Renal pelvic injection for drug delivery to the kidney

Abstract

Methods are disclosed for delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject with or without immunity against AAV, including locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein via renal pelvic injection, wherein the renal pelvic injection comprises direct renal pelvic injection or retrograde transureteral renal pelvic injection. Methods are also disclosed for treating a kidney disorder in a subject with or without immunity against AAV, comprising locally delivering to the kidney of the subject via renal pelvic injection, including direct renal pelvic injection and retrograde transureteral renal pelvic injection, an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject. The AAV can be AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, or AAVShH10.

Description

[0001] RENAL PELVIC INJECTION FOR DRUG DELIVERY TO THE KIDNEY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This claims the benefit of U.S. Provisional Application No. 63 / 527,248, filed July 17, 2023, which is incorporated by reference herein in its entirety.

[0004] FIELD OF THE DISCLOSURE

[0005] This relates to the field of methods of gene therapy, more specifically, this relates to viral vectors used in gene therapy to target the kidney and methods of delivering such viral vectors using renal pelvic injection to enhance the drug delivery to the kidney.

[0006] SEQUENCE LISTING

[0007] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence” (15,210 bytes), which was created on July 12, 2024 which is incorporated by reference herein.

[0008] BACKGROUND

[0009] Adeno-associated virus (AAV) vectors are a promising vector for in vivo gene therapy that can attain a high level of transduction in various organs. To date, eight commercial AAV vector gene therapy products have been approved by authorizing agencies in the United States, European countries and Japan. With the demonstration of the safety and efficacy of the first two local injection products, GLYBERA® for intramuscular injection to treat lipoprotein lipase deficiency and LUXTURNA® for subretinal injection to treat Leber congenital amaurosis type 2, the first intravenous injection product, ZOLGENSMA® for the treatment of spinal muscular atrophy, was approved by FDA in 2019. Three additional intravenous injection products have been approved by either or both of FDA and EMA for the treatment of hemophilia A, hemophilia B and Duchenne muscular dystrophy. Moreover, an intracranial injection AAV vector product has been approved by EMA for the treatment of aromatic L-amino acid decarboxylase (AADC) deficiency.

[0010] However, in contrast to the remarkable progress in the intravenous approaches to target the liver, central nervous system and skeletal muscles, it remains difficult to achieve renal transduction at a level similar to the one attained in other organs by systemic AAV vector administration even at a high dose. The intravenous administration of AAV vectors based on common serotypes can only achieves mesangial cell and interstitial cell transduction in the kidney, and the current AAV vector-mediated renal gene delivery methods are not capable of effectively transducing clinically relevant cell types including tubular epithelial cells and podocytes at satisfactory levels. Thus, a need remains for AAV vectors and methods that can be used for the gene therapy for kidney disease.

[0011] In addition to the above-described target organ-specific challenge in AAV vector-mediated renal gene delivery, the high prevalence of pre-existing anti-AAV neutralizing antibodies in humans (Boutin, Monteilhet et aL, Hum. Gene Ther. 2010; 21(6):704-12, 2010) has remained as the major universal limitation to be overcome in A AV-mediated gene therapy. Up to 70% of adult human population is positive for anti-AAV capsid neutralizing antibodies developed by natural infection that make the therapy ineffective; therefore, individuals with high levels of anti-AAV neutralizing antibodies have so far been excluded from the therapy-eligible groups and may continue not to benefit from the therapy. In addition, redosing for maintaining therapeutic effects is currently not possible due to the robust humoral immune responses of AAV vectors that occur upon the first vector administration and are difficult to prevent or attenuate. Thus, a need remains for AAV vectors and methods that can be used for the gene therapy for kidney disease for patients who have immunity against AAV.

[0012] SUMMARY OF THE DISCLOSURE

[0013] Methods arc disclosed for delivering a composition comprising an adcno-associatcd virus (AAV) to one or more kidney cell types in a subject with or without immunity against AAV, comprising locally delivering to the kidney of the subject with or without immunity against AAV via renal pelvic injection an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein. The renal pelvic injection includes, direct renal pelvic injection surgically or percutaneously and retrograde transureteral renal pelvic injection. In non-limiting examples, the AAV is AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, or AAVShHIO.

[0014] Methods are also disclosed for treating a kidney disorder in a subject with or without immunity against AAV, comprising locally delivering to the kidney of the subject with or without immunity against AAV via renal pelvic injection an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject with or without immunity against AAV. In non-limiting examples, the AAV is AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, or AAVShHIO.

[0015] The foregoing and other features and advantages of the invention will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

[0016] BRIEF DESCRIPTION OF THE FIGURES

[0017] Figures 1A-1B. Adeno-associated virus (AAV) Barcode-Seq identified robust AAV capsids that outperform AAV9 for renal transduction following renal pelvis (RP) administration. (A) Eight- week-old C57BL / 6J male mice were injected with AAV-CAG-Barcode library that covers 47 AAV capsids at a dose of 3xl0nvg / mouse (n=3 per group). Kidneys were harvested 6 weeks post-injection and relative transduction efficiency of each capsid was determined by AAV DNA / RNA Barcode-Seq. (B) Relative renal transduction efficiency of common vectors and those with significantly higher vector DNA delivery or significantly higher expression of vector genome transcripts than those of AAV9 by RP injection in mice. Dark and light gray bars arc those with and without statistical significance compared to the AAV9 values, respectively. AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, and AAV2.7m8 exhibit significant enhancement in kidney transduction at both DNA and RNA levels, whereas AAV3, AAV-LK03, and AAVShHIO, despite their significantly increased AAV vector genome delivery to the kidney, did not result in augmented transgene expression potentially due to epigenetic suppression of transgene expression in mice. Mean + s.e.m. For a full list of AAV capsids, see Figure 8.

[0018] Figures 2A-2C. Renal transduction of AAVKP1 was enhanced by renal pelvis (RP) injection. (A) Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato and AAVKP1-CAG- tdTomato at a dose of 3x10’1vg / mouse via renal pelvis (n=4 per group). Kidneys were harvested 2 weeks post-injection. (B) Quantification of viral genome in the kidney by quantitative PCR (qPCR). Vector genome copy numbers were expressed as double-stranded vector genome copy numbers per diploid genomic equivalent. Mean ± s.e.m. Unpaired t-test. ***, p<0,001. (C) Representative images of native tdTomato fluorescence in the kidney. Enhanced transduction was observed in the cortex of the kidney injected with AAVKPl-CAG-tdTomato. Scale bar, 1 mm.

[0019] Figures 3A-3B. AAVKP1 injected via renal pelvis (RP) transduced proximal tubule cells, but not podocytes. Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato and AAVKPl-CAG-tdTomato via renal pelvis at a dose of 3x10" vg / mouse (n=4 per group). Kidneys were harvested 2 weeks post-injection. (A) Representative images of native tdTomato fluorescence and LTL (proximal tubules), and quantification of tdTomato positive area in proximal tubules. Mean ± s.e.m. Unpaired t-test. ***, p<0,001. (B) Representative images of native tdTomato fluorescence and WT1 (podocyte nuclei). Scale bars, 50 pm. LTL, tetragonolobus lectin; WT1, Wilms tumor 1.

[0020] Figures 4A-4D. Limited extra-renal dissemination of AAVKP1 following renal pelvis (RP) injection prevented off-target liver transduction. Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato and AAVKPl-CAG-tdTomato at a dose of 3xl0uvg / mouse (A, B) and IxlO13vg / kg (C, D) (n=4 per group). (A) Viral genome in the liver was quantified by qPCR two weeks after injection. Vector genome copy numbers were expressed as double -stranded vector genome copy numbers per diploid genomic equivalent. (B) Representative images of native tdTomato fluorescence and DAPI in the liver. Reduced off-target liver transduction was observed in the liver of the mice injected with AAVKPl- CAG-tdTomato. Scale bar, 100 pm. (C) Viral genome in the kidney was quantified by qPCR 10 min after RP injection. (D) Blood vector concentration-time curves following RP injection. Area under the curve (AUC) was calculated using the trapezoid rule. Unpaired t-test. ****, p<0,0001; ***, p<0,001; *, p<0,05. Mean ± s.e.m.

[0021] Figure 5A-5E. Transcatheter renal pelvis (RP) injection was successfully performed without major complication. A 13-year-old female rhesus macaque (animal #1 in Table 2) was injected with PBS via left RP. The kidney was assessed 3 weeks post-injection. (A) Cystoscopic image of the catheter insertion through the orifice of the ureter. (B, C) A fluoroscopic image of transureteral retrograde renal pelvis injection. A contrast agent was injected to visualize the injection path. (D) Gross appearance of the injected left kidney. Scale bar, 1 cm. (E) A representative hematoxylin cosin (HE) staining image of the cortex of the left injected kidney. Scale bar, 1 mm. Figure 6A-6D. AAVKP1 injected via renal pelvis (RP) transduced proximal tubule cells in the kidney of non-human primates. A 3-year-old female rhesus macaque (animal #2 in Table 2) was injected with AAVKP1 -CAG-tdTomato via right RP at a dose of 1 .3x1013vg / animal. AAV vector transduction in the kidney and liver was assessed 3 weeks post-injection. (A) A representative image of native tdTomato fluorescence in the kidney. Scale bar, 1 cm. (B) Representative images of native tdTomato fluorescence and LTL (proximal tubules). (C) Representative images of native tdTomato fluorescence and WT1 (podocytes). (D) A representative image of native tdTomato fluorescence in the liver. Scale bars in B, C and D, 50 pm. LTL, tetragonolobus lectin; WT1, Wilms tumor 1.

[0022] Figures 7A-7I. Renal pelvis (RP) injection of AAVKP1 transduced the kidney of non-human primate (NHP) with ant-AAVKPl neutralizing antibody. A 15 and 3-year-old female rhesus macaques (animals #4 and #5 in Table 2) were injected with AAVKP1 -CAG-tdTomato via right RP at a dose of 1.2xl013and 6.3xl012vg / animal, respectively. AAV vector transduction in the kidney and liver was assessed 3 weeks post-injection. (A, E) Representative images of native tdTomato fluorescence in the kidney. Scale bar, 1 cm. (B, F) Representative images of native tdTomato fluorescence and LTL (proximal tubules). (C, G) Representative images of native tdTomato fluorescence and WT1 (podocytes). (D, H) A representative image of native tdTomato fluorescence in the liver. Scale bars in B, C, D, F, G and H, 50 pm. ETE, tetragonolobus lectin; WT1, Wilms tumor 1. Panels A to D are from Animal #4 and Panels E to H are from Animal #5, showing the same kidney and liver transduction profiles. (I) Blood vector concentration-time curves following RP injection.

