer structure, i.e., a non-layered (i.e., non-bilayer) morphology. Non-bilayer morphologies may include, for example, three-dimensional tubes, rods, cubic symmetric structures, etc., but are not limited to these. The non-layered morphology (i.e., non-bilayer structure) of the lipid particles can be determined using analytical techniques known and used by those skilled in the art. Such techniques include, but are not limited to, cryogenic transmission electron microscopy (“cryo-TEM”), differential scanning calorimetry (“DSC”), X-ray diffraction, etc. For example, the morphology (layered vs. non-layered) of lipid nanoparticles can be readily assessed and characterized using, for example, cryogenic TEM analysis, as described in US2010 / 0130588, the contents of which are incorporated herein by reference in their entirety.

[0366] In some further embodiments, the lipid nanoparticles having a non-layered morphology are electronically dense. In these embodiments, the lipid nanoparticles are structurally monolayered or multilayered. In some aspects, this disclosure provides a lipid nanoparticle formulation comprising multivesicular particles and / or foam-based particles.

[0367] Lipid nanoparticles can spontaneously form after mixing mRNA and lipids. Depending on the desired particle size distribution, the resulting nanoparticle mixture can be extruded through a membrane (e.g., 100 nm cutoff) using, for example, a heated barrel extruder (such as a LIPEX extruder (NORTHERN LIPIDS, INC)). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be achieved, for example, by dialysis or tangential flow filtration.

[0368] Typically, lipid nanoparticles can be formed by any method known in the art, including, for example, lipid nanoparticles can be formed by methods such as those described in US2013 / 0037977, US2010 / 0015218, US2013 / 0156845, US2013 / 0164400, US2012 / 0225129 and US2010. / The methods described in US 0130588 are used to prepare lipid nanoparticles, the contents of which are incorporated herein by reference in their entirety. In some embodiments, lipid nanoparticles can be prepared using continuous mixing, direct dilution, or in-line dilution. Methods and apparatus for preparing lipid nanoparticles using direct dilution and in-line dilution are described in US 2007 / 0042031, the contents of which are incorporated herein by reference in their entirety. Methods and apparatus for preparing lipid nanoparticles using stepwise dilution are described in US 2004 / 0142025, the contents of which are incorporated herein by reference in their entirety.

[0369] definition For convenience, the meanings of some terms and expressions used in this specification, embodiments, and appended claims are provided below. Unless otherwise stated or implied by the context, the following terms and expressions include the meanings provided below. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention, as the scope of the invention is defined only by the claims. Unless otherwise defined, all technical and scientific terms used herein will have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. If there is a significant difference between the use of a term in the art and its definition provided herein, the definition provided in the specification shall prevail.

[0370] The terms “reduce,” “reduction,” “lower,” or “inhibit” are used herein to refer to a reduction in a statistically significant amount. In some embodiments, “reduce,” “reduction,” or “lower” or “inhibit” generally refers to a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent) and may include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduce” or “inhibit” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. The reduction may preferably be reduced to a level within the generally accepted normal range, for example, within the normal range for an individual not suffering from a given condition.

[0371] The terms “increased,” “increased,” “enhanced,” or “activated” are used herein to refer to an increase in a statistically significant amount. In some implementations, the terms “increased,” “increased,” “enhanced,” or “activated” may refer to an increase of at least 10% compared to a reference level, such as at least about 20%, or at least about 30%, or at least about 40%, 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 at most and including 100%, or any increase between 10 and 100% compared to a reference level, 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 of 2 to 10 times or more compared to a reference level. In the context of a biomarker or symptom, “increased” means such a level is statistically significant.

[0372] As used herein, “subject” refers to a person or an animal. Generally, an animal is a vertebrate such as a primate, rodent, domesticated animal, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domesticated and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms “individual,” “patient,” and “subject” are used interchangeably herein.

[0373] Preferably, the subject is a mammal. Mammals can be humans, non-human primates, mice, rats, dogs, cats, horses, or cattle, but are not limited to these examples. Non-human mammals can be advantageously used as subjects representing animal models of kidney-related diseases. Subjects can be male or female.