[0023] Figure 8. Relative transduction efficiency of adeno-associated virus (AAV) capsids in the kidney following renal pelvis (RP) administration. A full set of the data obtained from the 47 AAV capsids is presented, a portion of which is shown in Figure 1. Eight-week-old C57BL / 6J male mice were injected with AAV-CAG-Barcode library at a dose of 3xl0nvg / mouse (n=3 per group). Kidneys were harvested 6 weeks post-injection and relative transduction efficiency of each capsid was determined by AAV DNA / RNA Barcode-Seq. AAV capsids that showed significantly higher and lower renal transduction than AAV9 were highlighted. AAV capsids that showed significantly higher renal transduction than AAV9 by RP injections have been selected for data presentation in Figures 1A-1B. Asterisks (*) indicate AAV capsids that showed significantly higher renal DNA transduction but significantly lower RNA transduction than AAV9. Mean ± s.e.m. Adjusted p<0.05 (Bonferroni correction) was considered as statistical significance.

[0024] Figure 9. Renal transduction of AAVKP1 was not enhanced by ischemia.

[0025] Eight-week-old C57BL / 6J male mice were injected with AAVKP1 -CAG-tdTomato at a dose of 3xl0nvg / mouse via renal pelvis with various ischemic time (0 min, 5 min, 10 min and 15 min). Kidneys were harvested 2 weeks post-injection. Representative images of native tdTomato fluorescence in the kidney are shown. Enhanced transduction in the cortex of the kidney was observed in the 5 min, 10 min and 15 min groups, but not in the 0 min group. Scale bar, 1 mm. Figures 10A-10B. Renal pelvis (RP) injection results in accumulation of 25 nm microspheres in the cortical interstitium. Eight-week-old C57BL / 6J male mice were injected with fluorescent 25 nm microspheres via the renal pelvis with (A) or without (B) blockade of the renal blood flow. There was no circulation time following RP injections. Kidneys were harvested following PBS perfusion. Representative images of microspheres and CD31 (endothelial cells) in the kidney are shown. Accumulation of microspheres was not observed without blockade of the renal blood flow. Scale bar, 50 pm.

[0026] Figures 11A-11C. Inflammation in the cortex of the kidney following RP injection of AAV- KPl-CAG-tdTomato in non-human primates (NHPs). Female rhesus macaques were injected with AAV- KPl-CAG-tdTomato (A, NHP #2 without neutralizing antibody and B, NHP #4 with neutralizing antibody) or vehicle (C, NHP #1). Kidneys were harvested 3 weeks post-injection. Representative HE staining images of the cortex of injected and contralateral kidneys arc shown. Scale bar, 100 pm.

[0027] DETAILED DESCRIPTION OF SEVERAL ASPECTS

[0028] Methods are disclosed for delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject with or without immunity against AAV. In further aspects, methods are disclosed for treating a kidney disorder in a subject with or without immunity against AAV, that includes locally delivering an AAV vector encoding a therapeutic protein to cells of kidney via renal pelvic injection comprising direct renal pelvic injection and retrograde transureteral renal pelvic injection.

[0029] Kidney disease, including chronic and acute disease, causes over 800,000 deaths worldwide each year. Acute kidney disease (AKD) involves loss of kidney function typically stemming from an acute causative event (e.g., sepsis, ischemia, trauma, and / or nephrotoxic drugs). In contrast, chronic kidney disease (CKD) involves progressive loss of kidney function over a period of months or years. The pathophysiology of kidney disease varies greatly depending on the type of disease. For example, multiple pathogenic processes such as inflammation, hypoxia, pro-fibrotic cell accumulation, extracellular matrix remodeling, and vascular drop-out have been proposed to be involved in CKD progression.

[0030] Disclosed herein is a comprehensive assessment of renal transduction efficiency of AAV capsids by RP injection. Nine AAV capsids (AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV7m8, AAV3, AAVLK03, and AAVShHIO) were identified that transduce the kidney more efficiently than AAV9 by RP injection at the vector genome DNA levels for all the nine capsids and at the vector transcripts levels for the first six capsids. The IV injection data are described elsewhere. Detailed analysis revealed that local injection of AAVKP1 enables gene transfer to proximal tubules while minimizing the off-target liver transduction. On the other hand, there was no advantage of local injection of AAV9 whose renal and hepatic transduction remained the same regardless of administration routes. In addition, disclosed herein is the experimental evidence that RP injection can successfully transduce kidney cells even in the presence of anti- AAV neutralizing antibodies, demonstrating that RP injection can overcome the limitation in AAV vector- mediated gene delivery posed by the presence of immunity against AAV.

[0031] The disclosed methods utilize direct administration to the renal pelvis. Renal pelvic injection includes direct renal pelvic injection and retrograde transureteral renal pelvis injection.

[0032] In some aspect, the method includes a) identifying a ureteral orifice and a ureter of the subject using a cystoscope; b) advancing a guidewire from the cystoscope through the ureteral orifice and through a ureter of the subject to the renal pelvis; c) removing the cystoscope; d) advancing a balloon catheter comprising a balloon over the guide wire to the proximal aspect of the ureter of the subject; e) inflating the balloon of the balloon catheter at a proximal aspect of the ureter adjacent to a ureteropelvic junction or at a ureteropelvic junction of the subject to occlude the ureter; f) measuring the volume of a renal pelvis of the subject using a contrast agent and fluoroscopy in the ureter occluded by the balloon catheter in step c); g) positioning a catheter and injecting the composition comprising the AAV vector through the catheter into the renal pelvis of the subject; and h) injecting a volume of the air through the catheter into the renal pelvis, thereby retrogradely delivering the composition comprising the AAV vector to renal parenchyma of the subject, under pressure. These steps can be performed in any order. In one aspect, steps a to h are performed in sequence. In another aspect, steps a to h are not performed in sequence. In some aspects, step f is performed independently, such as prior to step a, or following step c.

[0033] Renal pelvis injection can include the following steps: (1) a cystoscope is inserted into the bladder through the urethra to identify the ureteral orifices; (2) a guidewire is advanced from the cystoscope via the ureter (see, for example, Figure 5A) to the renal pelvis of the treatment side; (3) The scope is removed and a balloon catheter is advanced over the wire to the proximal aspect of the ureter; (4) the balloon of the catheter is inflated at the proximal aspect of the ureter adjacent to the ureteropelvic junction or at the ureteropelvic junction to achieve an adequate ureteral occlusion (see, for example, Figure 5B); (5) The volume of the renal pelvis is measured by injecting a contrast agent under a fluoroscopy with the ureter occluded by the balloon catheter; (6) the catheter is properly positioned for occlusion of the ureter (see, for example, Figure 5C); (7) a test agent (e.g., AAV vector solution) is injected, followed by air injection to retrogradely deliver an AAV vector solution to the renal parenchyma with a raised pressure inside the renal pelvis. In some aspects, the AAV vector solution is about 0.1 to about 1 .0 mL / kg body weight, such as about 0.1 to about 0.3 mL / kg, about 0.1 to about 0.4 mL / kg, about 0.1 to about 0.5 mL / kg, about 0.2 to about 0.4 mL / kg, about 0.2 to about 0.5 mL / kg, about 0.2 to about 0.6 mL / kg, about 0.3 to about 0.5 mL / kg, about 0.3 to about 0.6 mL / kg, about 0.3 to about 0.7 mL / kg, about 0.4 to about 0.6 mL / kg, about 0.4 to about 0.7 mL / kg, about 0.4 to about 0.8 mL / kg, about 0.5 to about 0.7 mL / kg, about 0.5 to about 0.8 mL / kg, about 0.5 to about 0.9 mL / kg, about 0.6 to about 0.8 mL / kg, about 0.6 to about 0.9 mL / kg, about 0.6 to about 1 mL / kg, about 0.7 to about 0.9 mL / kg, about 0.7 to about 1 mL / kg, or about 0.8 to about 1 mL / kg. Exemplary values are about 0.41, about 0.29, about 0.42, and about 0.45 mL / kg). In other aspects, the air is about 0.05 to about 0.4 mL / kg, such as about 0.05 to about 0.07 mL / kg, about 0.05 to about 0.08 mL / kg, about 0.05 to about 0.1 mL / kg, about 0.05 to about 0.2 mL / kg, about 0.06 to about 0.08 mL / kg, about 0.06 to about 0.09 mL / kg, about 0.06 to about 0.1 mL / kg, about 0.06 to about 0.2 mL / kg, about 0.07 to about 0.09 mL / kg, about 0.07 to about 0.1 mL / kg, about 0.07 to about 0.2 mL / kg, about 0.07 to about 0.3 mL / kg, about 0.08 to about 0.1 mL / kg, about 0.08 to about 0.2 mL / kg, about 0.08 to about 0.3 mL / kg, about 0.08 to about 0.4 mL / kg, about 0.09 to about 0.3 mL / kg, or about 0.09 to about 0.4 mL / kg, about 0. 1 to about 0.3 mL / kg, about 0.1 to about 0.4 mL / kg, about 0.2 to about 0.4 mL / kg. Exemplary values are about 0.06, 0.08, 0.15, 0.22, 0.24 mL / kg air. In some aspects, the air volume is equal to or greater than the pelvic volume plus the catheter dead space. One of skill in the art can readily determine the amount of AAV vector solution and the amount of air. (8) The consistent balloon occlusion is maintained during the dwelling time of the AAV vector solution in the kidney.

[0034] In some aspects, the method also includes (9) changing the position of the subject to the lateral decubitus (with the injected kidney side down) after a sufficient time, such as 5 minutes (for example, 3 to 10 minutes, such as 4 to 8 minutes, or 5 to 7 minutes) of the dwelling time and wait for another sufficient time, such as 5 min (for example, 3 to 10 minutes, such as 4 to 8 minutes, or 5 to 7 minutes) with ureter occlusion maintained with the inflated balloon.

[0035] In more aspects, (10) after a total of 10 min of the dwelling time (see also the times above), the position of the subject is changed to the supine position and the balloon is deflated; (11) the catheter position is assured by injecting a contrast agent; and (12) the balloon catheter is removed.

[0036] In further aspects, this procedure is performed with anesthesia.

[0037] In some aspects, the volume of the renal pelvis can be estimated morphologically using images obtained through computed tomography (CT), magnetic resonance imaging (MRI) or sonography using ultrasound (US). In brief, renal pelvis volume can be calculated using the formula: 0.524 (n / 6) *APD x L x T, where APD represents the anterior-posterior diameter, and L and T represent the long and transverse diameters of the renal pelvis, respectively. However, as the volume of renal pelvis of each subject is highly variable and the volume of agent / contrast to be used until effective retrograde reflux / influx to renal parenchyma is dependent on the pressure and resistance of the wall of the renal pelvis, morphologic volume estimation may not provide the most appropriate volume to be used for vector delivery.

[0038] In some aspects, the volume of the renal pelvis can be determined directly by injecting a contrast agent into the renal pelvic space at the time of the procedure. After the balloon occlusion of the catheter, gentle injection of the contrast agent is performed under a fluoroscopy to ensure the complete occlusion of the ureteral outflow from the renal pelvis. Then, continuous suction of the balloon catheter is performed over 30 to 60 seconds to withdraw the contrast agent and urine inside the renal pelvis and the luminal space of the balloon catheter. The contrast agent is then gently infused into the renal pelvis under a fluoroscopy or digital subtraction angiography (DSA) until renal parenchymal visualization is noted. The total volume of the injected contrast agent required for renal parenchymal visualization is recorded. By subtracting the pre- measured dead space of the balloon catheter from the total volume of the injected contrast agent, the renal pelvic volume under the balloon occlusion condition is determined.

[0039] In some aspects, for use in humans, the transverse diameter of renal pelvis is <1 cm for most adults. This means that normal renal pelvis volume in human is up to a few mLs (in most cases it is about 1 mL or less) and the volume of air for injection is up to a few mLs. Thus in some embodiments about 0.5 to about 1 mL of the composition comprising the AAV vector, such as about 0.5, about 0.6, about 0.7, about 0.8, about 0.9 or about 1 mL of the composition comprising the AAV vector is utilized. In some aspect, it is about ImL or less

[0040] In some aspects, the volume of the air injected through the balloon catheter following the completion of injection of AAV vector solution is about 0.05 to 0.4 mL / kg, which is equal to the dead space of the balloon catheter + up to 2.5 times the volume of the renal pelvis. The volume of the normal renal pelvis in human is up to 10 mL but it depends on age. As the renal pelvic wall is highly elastic and the significantly lower hydraulic resistance of the air compared with contrast agent, more volume of air is necessary than the renal pelvic volume determined by the contrast agent. The mean maximum percent increase of normal renal pelvic volume with diuresis for non-hydronephrotic patient cohort is 88%, ranging from 33 to 134% (see Koff, et al., J. Urol. 174(1 ): 303— 7. 2005), although the data is limited to pediatric patients. Thus, air corresponding to 150 to 250% of the renal pelvic volume determined by the abovedescribed method (i.e., 50-150% more volume) can be used in addition to the dead space of the balloon catheter.

[0041] In some aspects, the pressure in the renal pelvis can be measured during the injection of AAV vector solution using a pressure transducer connected to the catheter engaged in the pelvic space. The pressure may be elevated during the injection of AAV vector solution to approximately 30 to 70 cmtLO or approximately 22 to 52 mmHg. However, high pressures such as 130 cmH20 or 95 mmHg or greater are generally not required for achieving AAV vector transduction in the kidney.

[0042] In some aspects, the retrograde pelvic injection method may utilize “pyelovenous back flow” to retrogradely introduce the AAV vector solution into the renal parenchyma under balloon occluded condition. Pyelovenous back flow can occur more frequently with approximately 40 crnHiO (30 mmHg) or higher intrapelvic pressure, while normal intrapelvic pressure is approximately 15 cmH2O (or 1 1 mmHg). Thus, in aspects, the pressure is kept high enough to achieve this pyelovenous back flow; however, the pressure may not be greater than 70 cmHzO (or 52 mmHg) as it may cause rupture of the renal pelvis. In some aspects, the pressure is about 30 cmHjO to about 70 cmHiO. In more aspects, the pressure is about 30 to about 45 cmHiO, about 30 to about 50 cmHiO, about 30 to about 60 cmHzO, about 35 to about 50 cmHzO, about 35 to about 55 cmHzO, about 35 to about 60 cmH2O, about 35 to about 65 cmH2O, about 40 to about 55 cmHjO, about 40 to about 60 cmH2O, about 40 to about 65 cmH2O, about 45 to about 60 cmH2O, about 45 to about 65 cmH2O, about 40 to about 70 cmftO, about 45 to about 70 cmH2O, about 50 to about 70 cmlLO, or about 55 to about 70 cmH2O. In some aspects, the desired pressure may be maintained between 35 to 65 cmHzO (or about 26 to 48 mmHg), a higher end of this pressure range can be used at the time of introduction and a lower end of this pressure range can be used at the time of maintenance / dwelling.

[0043] In more aspects, that AAV vector composition can be included up to about 1 x 1014vg / mL but the concentrations above about 1 x 1013vg / mL can lead to vector particle aggregation. In some aspects, the AAV is administered at a dose of about 1 x 1011to about 1 x 1013viral genomes (vg) per kilogram (kg). In some examples, the AAV is administered at a dose of about 1 x 1012to about 8 x 1013vg / kg. In other examples, the AAV is administered at a dose of about 1 x 1013to about 6 x 1013vg / kg. In specific nonlimiting examples, the AAV is administered at a dose of at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, at least about 5 x 1012, at least about 1 x 1013, at least about 5 x 1013, or at least about 1 x 1014vg / kg. In other non-limiting examples, the AAV is administered at a dose of no more than about 5 x 1011, no more than about 1 x 1012, no more than about 5 x 1012, no more than about 1 x 1013, no more than about 5 x 1013, or no more than about 1 x 1014vg / kg. In one non-limiting example, the AAV is administered at a dose of about 1 x 1012vg / kg.

[0044] Terms

[0045] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “a vector” includes singular or plural vectors and can be considered equivalent to the phrase “at least one vector.” As used herein, the term “comprises” means “includes.” The term “about” indicates within five percent. It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0046] The term “AAV capsid” as used herein means any assembled AAV capsid that comprises or derives from a specific type of AAV viral protein shell (i.e., capsid).

[0047] The term “AAV vector” as used herein means any vector that comprises or derives from components of AAV and is suitable to infect mammalian cells, including human cells, of any of a number of tissue types, such as brain, heart, lung, skeletal muscle, liver, kidney, spleen, or pancreas, whether in vitro or in vivo. The term “AAV vector” may be used to refer to an AAV type viral particle (or virion) comprising at least a nucleic acid molecule encoding a protein or nucleic acid molecule of interest.

[0048] The term “administration” refers to providing or giving a subject an agent by any effective route. Exemplary routes of administration include, but arc not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, intrathecal and intratumoral), sublingual, rectal, transdermal, intranasal, intraductal, vaginal and inhalation routes. In some aspects, administration is to a urinary tract via direct renal pelvic injection surgically or percutaneously or retrograde transureteral renal pelvic injection.

[0049] An “agent” includes any vector, polypeptide, compound, small molecule, organic compound, salt, polynucleotide, or other molecule of interest. An agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A “therapeutic agent” is a substance that demonstrates some therapeutic effect by restoring or maintaining health, such as by alleviating the symptoms associated with a disease or physiological disorder, or delaying (including preventing) progression or onset of a disease, such as a kidney disease such as chronic kidney disease (CKD). An agent can be an AAV vector.

[0050] An “animal” is a living multi-cellular vertebrate organism, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects, including non-human primates.

[0051] The term “conservative substitutions” of a polypeptide that involve the substitution of one or more amino acids for amino acids having similar biochemical properties that do not result in change or loss of a biological or biochemical function of the polypeptide are designated “conservative” substitutions. These conservative substitutions are likely to have minimal impact on the activity of the resultant protein. Table A shows amino acids that can be substituted for an original amino acid in a protein, and which are regarded as conservative substitutions.

[0052] TABLE A

[0053] Original Residue Conservative Substitutions

[0054] Ala ser

[0055] Arg lys

[0056] Asn gin; his

[0057] Asp glu

[0058] Cys ser

[0059] Gin asn

[0060] Glu asp

[0061] Gly pro

[0062] His asn; gin lie leu; val

[0063] Leu ile; val

[0064] Lys arg; gin; glu

[0065] Met leu; ile

[0066] Phe met; leu; tyr

[0067] Ser thr

[0068] Thr ser Trp tyr

[0069] Tyr trp; phe

[0070] Vai ile; leu

[0071] One or more conservative changes, or up to ten conservative changes (such as two substituted amino acids, three substituted amino acids, four substituted amino acids, or five substituted amino acids, etc.) can be made in the polypeptide without changing a biochemical function of the protein, such as a capsid protein, such as the affinity for a particular cell type.

[0072] “Chronic kidney disease” or “CKD” is a condition resulting in progressive loss of kidney function over a period of months or years. In several aspects, a subject with CKD is one having a glomerular filtration rate (GFR) of less than 60 mL / min / 1.73 m2for three consecutive months (sec also, the National Kidney Foundation’s guidelines for diagnosing CKD (Levey et al., Ann Intern. Med., 139: 137-147, 2003), incorporated by reference herein in its entirety). CKD is often diagnosed in the course of screening individuals known to be at risk of CKD, such as those with high blood pressure or diabetes, or those with a family history of CKD. CKD may also be identified when it leads to one of its recognized complications, such as cardiovascular disease, anemia or pericarditis.

[0073] The severity of CKD can be classified in five stages based on level of kidney function, with stage 1 being the mildest and usually causing few symptoms and stage 5 being a severe illness with poor life expectancy. The stages include:

[0074] Stage 1: Slightly diminished function; kidney damage with normal or relatively high GFR (>90 mL / min / 1.73 nr). Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.

[0075] Stage 2. Mild reduction in GFR (60-89 mL / min / 1.73 m2) with kidney damage.

[0076] Stage 3. Moderate reduction in GFR (30-59 mL / min / 1.73 m2).

[0077] Stage 4. Severe reduction in GFR (15-29 mL / min / 1.73 m2). Preparation for renal replacement therapy.

[0078] Stage 5. Established kidney failure (GFR <15 mL / min / 1.73 nr, permanent renal replacement therapy (RRT), or end stage renal disease (ESRD). Stage 5 CKD is also known as end stage renal disease (ESRD) or end-stage kidney disease (ESKD).

[0079] Methods of identifying and treating a subject with CKD are known to the person of ordinary skill in the art. As used herein, CKD does not include acute kidney disease, the etiology of which typically includes an acute causative event (e.g., sepsis, ischemia, trauma, and / or nephrotoxic drugs) which leads to onset of kidney disease in less than three months. (See, e.g., Levey et al., Ann Intern. Med., 139: 137-147, 2003, Brenner and Rector, Eds., The Kidney, 9thedition. Philadelphia, Elsevier, Saunders. 2001; Lameire et al., Lancet, 382: 168-179, 2013; and Jo et al., Clin J Am Soc Nephrol, 2: 356-365, 2007, each of which is incorporated by reference herein in its entirety.)