[0374] Subjects may be those who have been previously diagnosed with, or are identified as having, or are currently suffering from, a condition requiring treatment (e.g., kidney-related disease) or one or more complications associated with such condition, and optionally have received treatment for kidney-related disease or one or more complications associated with kidney-related disease. Alternatively, subjects may be those who have not been previously diagnosed with kidney-related disease or one or more complications associated with kidney-related disease. For example, subjects may be those who exhibit one or more risk factors for kidney-related disease or one or more complications associated with kidney-related disease, or subjects who do not exhibit risk factors.

[0375] "Subjects who need treatment for a specific condition" can be subjects who have the condition, have been diagnosed with the condition, or are at risk of developing the condition.

[0376] The term "expression" refers to the cellular processes involved in the production of RNA and proteins, and, where appropriate, the secretion of proteins, including, but not limited to, transcription, transcript processing, translation, and protein folding, modification, and processing. Expression can refer to the transcription and stable accumulation of sense (e.g., mRNA) or antisense RNA derived from one or more nucleic acid fragments and / or the translation of mRNA into polypeptides.

[0377] "Expression products" include RNA transcribed from a gene and polypeptides obtained by translating mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operatively linked to a suitable regulatory sequence. Genes may or may not include regions before and after the coding region, such as the 5' untranslated (5'UTR) or "leader" sequence and the 3'UTR or "tail" sequence, as well as intercalation sequences (introns) between individual coding regions (exons).

[0378] In some embodiments, the methods described herein involve measuring, detecting, or determining the level of at least one biomarker. As used herein, the terms “detection” or “measurement” refer to observing a signal from, for example, a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a specific labeled moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescence, photochemical, biochemical, immunochemical, electrical, optical, or chemical methods. In some embodiments in any aspect, the measurement may be a quantitative observation.

[0379] In some implementations, the methods described herein are performed sequentially, i.e., step (a), then step (b), then step (c), and so on for all other steps.

[0380] In some embodiments of any aspect, the peptide, nucleic acid, or cell described herein may be engineered. As used herein, “engineered” means an aspect that has been artificially manipulated. For example, a peptide is considered “engineered” when at least one aspect of it (e.g., its sequence) has been artificially manipulated to differ from its naturally occurring aspect. By convention and as understood by those skilled in the art, progeny of engineered cells are generally still referred to as “engineered” even if the actual manipulation is performed on a prior entity.

[0381] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g., at least one transgene) is contained in a vector. In some aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operatively linked to a vector. As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. Furthermore, as used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring a transgene into a host cell. As used herein, a vector can be viral or non-viral. The term "vector" encompasses any genetic element capable of replicating when associated with a suitable control element and capable of transferring a gene sequence into a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, bacteriophages, transposons, granules, chromosomes, viruses, microvirus particles, etc.

[0382] In some embodiments of any aspect, the vector is recombinant, for example, it contains sequences derived from at least two different sources. In some embodiments of any aspect, the vector contains sequences derived from at least two different species. In some embodiments of any aspect, the vector contains sequences derived from at least two different genes, for example, it contains a fusion protein or a nucleic acid encoding an expression product, said nucleic acid being operatively linked to at least one non-natural (e.g., heterologous) genetic control element (e.g., promoter, repressor, activator, enhancer, response element, etc.).

[0383] In some embodiments of any aspect, the vectors or nucleic acids described herein are codon-optimized, for example, the natural or wild-type sequence of the nucleic acid has been altered or engineered to include alternative codons, such that the altered or engineered nucleic acid encodes a polypeptide expression product identical to the natural / wild-type sequence, but transcribed and / or translated with improved efficiency in the desired expression system. In some embodiments of any aspect, the expression system is an organism (or cells obtained from such an organism) other than the natural / wild-type sequence source. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in mammals or mammalian cells (e.g., mouse, rat, or human cells). In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in human cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in yeast or yeast cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in bacterial cells. In some embodiments of any aspect, the vectors and / or nucleic acid sequences described herein are codon-optimized for expression in... E. coli Expressed in cells.