[0080] The condition “Alport syndrome” is an inherited form of nephritis caused by mutation in the COL4A3, COL4A4, and / or COL4A5 genes for collagen and is sometimes referred to as hereditary nephritis, hemorrhagic familial nephritis, or hereditary deafness and nephritis. The term encompasses the specific individual forms of X-linked Alport syndrome (XLAS), autosomal recessive Alport syndrome (ARAS), and autosomal dominant Alport syndrome (ADAS).

[0081] A “control” is a reference standard. In some aspects, the control is a negative control sample obtained from a healthy patient. In other aspects, the control is a positive control, such as a sample obtained from a patient diagnosed with CKD or a dyslipidemic or vascular disorder. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of CKD or dyslipidemic or vascular disorder patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

[0082] A “difference” between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 68%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, or greater than 500%.

[0083] The term “detarget” refers to less than about 0.05 (5%), less than about 0.1 (10%) or less than about 0.2 (20%) of AAV vector genomes delivered to the organ of interest or AAV vector genome transcripts (transgene expression) compared to those of a benchmark (control) AAV. In some aspects, AAV9 as the benchmark (control) AAV.

[0084] The term “expressed” refers to the transcription of a nucleic acid into RNA or the translation of a nucleic acid into a protein. RNA may be expressed and remain intracellular or be secreted into the extracellular matrix or medium. Proteins may be expressed and remain intracellular, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

[0085] The term “expression control sequences” refers to nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signal for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0086] A “heterologous” sequence is a sequence that is not normally (in the wild-type sequence) found adjacent to a second sequence. In one aspect, the sequence is from a different genetic source, such as a virus or organism, than the second sequence.

[0087] An “isolated” biological component (such as a nucleic acid, peptide or protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, z.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides and proteins which have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. An isolated cell type has been substantially separated from other cell types, such as a different cell type that occurs in an organ. A purified cell or component can be at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0088] The” kidney” is an organ found in vertebrates that participates in the control of the volume of various body fluids, fluid osmolality, acid-base balance, various electrolyte concentrations, and removal of toxins. The kidneys receive blood from the paired renal arteries; blood exits into the paired renal veins. Each kidney is attached to a ureter, a tube that carries excreted urine to the bladder. Filtration occurs in the glomerulus. One-fifth of the blood volume that enters the kidneys is filtered. Examples of substances reabsorbed are solute-free water, sodium, bicarbonate, glucose, and amino acids. Examples of substances secreted are hydrogen, ammonium, potassium and uric acid. The nephron is the structural and functional unit of the kidney. Each adult human kidney contains around 1 million nephrons, while a mouse kidney contains only about 12,500 nephrons. The kidneys also carry out functions independent of the nephrons. For example, they convert a precursor of vitamin D to its active form, calcitriol; and synthesize the hormones erythropoietin and renin. The kidney is comprised of the following components: (1) nephron, (2) interstitium and (3) vasculature. The (1) nephron comprises glomerular endothelium, mesangial cells, podocytes, parietal epithelial cells, renal tubules (proximal tubules, Loop of Henle cells, distal tubules, collecting duct cells, and parietal epithelial cells). The (2) interstitium comprises fibroblasts, myofibroblasts, and pericytes. The (3) vasculature comprises vascular endothelial cells, vascular smooth muscle cells. The kidney also contains blood cells or bone marrow-derived cells, including lymphocytes, monocytes, neutrophils, dendritic cells, mast cells and resident macrophages. The kidney also contains blood cells, including lymphocytes, monocytes, neutrophils and resident macrophages. The kidney also contains specialized endocrine cells that produce erythropoietin, renin and calcitriol. “Podocytes” are cells in Bowman's capsule in the kidneys that wrap around capillaries of the glomerulus. Podocytes make up the epithelial lining of Bowman's capsule of the kidney, which filters the blood. Although various viscera have epithelial layers, the name “visceral epithelial cells” usually refers specifically to podocytes, which are specialized epithelial cells that reside in the visceral layer of the capsule. The podocytes have long foot processes called pedicels. These pedicels wrap around the capillaries and leave slits between them. Blood is filtered through these slits, each known as a filtration slit, slit diaphragm, or slit pore. “Epithelial cells” form the lining of the urinary tract. “Renal tubular epithelial cells” line the collecting ducts and the distal and proximal tubules of the kidney. In the kidney, the “proximal tube” is the segment of the nephron in kidneys which begins from the renal pole of the Bowman's capsule to the beginning of loop of Henle. It can be further classified into the proximal convoluted tubule and the proximal straight tubule. “Proximal tubule cells” are the cells from the proximal tubule. The luminal surface of the epithelial cells of this segment of the nephron is covered with densely packed microvilli forming a border that increases the luminal surface area of the cells, which are also densely packed with mitochondria. Cuboidal epithelial cells lining the proximal tubule have extensive lateral interdigitations between neighboring cells, which lend an appearance of having no discrete cell margins when viewed with a light microscope. A “nephron” is a microscopic structural and functional unit of the kidney composed of a renal corpuscle and a renal tubule. The renal corpuscle consists of a tuft of capillaries called a glomerulus and a cup-shaped structure called Bowman's capsule. The renal tubule extends from the capsule. The capsule and tubule are connected and are composed of epithelial cells with a lumen. A healthy adult has 1 to 1.5 million nephrons in each kidney.

[0089] The term “mammal” includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.

[0090] A “nucleic acid molecule” is a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include non-naturally occurring synthetic analogs, such as, for example and without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term “oligonucleotide” typically refers to short polynucleotides, generally no greater than about 50 nucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

[0091] Conventional notation is used herein to describe nucleotide sequences: the left-hand end of a singlestranded nucleotide sequence is the 5'-end; the left-hand direction of a double-stranded nucleotide sequence is referred to as the 5'-direction. The direction of 5' to 3’ addition of nucleotides to nascent RNA transcripts is referred to as the transcription direction. The DNA strand having the same sequence as an mRNA is referred to as the “coding strand;” sequences on the DNA strand having the same sequence as an mRNA transcribed from that DNA and which are located 5’ to the 5'-end of the RNA transcript are referred to as “upstream sequences;” sequences on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the coding RNA transcript are referred to as “downstream sequences.” With regard to nucleotide molecules, the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0092] In addition, a “recombinant nucleic acid” refers to a nucleic acid having nucleotide sequences that are not naturally joined together. This includes nucleic acid vectors comprising an amplified or assembled nucleic acid which can be used to transform a suitable host cell. A host cell that comprises the recombinant nucleic acid is referred to as a “recombinant host cell.” The gene is then expressed in the recombinant host cell to produce, such as a “recombinant polypeptide.” A recombinant nucleic acid may serve a non-coding function (such as a promoter, origin of replication, ribosome-binding site, etc.) as well.

[0093] For sequence comparison of nucleic acid sequences, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters are used. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Naf 1. Acad. Sci. USA 85:2444, 1988, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see for example, Current Protocols in Molecular Biology (Ausubel et al., eds 1995 supplement)).

[0094] One example of a useful algorithm is PILEUP. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used is similar to the method described by Higgins & Sharp, CABIOS 5: 151 -153, 1989. Using PILEUP, a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP can be obtained from the GCG sequence analysis software package, such as version 7.0 (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984.

[0095] Another example of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and the BLAST 2.0 algorithm, which are described in Altschul et al., J. Mol. Biol. 215:403-410, 1990 and Altschul et al.. Nucleic Acids Res. 25:3389-3402, 1977. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLASTP program (for amino acid sequences) uses as defaults a word length (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915, 1989).

[0096] A first nucleic acid sequence is “operably linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0097] A “pharmaceutically acceptable carrier” of use includes conventional excipients. Remington’s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975), describes compositions and formulations suitable for pharmaceutical delivery of the fusion proteins herein disclosed. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0098] A “polypeptide” or “protein” is a polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms "polypeptide" or “protein” as used herein is intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A “therapeutic protein” is a protein that, when expressed in a subject, results in an improvement of a sign or a symptom of a particular disorder in that subject, such as a kidney. In some aspects, administration of a “therapeutic protein” results in an improvement of a sign or a symptom of a kidney disorder in a subject, such as, but not limited to AKD and CKD. Administration of a therapeutic protein can result in improvement of systemic disease in a subject, such as, inborn errors of metabolism, autoimmune diseases, hypertension and diabetes.

[0099] “Preventing” a disease (such as a kidney disease) refers to inhibiting the full development of a disease. “Treating” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease.

[0100] A “promoter” refers to an array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Also included are those promoter elements which are sufficient to render promoterdependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters are included (see for example, Bitter et al., Methods in Enzymology 153:516-544, 1987). The promoter can direct expression in the cells of the kidney.

[0101] A “recombinant nucleic acid molecule” refers to one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, such as by genetic engineering techniques. Similarly, a recombinant protein is one encoded for by a recombinant nucleic acid molecule. In addition, a recombinant virus is a virus comprising sequence (such as genomic sequence) that is non-naturally occurring or made by artificial combination of at least two sequences of different origin. The term “recombinant” also includes nucleic acids, proteins and viruses that have been altered solely by addition, substitution, or deletion of a portion of a natural nucleic acid molecule, protein or virus. As used herein, “recombinant AAV” refers to an AAV particle in which a recombinant nucleic acid molecule (such as a recombinant nucleic acid molecule encoding a therapeutic protein) has been packaged.

[0102] A “subject” refers to any mammal, such as humans, non-human primates, pigs, sheep, cows, rodents and the like which is to be the recipient of the particular treatment. In two non-limiting examples, a subject is a human subject or a non-human primate subject. The subject can have a kidney disease.

[0103] A “therapeutically effective amount” refers to a quantity of a specified pharmaceutical or therapeutic agent (e.g. a recombinant AAV) sufficient to achieve a desired effect in a subject, or in a cell, being treated with the agent, such as increasing kidney function or another desired effect. The effective amount of the agent will be dependent on several factors, including, but not limited to the subject or cells being treated, and the manner of administration of the therapeutic composition.

[0104] A virus or vector “transduces” a cell when it transfers nucleic acid into the cell. A cell is “transformed” or “transfected” by a nucleic acid transduced into the cell when the DNA becomes stably maintained by the cell, either by incorporation of the nucleic acid into the cellular genome, or by episomal persistence.

[0105] Numerous methods of transfection are known to those skilled in the art, such as: chemical methods (e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope / capsid-DNA complexes) and by biological infection by viruses such as recombinant viruses {Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)}. In the case of infection by viruses, the infecting virus particles are absorbed by the target cells, resulting in integration of the viral genome into the cellular DNA or persistent presence of viral genetic components as cpisomcs.

[0106] Genetic modification of the target cell is an indicium of successful transfection. "Genetically modified cells" refers to cells whose genotypes have been altered as a result of cellular uptakes of exogenous nucleotide sequence by transfection. A reference to a transfected cell or a genetically modified cell includes both the particular cell into which a vector or polynucleotide is introduced and progeny of that cell.

[0107] A “transgene” refers to an exogenous gene supplied by a vector or a virus, such as an AAV.

[0108] AAV Vectors and Their Use

[0109] AAV vectors including a capsid protein are disclosed herein that can transduce cells of the kidney, such as of podocytes, mesangial cells, tubular epithelial cells, collecting duct cells, interstitial cells, and vascular cells. In some aspects, these AAV vector including a capsid protein are used for treating kidney disorders. The current disclosure also includes variants of the capsid polypeptides disclosed herein. Variant sequences include those sequences wherein one or more peptides or nucleotides of the sequence have been substituted, deleted, and / or inserted.

[0110] Additionally, the AAVs disclosed herein may be derived from various serotypes, including combinations of capsid serotypes and viral genome serotypes (e.g., "pseudotyped" AAV) or from various genomes (e.g., single-stranded or self-complementary). In particular aspects, the AAV vectors disclosed herein may comprise desired proteins or protein variants. A “variant” as used herein, refers to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both.

[0111] Adeno-associated viral vectors can be used in the methods disclosed herein. AAV belongs to the family Parvoviridae and the genus Dependovirus. AAV is a small, non-enveloped virus that packages a linear, single-stranded DNA genome. Both sense and antisense strands of AAV DNA are packaged into AAV capsids with equal frequency. In some aspects the AAV DNA includes a nucleic acid encoding a therapeutic protein. In other aspects, the AAV DNA includes a nucleic acid encoding an inhibitory RNA, such as a short hairpin (sh)RNA, an antisense RNA, or a small inhibitory interfering (si)RNA. The nucleic acid can also encode Cas9, gRNAs, or other components of a CRISPR / Cas9 system. The nucleic acid can by itself serves as homology-directed repair DNA template

[0112] The AAV genome is characterized by two inverted terminal repeats (ITRs) that flank two open reading frames (ORFs). In the AAV2 genome, for example, the first 125 nucleotides of the ITR are a palindrome, which folds upon itself to maximize base pairing and forms a T-shaped hairpin structure. The other 20 bases of the ITR, called the D sequence, remain unpaired. The ITRs are cis-acting sequences important for AAV DNA replication; the ITR is the origin of replication and serves as a primer for second- strand synthesis by DNA polymerase. The double-stranded DNA formed during this synthesis, which is called replicating-form monomer, is used for a second round of self-priming replication and forms a replicating-form dimer. These double-stranded intermediates arc processed via a strand displacement mechanism, resulting in single-stranded DNA used for packaging and double-stranded DNA used for transcription. Located within the ITR are the Rep binding elements and a terminal resolution site (TRS ). These features are used by the viral regulatory protein Rep during AAV replication to process the doublestranded intermediates. In addition to their role in AAV replication, the ITR is also essential for AAV genome packaging, transcription, negative regulation under non-permissive conditions, and site-specific integration (Daya and Berns, Clin Microbiol Rev 2 l(4):583-593, 2008). In some aspects, these elements are included in the AAV vector.

[0113] The left ORF of AAV contains the rep gene, which encodes four proteins - Rep78, Rep 68, Rep52 and Rep40. The right ORF contains the cap gene, which produces three viral capsid proteins (VPl, VP2 and VP3). The right ORF also contains two additional frame-shifted ORFs for the membrane-associated accessory protein (MAAP) and the assembly-activating protein (AAP). The AAV capsid contains 60 viral capsid proteins arranged into an icosahcdral symmetry. VPl, VP2 and VP3 arc present in a 1 :1: 10 molar ratio (Daya and Berns, Clin Microbiol Rev 21(4):583-593, 2008). In some aspects, these elements are included in the AAV vector. The AAV vector is packaged in the capsid proteins, which can target specific cell types, such as podocytes, mesangial cells, tubular epithelial cells, collecting duct cells, interstitial cells, and vascular cells .

[0114] In some aspects, a recombinant adeno-associated virus (rAAV) is generated having a capsid of interest. In AAV, the capsid includes VPl, VP2, and VP3. In some non-limiting examples, to produce a vector, a host cell which can be cultured that contains a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein of interest, or fragment thereof; a functional rep gene; a minigene composed of, at a minimum, AAV inverted terminal repeats (ITRs) and a transgene, such as a transgene encoding a therapeutic protein; and sufficient helper functions to permit packaging in the capsid protein. The components required to be cultured in the host cell to package an AAV minigene in an AAV capsid may be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigene, rep sequences, cap sequences, and / or helper functions) may be provided by a stable host cell which has been engineered to contain one or more of the required components using methods known to those of skill in the art. In some aspects, a stable host cell will contain the required component(s) under the control of an inducible promoter. However, the required component(s) can be under the control of a constitutive promoter. Promoters of use include, but are not limited to, the CMV promoter, the CAG promoter, the CB promoter, the SV40 promoter, the ubiquitin promoter, the EFl a promoter, the GAPDH promoter, the PGK promoter, the RSV promoter, and the |3-actin promoter.

[0115] In still another alternative, a selected stable host cell may contain selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contains the Rep and / or Cap protein-coding sequences under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0116] The minigene, Rep-coding sequences, Cap-coding sequences, and helper functions required for producing a rAAV can be delivered to the packaging host cell in the form of any genetic element which transfer the sequences carried thereon. The selected genetic element may be delivered by any suitable method, including those described herein. The methods used to construct vectors are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thEdition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 11724. Similarly, methods of generating rAAV virions are well known and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745.

[0117] The AAV vectors disclosed herein can include a promoter operably linked to a therapeutic protein of interest. In some aspects, the promoter is a ubiquitous promoter such as the CMV promoter, the CAG promoter, the CB promoter, the SV40 promoter, the ubiquitin promoter, the EFloc promoter, the GAPDH promoter, the PGK promoter, the RSV promoter, and the [I-actin promoter. In some aspects, the promoter is a kidney-specific or cell type-specific promoter, such as the podocin (NPHS2) promoter, the nephrin (NPHS1) promoter, the synaptopodin promoter, the WT1 promoter, the podocalyxin prompter, the PAX8 promoter, the gamma-glutamyltransferase (GGT) promoter, the sodium-glucose cotransporter-2 (SGLT2) promoter, the kidney androgen-regulated protein (KAP) promoter, the megalin promoter, the carbonic anhydrase II (CAII) promoter, the organic anion transporter 1 (OAT1, SLC22A6) promoter, the sodiumphosphate cotransporter 2a (NPT2a) promoter, the E-cadherin promoter, the kidney-specific cadherin promoter, the Na-K-Cl cotransporter 2 (NKCC2, SLC12A1) promoter, the WNK1 promoter, the WNK4 promoter, the v-type proton ATPase subunit Bl (ATP6V1B1) promoter, the thiazide-sensitive Na-Cl cotransporter (NCC) promoter, the calcium-sensing receptor (CaSR) promoter, the epithelial sodium channel (ENaC) promoter, the uromodulin promoter, the aquaporin 1 (AQP1) promoter, the aquaporin 2 (AQP2) promoter, the vasopressin receptor 2 (V2R) promoter, the transient receptor potential vanilloid 4 (TRPV4) promoter, the renin promoter, the parathyroid hormone receptor promoter, the smooth muscle alpha-actin (ACTA2) promoter, the COL4A1 promoter, the platelet-derived growth factor receptor beta (PDGFRfJ) promoter, the smooth muscle protein 22-a (SM22a) promoter, the smooth muscle myosin heavy chain (SM- MHC) promoter, the neuron-glial antigen 2 (NG2, chondroitin sulphate proteoglycan 4 (CSPG4)) promoter, the desmin promoter, the regulator of G-protein signaling-5 (RGS5) promoter, the fibroblast-specific protein 1 (FSP1) promoter, the transcription factor 21 (TCF21) promoter, the promoter the COL1A2 promoter, the COL3A1 promoter, the vimentin promoter, the TIE2 promoter, the von Willebrand factor (vWF) promoter, the CD31 promoter and the VE-cadherin promoter.

[0118] In some aspects, the AAV genome is modified to include a gene encoding a selectable marker, which includes, but are not limited to, a protein whose expression can be readily detected such as a fluorescent or luminescent protein or an enzyme that acts on a substrate to produce a colored, fluorescent, or luminescent substance ("detectable markers"). There are other genes of use, such as genes that encode drug resistance or provide a function that can be used to purify cells. Selectable markers include neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyl transferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine - guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP) blue, sapphire, yellow, red, orange, and cyan fluorescent proteins and variants of any of these. Luminescent proteins such as luciferase (e.g., firefly or Renilla luciferase) are also selectable makers.

[0119] Polypeptide sequences of the current disclosure can also be defined in terms of particular identity and / or similarity with certain polypeptides described herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and / or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence disclosed herein. The sequence identify can be about 95%, 96%, 97% 98%, or 99%. Unless otherwise specified, as used herein percent sequence identity and / or similarity of two sequences can be determined using the algorithm of Karlin and Altschul, Proc Natl Acad Sci U S A. 87(6):2264-8 1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul el al. (1990). BLAST searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (Nucleic Acids Res. 25( 17) : 3389- 402,1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used.

[0120] The vectors disclosed herein can contain nucleic acid sequences encoding an intact AAV capsid which may be from a single AAV serotype.

[0121] In an aspect, the methods may comprise an adeno-associated virus (AAV) vector as the gene therapy agent. The AAV vector can transduce one or more kidney cell types, such as cells constituting a nephron, cells in the renal interstitium and cells constituting the vasculature in the kidney, These cells include, but are not limited to, mesangial cells, glomerular endothelial cells, podocytes, parietal epithelial cells in Bowman’ s capsule, proximal tubule cells, Loop of Henle cells, distal tubule cells, collecting duct cells, fibroblasts, myofibroblasts, pericytes, vascular endothelial cells, and vascular smooth muscle cells. In the method described herein. The AAV vector comprises a capsid protein comprising a capsid sequence selected from the group of AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, or AAVShHIO capsid protein or a variant thereof exhibiting at least 70% identity thereto. The sequence identify can be about 95%, 96%, 97% 98%, or 99%. In the case the AAV vector transduces duct cells, the AAV vector comprises a capsid protein comprising a capsid sequence selected from the group of AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, AAVShHIO capsid protein.