[0384] As used herein, the term "expression vector" refers to a vector that directs the expression of RNA or polypeptide derived from a sequence linked to a transcriptional regulatory sequence on the vector. The expressed sequence is typically, but not necessarily, heterologous to the cell. Expression vectors may contain additional elements; for example, an expression vector may have two replication systems, thereby allowing it to be maintained in two organisms, such as being expressed in human cells and cloned and amplified in a prokaryotic host.

[0385] As used herein, the term "viral vector" refers to a nucleic acid vector construct that contains at least one virally derived element and has the ability to be packaged into viral vector particles. Viral vectors may contain nucleic acids encoding polypeptides as described herein in place of non-essential viral genes. Vectors and / or particles can be used for the purpose of transferring any nucleic acid into cells in vitro or in vivo. Many forms of viral vectors are known in the art. Non-limiting examples of viral vectors of the present invention include AAV vectors, adenovirus vectors, lentiviral vectors, retroviral vectors, herpesvirus vectors, alphavirus vectors, poxvirus vectors, baculovirus vectors, and chimeric virus vectors.

[0386] As used herein, the terms “treat,” “treatment,” “treating,” or “improvement” refer to therapeutic treatment aimed at reversing, alleviating, improving, inhibiting, slowing, or interrupting the progression or severity of a condition associated with a disease (e.g., kidney-related disease). The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a kidney-related disease. Treatment is generally “effective” if one or more symptoms are reduced or clinical markers are decreased. Alternatively, treatment is “effective” if the progression of the disease is slowed or stopped. That is, “treatment” includes not only improvement in symptoms or markers but also cessation or at least slowing of symptom progression or worsening compared to the expected outcome without treatment. Beneficial or desired clinical outcomes include, but are not limited to, relief of one or more symptoms, reduction in the severity of the disease, a stable (i.e., non-worsening) state of the disease, delay or slowing of disease progression, improvement or mitigation of the disease state, remission (whether partial or complete remission), and / or a reduction in mortality (whether detectable or undetectable). The term “treatment” for a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

[0387] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). The expression "pharmaceutically acceptable" is used herein to mean those compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, and in proportion to a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a carrier other than water. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, a pharmaceutically acceptable carrier may be an artificial or engineered carrier, such as a carrier for which the active ingredient is not found in nature.

[0388] As used herein, the term "administration" means placing a compound disclosed herein into a subject by some method or route, thereby delivering the agent at least partially at the desired site. Pharmaceutical compositions comprising compounds disclosed herein may be administered via the retrograde ureteral route. Such activities may be performed, for example, by a medical professional and / or a subject receiving treatment.

[0389] The terms “statistically significant” or “significantly” refer to statistical significance and usually indicate a difference of two standard deviations (2SD) or greater.

[0390] Except where described in the operational examples or elsewhere, all figures for the amount of expressed components or reaction conditions used herein should be understood to be modified by the term “about” in all cases. When used with percentages, the term “about” may refer to ±1%.

[0391] As used herein, the term "includes" means that other elements may exist in addition to the defined elements presented. The use of "includes" implies inclusion rather than limitation.

[0392] The term “composed of” means the compositions, methods and their respective components as described herein, excluding any elements not described in the description of the embodiments.

[0393] As used herein, the term "consistently of" refers to those elements required for a given embodiment. This term allows for the presence of additional elements, but these elements do not materially affect the basic and novel or functional features of that embodiment of the invention.

[0394] It should be understood that each of the individual values ​​described in this article can be used to define the lower and upper limits of a range.

[0395] Unless the context clearly indicates otherwise, the singular forms “a / an,” “a kind,” and “the” include the plural referent. Similarly, the word “or” is intended to include “and,” unless the context clearly indicates otherwise. Suitable methods and materials will be described below, but similar or equivalent methods and materials may be used in practice or testing of this disclosure. The abbreviation “eg” is derived from the Latin *exempli gratia* and is used herein to denote a non-limiting instance. Therefore, the abbreviation “eg” is synonymous with the term “for example.”