[0122] Furthermore, the AAV vector may detarget at least one other cell type, such as liver cells, resulting in transduction of the cell type less than about 0.25 times that of AAV9 delivered at the delivery point. The AAV vector may detarget at least one cell type, resulting in transduction of at least one cell type less than about 0.20, 0.15 or 0.10, or 0.05 times that of AAV9 delivered at the delivery point. Yet further, the AAV vector may disseminate to at least one of the liver, heart, and kidney, and pancreas, to a level less than about 0.1 times that of AAV9 delivered at the delivery point. The AAV vector may disseminate to at least one of the liver, heart or pancreas at a level less than about 0.09, 0.08, 0.07, 0.06, or 0.05 times that of AAV9 delivered at the delivery point.

[0123] In some aspects, AAV vector described herein may target kidney tissue. Accordingly, in some aspects, AAV described herein may be useful for treatment of kidney disease. As used herein a “kidney disease” is a disease or condition of the kidney. A kidney disease may be of a genetic origin, either inherited or acquired through a somatic mutation. A kidney disease may be a cancer of the kidney, including but not limited to renal cell cancer, clear cell cancer, papillary cancer type 1, papillary cancer type 2, chromophobe cancer, oncocytic cell cancer, collecting duct cancer, transitional cell cancer of the renal pelvis and Wilm's tumor. Further non- limiting examples of kidney disease include Abderhalden-Kaufmann-Lignac syndrome (nephropathic cystinosis), acetaminophen-induced nephrotoxicity, acute kidney failure / acute kidney injury, acute lobar nephronia, acute phosphate nephropathy, acute tubular necrosis, adenine phosphoribosyltransferase deficiency, adenovirus nephritis, Alagille syndrome, alkaptonuria, Alport syndrome, Alstrom syndrome, amyloidosis, ANCA vasculitis related to endocarditis and other infections, angiomyolipoma, anabolic steroid abuse, analgesic nephropathy, anorexia nervosa and kidney disease, anticoagulant-related nephropathy, angiotensin II type 1 receptor antibody-mediated focal segmental glomerulosclerosis, antiphospholipid syndrome, anti-LRP2 nephropathy (anti-brush border nephropathy), anti-TNF-a therapy-related glomerulonephritis, APOL1 mutations, apparent mineralocorticoid excess syndrome, APRT deficiency and 2,8-dihydroxyadenine nephropathy, aristolochic acid nephropathy, arteriovenous malformations and fistulas of the urologic tract, autosomal dominant hypocalcemia, Balkan endemic nephropathy, Bardet-Biedl syndrome, Bartter Syndrome, bath salts and acute kidney injury, beer potomania, beeturia, 0-thalassemia renal disease, bile cast nephropathy, Birt-Hogg-Dube syndrome, BK Polyoma, bodybuilder's glomerulopathy, border-crossers' nephropathy, bourbon virus and acute kidney injury, brash syndrome, burnt sugarcane harvesting and acute renal dysfunction, Byetta and renal failure, Clq nephropathy, C3 glomerulopathy, C3 glomerulopathy with monoclonal gammopathy, C4 glomerulopathy, CAKUT (congenital anomalies of the kidney and urologic tract), calyceal diverticulum, calcineurin inhibitor nephrotoxicity, callilepsis laureola poisoning, cannabinoid hyperemesis acute renal failure, capillary leak syndrome, carbon disulfide and renal injury, carcinoid syndrome and renal failure, cardiorenal syndrome, cardiorenal syndrome, Carfilzomib-induced renal injury, CFHR5 nephropathy, Charcot-Marie -Tooth disease with glomerulopathy, Chinese herbal medicines and nephrotoxicity, cherry concentrate and acute kidney injury, Chinese herbal medicine nephropathy, cholesterol emboli, Churg- Strauss syndrome, chyluria, ciliopathy, cisplatin nephrotoxicity, cocaine and the kidney, cold diuresis, colistin nephrotoxicity, collagenofibrotic glomerulopathy, collagen-related kidney diseases, collapsing glomerulopathy (collapsing glomerulopathy related to CMV), combination antiretroviral (CART) related- nephropathy, congenital adrenal hyperplasia, 17-alpha-hydroxylase / 7 20-lyase deficiency, congenital anomalies of the kidney and urinary tract (CAKUT), congenital nephrotic syndrome, congestive renal failure, conorenal syndrome (Mainzer-Saldino syndrome or Saldino-Mainzer disease), contrast nephropathy, copper sulfate intoxication, coronavirus (COVID-19) associated kidney failure and kidney disease, cortical necrosis, Crizotinib-related acute kidney injury, crying kidneys, cryocry stalglobulinemia, cryoglobulinemia, crystalglobulin-induced nephropathy, crystal-induced acute kidney injury, crystal-storing histiocytosis, crystal meth-associated cortical necrosis, cystic kidney disease-acquired, cystic kidney disease-localized, cystinosis, cystinuria, Dasatinib-induced nephrotic -range proteinuria, Deferasirox (Exjade) nephrotoxicity, dense deposit disease (MPGN Type 2), Dent disease (X-linked recessive nephrolithiasis), DHA crystalline nephropathy, dialysis disequilibrium syndrome, diabetes and diabetic kidney disease, diabetes insipidus, distal renal tubular acidosis, dietary supplements and renal failure, diffuse mesangial sclerosis, diuresis, djcnkol bean poisoning (djcnkolism), Down syndrome and kidney disease, drugs of abuse and kidney disease, duplicated ureter, EAST syndrome, eating disorders and kidney disease, EBOLA and the kidney, ectopic kidney, ectopic ureter, edema, Erdheim-Chester disease, Fabry's Disease, familial amyloidosis, familial hypocalciuric hypercalcemia, Fanconi syndrome, Fraser syndrome, fibronectin glomerulopathy, fibrillary glomerulonephritis and immunotactoid glomerulopathy, Fraley syndrome, focal segmental glomerulosclerosis (focal sclerosis, focal glomerulosclerosis), focal segmental glomerulosclerosis (familial) with complete heart block, Galloway Mowat syndrome, giant cell (temporal) arteritis with kidney involvement, gestational hypertension, Gitelman syndrome, glomerular diseases, glomerular tubular reflux, glycosuria, Goodpasture syndrome, green smoothie cleanse nephropathy, HANAC syndrome, Harvoni (Ledipasvir with Sofosbuvirj-induced renal injury, hair dye ingestion and acute kidney injury, hantavirus infection podocytopathy, heat stress nephropathy, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), hemophagocytic syndrome, hemorrhagic cystitis, hemorrhagic fever with renal syndrome (HFRS, hantavirus renal disease, Korean hemorrhagic fever, epidemic hemorrhagic fever, nephropathis epidemica), hemosiderinuria, hemosiderosis related to paroxysmal nocturnal hemoglobinuria and hemolytic anemia, hepatic glomerulopathy, hepatic veno-occlusive disease, sinusoidal obstruction syndrome, hepatitis C-associated renal disease, hepatocyte nuclear factor 1 [3-associated kidney disease, hepatorenal syndrome, herbal supplements and kidney disease, high altitude renal syndrome, high blood pressure and kidney disease, HTV-associated immune complex kidney disease (HIVTCK), HTV-associated nephropathy (HIV AN), hereditary interstitial kidney disease, holocaust kidney disease, HNFlb-related autosomal dominant tubulointerstitial kidney disease, horseshoe kidney (renal fusion), Hunner's ulcer, hyperaldosteronism, hypercalcemia, hyperkalemia, hypermagnesemia, hypernatremia, hyperoxaluria, hyperphosphatemia, hypocalcemia, hypokalemia (hypokalemia-induced renal dysfunction), hypokalemic periodic paralysis, hypomagnesemia, hyponatremia, hypophosphatemia, hypophosphatemia in users of cannabis, hypertension, hypertension, monogenic, iced tea nephropathy, Ifosfamide nephrotoxicity, IgA nephropathy, IgG4 nephropathy, immersion diuresis, immune-checkpoint therapy-related interstitial nephritis, Infliximab-rclatcd renal disease, interstitial cystitis (painful bladder syndrome), interstitial nephritis, interstitial nephritis (karyomegalic), Ivemark's syndrome, JC virus nephropathy, Joubert syndrome, ketamine-associated bladder dysfunction, kidney stones (nephrolithiasis), kidney stones due to light chains, Kimura disease, kombucha tea toxicity, lead nephropathy and lead-related nephrotoxicity, lecithin cholesterol acyltransferase deficiency (LCAT deficiency), leptospirosis renal disease, light chain deposition disease (monoclonal immunoglobulin deposition disease), Liddle syndrome, Lightwood-Albright syndrome, lipoprotein glomerulopathy, lithium nephrotoxicity, LMX1B mutations-caused hereditary FSGS, loin pain hematuria, Lowe syndrome, lupus (systemic lupus erythematosis), lupus kidney disease (lupus nephritis), lupus nephritis with antineutrophil cytoplasmic antibody seropositivity, lupus podocytopathy, lupus-like nephritis, Lyme disease-associated glomerulonephritis, lysinuric protein intolerance, lysozyme nephropathy, malarial nephropathy, malignancy-associated renal disease, malignant hypertension, malakoplakia, Marfan syndrome and kidney disease, Mckittrick-Wheelock syndrome, MDMA (Molly; Ecstasy; 3,4-mcthylcncdioxymcthamphctaminc) and kidney failure, meatal stenosis, medullary cystic kidney disease (uromodulin-associated nephropathy, juvenile hyperuricemic nephropathy type 1), medullary sponge kidney, megaureter, melamine toxicity and the kidney, MELAS syndrome, membranoproliferative glomerulonephritis, membranous nephropathy, membranous nephropathy with spherules, membranous-like glomerulopathy with masked IgG kappa deposits, mercury poisoning, Mesoamerican nephropathy, metabolic acidosis, metabolic alkalosis, Methotrexate-related renal failure, microscopic polyangiitis, milk- alkali syndrome, minimal change disease, monoclonal gammopathy of renal significance, dysproteinemia, mouthwash toxicity, MUC1 nephropathy, multicystic dysplastic kidney, multiple myeloma, myeloproliferative neoplasms and glomerulopathy, nail-patella syndrome, nephrocalcinosis, nephrocystin-1 gene deletions and ESRD, nephrogenic systemic fibrosis, nephronophthisis due to nephrocystin-1 gene deletions, nephroptosis (floating kidney, renal ptosis), nephrotic syndrome, neurogenic bladder, 9 / 11 and kidney disease, nodular glomerulosclerosis, non-gonococcal urinary tract infection, nuclear pore protein complex mutations and nephropathy, nutcracker syndrome, oligomeganephronia, orofaciodigital syndrome, orotic aciduria, orthostatic hypotension, orthostatic proteinuria, osmotic diuresis, osmotic nephrosis, ovarian hyperstimulation syndrome, oxalate nephropathy, pancreatitis and acute kidney injury, page kidney, papillary necrosis, papillorenal syndrome (renal-coloboma syndrome, isolated renal hypoplasia), PARN mutations and kidney disease, parvovirus B19 and the kidney, parvovirus infection causing interstitial fibrosis and acute kidney injury, perfluorinated chemicals and kidney disease, peritoneal-renal syndrome, POEM syndrome, posterior urethral valve, podocyte infolding glomerulopathy, post-infectious glomerulonephritis (post-streptococcal glomerulonephritis), post-infectious glomerulonephritis (atypical), polyarteritis nodosa, polycystic kidney disease, post-obstructive diuresis , preeclampsia, Propofol infusion syndrome, proliferative glomerulonephritis with monoclonal IgG deposits (Nasr disease), Propolis (honeybee resin) related renal failure, proteinuria, proximal renal tubular acidosis, pseudohyperaldosteronism, pseudohypobicarbonatemia, pseudohypoparathyroidism, pseudoporphyria, psoriasis and kidney disease, pulmonary-renal syndrome, purple urine bag syndrome, pyelonephritis, pyonephrosis, pyridium and kidney failure, quail poisoning and rhabdomyolysis, radiation nephropathy, Ranolazine and the kidney, refeeding syndrome, reflux nephropathy, rapidly progressive glomerulonephritis, REN mutations causing autosomal dominant tubulointerstitial kidney disease, renal abscess (perinephric abscess), renal agenesis, renal arcuate vein microthrombi-associated acute kidney injury, renal artery aneurysm, renal artery dissection (spontaneous), renal artery stenosis, renal cell cancer, renal cyst, renal hyperuricemia with exercise-induced acute renal failure, renal infarction, renal infarction due to amyloidosis, renal osteodystrophy, renal sinus lipomatosis, renal tubular acidosis, renin mutations and autosomal dominant tubulointerstitial kidney disease, renin secreting tumors (juxtaglomerular cell tumor), reset osmostat, retrocaval ureter, retroperitoneal fibrosis, rhabdomyolysis (rhabdomyolysis related to bariatric surgery), rheumatoid arthritis-associated renal disease, Rubraca (Rucaparib)-related increase in creatinine, sarcoidosis renal disease, salt wasting (renal and cerebral), Saturday night acute kidney injury, schistosomiasis and glomerular disease, Schimke immuno-osseous dysplasia, scleroderma renal crisis, Serpentine Fibula-polycystic kidney syndrome, steroid-resistant nephrotic syndrome (Exncr Syndrome), Sezary syndrome, sickle cell nephropathy, silica exposure and chronic kidney disease, Sjogren's syndrome and renal disease, Sri Lankan farmers' kidney disease, star fruit nephrotoxicity, synthetic cannabinoid use and acute kidney injury, kidney disease following hematopoietic cell transplantation (kidney disease related to stem cell transplantation), TAFRO syndrome, tea and toast hyponatremia, Tenofovir-induced nephrotoxicity, thrombotic microangiopathy associated with monoclonal gammopathy, trench nephritis, TREX1 mutation causing autosomal dominant thrombotic microangiopathy and CKD, thin basement membrane disease (Benign Familial Hematuria), trigonitis, tuberculosis (genitourinary), tuberous sclerosis, tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD) (TSC2 / PKD1 contiguous gene syndrome), tubulointerstitial kidney disease, tubular dysgenesis, immune complex tubulointerstitial nephritis due to autoantibodies to the proximal tubule brush border, tumor lysis syndrome, uremia, uremic encephalopathy and cortical blindness, uremic optic neuropathy, ureteritis cystica, ureterocele, urethral caruncle, urethral stricture, urinary incontinence, urinary tract infection, urinary tract obstruction, urogenital fistula, uromodulin-associated kidney disease, vancomycin-associated cast nephropathy, vasomotor nephropathy, vesicointestinal fistula, vesicoureteral reflux, VGEF inhibition and renal thrombotic microangiopathy, volatile anesthetics and acute kidney injury, von Hippel-Lindau disease, Waldenstrom's macroglobulinemic glomerulonephritis, warfarin-related nephropathy, wasp stings and acute kidney injury, Wegener's granulomatosis (granulomatosis with polyangiitis), West Nile virus and chronic kidney disease, Wunderlich syndrome and Zellweger syndrome (cerebrohepatorenal syndrome). In one aspect, the disease is CKD. In other aspects, the kidney disease is polycystic kidney disease, end stage kidney disease, or renal failure.

[0124] In some aspects, the kidney disease is polycystic kidney disease, end stage kidney disease, renal failure, an inherited monogenic disorder of the kidney, nephrolithiasis, nephronophthisis, Cystinuria disease, Dents disease, Fanconi syndrome, diabetic nephropathy, polycystic kidney disease, Alport syndrome, steroid resistant nephrotic syndrome, acute kidney injuries, thin basement membrane syndrome, Gitelman syndrome, Barttcr syndrome, a collagen-related kidney diseases, Lowe Syndrome, Liddle’s syndrome, Hereditary Interstitial Kidney Disease, Tuberous Sclerosis, Cystinosis, Fabry Disease, steroid-resistant nephrotic syndrome, distal renal tubular acidosis, familial amyloidosis, congenital nephrotic syndrome, tubulointerstitial kidney disease, proximal renal tubular acidosis, or an acute kidney injury. In other aspects, the kidney disease is glomerular disease, tubulointerstitial disease, renal vascular disease, genetic kidney disease, autoimmune kidney disease, infectious kidney disease, inborn errors of metabolism, renal cancer, acute kidney injury, chronic kidney disease, end stage kidney disease, ciliopathy including polycystic kidney disease, nephronophthisis, Bardet-Biedl syndrome and Meckel-Gruber syndrome, collagenopathy including Alport syndrome, congenital nephrotic syndrome, steroid resistant nephrotic syndrome, glomerulonephritis, nephrolithiasis including cystinuria and Dent disease, renal tubular acidosis, Fanconi syndrome, hereditary interstitial kidney disease, diabetes, diabetic nephropathy, thin basement membrane syndrome, nephrotic diabetes insipidus, hereditary tyrosinemia, cystinosis, Fabry disease, Gitelman syndrome, Bartter syndromes, Lowe syndrome, Liddle’s syndrome, or steroid-resistant nephrotic syndrome.

[0125] In some aspects, the AAV vector can further comprise a polynucleotide the translation product of which is a protein of therapeutic interest. By way of nonlimiting examples, the protein of interest can be collagen type 4 alpha 3, collagen type 4 alpha 4, collagen type 4 alpha 5, podocin, nephrin, rBAT, bn +amino acid transporter, nephrocystin 1, CLCN5, polycystin-1, polycystin- 2, BBS. When the proteins of interest are too large to be packaged, dual AAV vector approaches based on mRNA trans-splicing approaches, overlapping AAV vector approaches, intein-based protein splicing approaches can be employed. The AAV vector can include a promoter operably linked to a nucleic acid molecule encoding a therapeutic protein. Promoters can be ubiquitous promotors including the CMV promoter and CAG promoter. Promoters also include, but are not limited to, the promoter for sodium-dependent phosphate transporter type 2a (NPT2a) or gamma-glutamyl transpeptidase for expression in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) for expression in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) for expression in the collecting duct. These promoters can be used to target proteins for the treatment of kidney tubule disease (see Watanabe et al., PLoS One. 2017; 12(3): e0168638). Promoters also include, but are not limited to, the promoter for podocin, nephrin and podocalyxin for expression in podocytes. These promoters can be used to target proteins for the treatment of podocytopathies. Promoters also include, but are not limited to, the promoter for neuron-glial antigen 2 and platelet-derived growth factor receptor beta (PDGFR[3) for expression in pericytes. These promoters can be used to target proteins for the treatment of inflammation and fibrosis in the kidney. Therapeutic proteins include any protein of use in treating a kidney disorder in a subject, including, collagen type 4 alpha 3, collagen type 4 alpha 4, collagen type 4 alpha 5, podocin, nephrin, rBAT, b0 +amino acid transporter, nephrocystin 1, CLCN5, polycystin-1, polycystin-2, or BBS.

[0126] In some aspects, the AAV vector can further comprise a polynucleotide that expresses functional RNA molecule such as tRNA, snRNA, snoRNA, shRNA, miRNA, IncRNA, sgRNA, dgRNA, crRNA, antisense RNA, ribozymes, antagomir, which are of therapeutic interest. By way of nonlimiting examples, the functional RNA of interest can be siRNA, shRNA, or miRNA that target connective tissue growth factor, apolipoprotein LI, or vascular endothelial growth factor, miRs such as miR-29 and miR-146a that are over- expressed to compensate their downregulation, and antagomirs such as those that silence upregulated miR- 21 or miR-126. The AAV vector can include a promoter operably linked to a nucleic acid molecule that transcribes a functional RNA molecule or a set of functional RNA molecules that have therapeutic potential. A promoter of use includes a ubiquitous RNA polymerase III promotor, including the small nuclear RNA U6 promoter and the Hl promoter, and a ubiquitous RNA polymerase II promotor, including, but not limited to, the CMV and CAG promoters. Promoters of use also include, but are not limited to, the promoter for sodium-dependent phosphate transporter type 2a (NPT2a) or gamma-glutamyl transpeptidase for expression in the proximal tubule, sodium-potassium-2-chloride cotransporter (NKCC2) for expression in the thick ascending limb of Henle (TALH), and aquaporin 2 (AQP2) for expression in the collecting duct. Promoters also include, but are not limited to, the promoter for podocin, nephrin and podocalyxin for expression in podocytes. Promoters of use also include, but arc not limited to, the promoter for neuron-glial antigen 2 and platelet-derived growth factor receptor beta (PDGFR|3) for expression in pericytes. Therapeutic functional RNA molecules include any RNAs of use in treating a kidney disorder in a subject, including, but not limited to, miR-29 and an antagomir against miR-21.