[0396] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements visible herein. For convenience and / or patentability reasons, one or more members of a group may be included in or removed from the group. When any such inclusion or removal occurs, the specification herein is deemed to contain the modified group, thereby satisfying the written description of all Markush groups as used in the appended claims.

[0397] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application should have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains. It should be understood that the invention is not limited to the specific methods, schemes, and reagents described herein and therefore may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the claims. Definitions of commonly used terms in cell biology, immunology, and molecular biology can be found in *The Merck Manual of Diagnosis and Therapy*, 20th edition, Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); *The Encyclopedia of Molecular Cell Biology and Molecular Medicine*, edited by Robert S. Porter et al., 1999-2012, Blackwell Science Ltd. (ISBN 9783527600908); and *Molecular Biology and Biotechnology: A Comprehensive Desk Reference*, edited by Robert A. Meyers, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); *Immunology*, Werner Luttmann, Elsevier, 2006; and Janeway's *Immunobiology*. Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), WW Norton&Company, 2016 (ISBN0815345054, 978-0815345053); Lewin's Genes XI, Jones&Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, MolecularCloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012) (ISBN 1936113414); Davis et al., BasicMethods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, JonLorsch (editor) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (Editor), John Wiley and Sons, 2014 (ISBN047150338X, 9780471503385); Current Protocols in Protein Science (CPPS), JohnE. Coligan (Editor), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (edited) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety.

[0398] In any of the embodiments described herein, the disclosure does not relate to methods of cloning humans, methods of modifying the genetic characteristics of human germlines, methods of using human embryos for industrial or commercial purposes or methods of altering the genetic characteristics of animals that may cause suffering to the animals but have no substantial medical benefit to humans or animals, and animals produced by such methods.

[0399] Other terms are defined in the description of various aspects of the invention herein.

[0400] All patents and other publications cited throughout this application, including references, granted patents, published patent applications, and co-pending patent applications, are expressly incorporated herein by reference for the purpose of describing and disclosing methods that may be used in conjunction with the techniques described herein, as described in such publications. These publications are provided solely because of their publication prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventor has no right to claim prior disclosure by virtue of a prior invention or for any other reason. All statements regarding the dates of these documents or representations regarding their contents are based on information available to the applicant and do not constitute any admission of the accuracy of the dates or contents of these documents.

[0401] The description of embodiments of this disclosure is not intended to be exhaustive, nor is it intended to limit this disclosure to the precise forms disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications can be made within the scope of this disclosure, as will be recognized by those skilled in the art. For example, although method steps or functions are presented in a given order, alternative embodiments may implement functions in a different order, or functions may be implemented substantially simultaneously. The teachings of this disclosure provided herein can be applied, as appropriate, to other procedures or methods. The various embodiments described herein can be combined to provide further embodiments. If desired, aspects of this disclosure may be modified to incorporate the compositions, functions, and concepts of the foregoing references and applications to provide further embodiments of this disclosure. These and other changes may be made to this disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0402] Specific elements of any of the foregoing embodiments may be combined or substituted with elements of other embodiments. Furthermore, while advantages relating to certain embodiments of this disclosure have been described in the context of these embodiments, other embodiments may also possess such advantages, and not all embodiments necessarily need to exhibit such advantages to fall within the scope of this disclosure.

[0403] Some implementations of the technology described in this article can be defined according to any of the following numbered paragraphs: 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 rAAV is administered to the renal pelvis of the kidney via a catheter at a rate of approximately 0.13 mL / kg to approximately 0.33 mL / kg (kg being the subject's body weight), wherein the nephrons of the kidney are transduced with high efficiency by rAAV.

[0404] 2. The method of paragraph 1, wherein a solution containing rAAV is administered to the kidney for about 0.5 minutes to about 60 minutes.

[0405] 3. The method of paragraph 1, wherein a solution containing rAAV is administered to the kidney for about 1 ...

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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