[0127] The disclosed AAV vectors can be used in a method of gene therapy for a disorder of the kidney in a subject. The AAV vectors comprise a capsid protein sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 15, SEQ ID NO: 6, or a variant thereof exhibiting at least 70% identity thereto. The sequence identity can be about 95%, 96%, 97% 98%, or 99%. SEQ ID NOs: 1-6 are the amino acid sequence of AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, and AAVShHIO capsid proteins, and are shown below:

[0128] AAVKP1

[0129] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPGNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDTDSAADPQPLGEPPA APSGLGTGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVK EVTQNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAM GRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSG TTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLV NPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNL QSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIK NTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNT EGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1)

[0130] AAVKP2

[0131] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGE PVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVL EPLGLVEEGAKTAPGKKRPVEHSPVEPDSSSGIGKTGQQPAKKRLNFGQTGDADSVPDPQPLGQPP AAPSGLGTNTMATGSGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNH LYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNEGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQA VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTS GTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSL VNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANN LQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMI KNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVD TNGVYSEPRPIGTRYLTRPL (SEQ ID NO: 2)

[0132] AAVKP3

[0133] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPL GLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAP SGVGPNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYK QISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEV TQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTN QSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGP AMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSN TAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPV PADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVY SEPRPIGTRYLTRNL (SEQ ID NO: 3)

[0134] AAVDJ

[0135] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPL GLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAP SGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKE VTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTN TQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPG PAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTP VPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGV

[0136] YSEPRPIGTRYLTRNL (SEQ ID NO: 4)

[0137] AAV2G9

[0138] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSVAGPSNMAVQGRNWLPGPCYRQQRVSKTSADNNNSEFAWTGATKYHLNGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQ AATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVP ANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 5)

[0139] AAV2.7m8

[0140] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLA LGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPP QILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDF TVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 6).

[0141] AAV3

[0142] MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGE PVNEADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILE PLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPA APTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKE VTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSG TTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLV NPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNL QSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIK NTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDT NGVYSEPRPIGTRYLTRNL (SEQ ID NO: 7).

[0143] AAVLK03

[0144] MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGE PVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLE PLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPA APTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHL YKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKE VTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAV GRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSG TTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLV NPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNL QSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIK NTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDT NGVYSEPRPIGTRYLTRPL (SEQ ID NO: 8).

[0145] AAVShHIO

[0146] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGE PVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVL EPFGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPA TPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNVQV KEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQA VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQDQSG SAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIIN PGTAMASHKDDKNKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNL QSSSTDPATGDVHVMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIK NTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVD NNGLYTEPRPIGTRYLTRPL (SEQ ID NO: 9).

[0147] The disclosed AAV can be delivered to the kidney of a subject by renal pelvic injection comprising direct renal pelvic injection and retrograde transureteral renal pelvic injection. Pharmaceutical compositions can be produced that contain the AAV and a pharmaceutically acceptable excipient. Such excipients include any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A thorough discussion of pharmaceutically acceptable excipients is available in Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23rdEdition (2021).

[0148] In some aspects, the excipients confer a protective effect on the AAV virion such that loss of AAV virions, as well as transduceability resulting from formulation procedures, packaging, storage, transport, and the like, is minimized. These excipient compositions are therefore considered "virion-stabilizing" in the sense that they provide higher AAV virion titers and higher transduceability levels than their non-protected counterparts, as measured using standard assays, see, for example, Published U.S. Application No. 2012 / 0219528. These compositions therefore demonstrate "enhanced transduceability levels" as compared to compositions lacking the particular excipients described herein, and are therefore more stable than their nonprotected counterparts.

[0149] Exemplary excipients that can be used to protect an AAV virion from activity degradative conditions include, but are not limited to, detergents, proteins, e.g., ovalbumin and bovine serum albumin, amino acids, e.g., glycine, polyhydric and dihydric alcohols, such as but not limited to polyethylene glycols (PEG) of varying molecular weights, such as PEG-200, PEG-400, PEG-600, PEG-1000, PEG-1450, PEG- 3350, PEG-6000, PEG-8000 and any molecular weights in between these values, with molecular weights of 1500 to 6000 preferred, propylene glycols (PG), sugar alcohols, such as a carbohydrate, preferably, sorbitol. The detergent, when present, can be an anionic, a cationic, a zwitterionic or a nonionic detergent. An exemplary detergent is a nonionic detergent. One suitable type of nonionic detergent is a sorbitan ester, e.g., polyoxyethylenesorbitan monolaurate (TWEEN®-20) polyoxyethylenesorbitan monopalmitate (TWEEN®- 40), polyoxyethylenesorbitan monostearate (TWEEN®-60), polyoxyethylenesorbitan tristearate (TWEEN®- 65), polyoxyethylenesorbitan monooleate (TWEEN®-80), polyoxyethylenesorbitan trioleate (TWEEN®- 85), such as TWEEN®-20 and / or TWEEN®-80. These excipients are commercially available from a number of vendors, such as Sigma, St. Louis, Mo. The excipient can be Pluronic F68.

[0150] The amount of the various excipients present in any of the disclosed compositions varies and is readily determined by one of skill in the art. For example, a protein excipient, such as BSA, if present, will can be present at a concentration of between 1.0 weight (wt.) % to about 20 wt. %, preferably 10 wt. %. If an amino acid such as glycine is used in the formulations, it can be present at a concentration of about 1 wt. % to about 5 wt. %. A carbohydrate, such as sorbitol, if present, can be present at a concentration of about 0.1 wt % to about 10 wt. %, such as between about 0.5 wt. % to about 15 wt. %, or about 1 wt. % to about 5 wt. %. If polyethylene glycol is present, it can generally be present on the order of about 2 wt. % to about 40 wt. %, such as about 10 wt. % top about 25 wt. %. If propylene glycol is used in the subject formulations, it will typically be present at a concentration of about 2 wt. % to about 60 wt. %, such as about 5 wt. % to about 30 wt. %. If a detergent such as a sorbitan ester (TWEEN®) is present, it can be present at a concentration of about 0.05 wt. % to about 5 wt. %, such as between about 0.1 wt. % and about 1 wt %, see U.S. Published Patent Application No. 2012 / 0219528, which is incorporated herein by reference. The excipient can be Pluronic F68 (for example, at 0.001%). In one example, an aqueous virion-stabilizing formulation comprises a carbohydrate, such as sorbitol, at a concentration of between about 0.1 wt. % to about 10 wt. %, such as between about 1 wt. % to about 5 wt. %, and a detergent, such as a sorbitan ester (TWEEN®) at a concentration of between about 0.05 wt. % and about 5 wt. %, such as between about 0.1 wt. % and about 1 wt. %. Virions are generally present in the composition in an amount sufficient to provide a therapeutic effect when given in one or more doses, as defined above.

[0151] Appropriate doses depend on the subject being treated (e.g., human or nonhuman primate or other mammal), age and general condition of the subject to be treated, the severity of the condition being treated, the mode of administration of the AAV vector / virion, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through clinical trials. The method can include measuring an outcome, such as kidney function. The method can include administering other therapeutic agents, such as an AAV vector transduction-enhancing agents including histone deacetylase inhibitors, proteasome inhibitors, immunosuppressive agents including corticosteroids, calcineurin inhibitors and sirolimus, and agents that degrade immunoglobulins including IdeS and IdeZ. The method can also combine with ultrasound-targeted microbubble destruction that can enhance AAV vector transduction. The method can also include having the subject make lifestyle modifications.

[0152] For example, for in vivo injection, i.e., injection directly to the subject, a therapeutically effective dose will be on the order of from about 105to 1016of the AAV virions, such as 108to 1014AAV virions. The dose, of course, depends on the efficiency of transduction, promoter strength, the stability of the message and the protein encoded thereby, and clinical factors. Effective dosages can be readily established using dose response curves.

[0153] Dosage treatment may be a single dose schedule or a multiple dose schedule to ultimately deliver the amount specified above. Moreover, the subject may be administered as many doses as appropriate. Thus, the subject may be given, e.g., 105to 1016AAV virions in a single dose, or two, three, four, five, six or more doses that collectively result in delivery of, e.g., 105to 1016AAV virions. One of skill in the art can readily determine an appropriate number of doses to administer.

[0154] In some aspects, the AAV is administered at a dose of about 1 x 1011to about 1 x 1014viral genomes (vg) per kilogram (kg). In some examples, the AAV is administered at a dose of about 1 x 1012to about 8 x 1013vg / kg. In other examples, the AAV is administered at a dose of about 1 x 1013to about 6 x 1013vg / kg. In specific non-limiting examples, the AAV is administered at a dose of at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, at least about 5 x 1012, at least about 1 x 1013, at least about 5 x 1013, or at least about 1 x IO14vg / kg. Tn other non-limiting examples, the AAV is administered at a dose of no more than about 5 x 1011, no more than about 1 x 1012, no more than about 5 x 1012, no more than about 1 x TO13, no more than about 5 x 1013, or no more than about 1 x TO14vg / kg. In one non-limiting example, the AAV is administered at a dose of about 1 x 1012vg / kg. The AAV can be administered in a single dose, or in multiple doses (such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses) as needed for the desired therapeutic results. Administration can be via renal pelvic injection comprising direct renal pelvic injection and retrograde transureteral renal pelvic injection.

[0155] EXAMPLES

[0156] Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy. However, gene transfer to the kidney, especially clinically relevant cell types such as proximal renal tubule cells and podocytes has been unsuccessful. An unbiased side-by-side comparison of 47 AAV serotypes and mutants using AAV Barcode-Seq identified nine AAV capsids that exhibited remarkable enhancement of gene delivery to the kidney when delivered by renal pelvis (RP) injection. Among the nine AAV capsids, i.e., AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9 AAV2.7m8, AAV3, AAVLK03, and AAVShHIO, the first six capsids exhibited remarkable enhancement of transgene expression in mice and the other three capsids have the potential to show remarkable expression in primates based on the reported epigenetic mechanism of suppression of transgene expression, which is AAV capsid dependent. Among the nine AAV capsids, AAVKP1 was selected as the representative AAV capsids exhibiting this unique attribute. Detailed analyses were performed using this capsid in comparison to AAV9. It was demonstrated that, although both AAVKP1 and AAV9 transduced mesangial cells in the glomeruli effectively by both intravenous (IV) and renal pelvic (RP) injections (the latter is also called renal pelvis injection), RP injection of AAVKP1, but not AAV9, remarkably enhanced proximal renal tubule transduction while minimizing off-target liver transduction. It was also demonstrated that the effective proximal renal tubule transduction with AAVKP1 by RP injection is the case in rhesus macaque. Moreover, evidence is provided that these contrasting observations between AAVKP1 and AAV9 are attributed to their distinct pharmacokinetics, i.e., AAVKPl ’s propensity to accumulate at the injection site and AAV9’s delayed blood clearance. Thus, context-dependent capsid engineering and selection of vector administration routes were used to provide unexpected superior AAV vector-mediated renal gene delivery. See also U.S. Provisional Application No. 63 / 462,492, filed April 27, 2023, incorporated herein by reference.

[0157] Here, it is shown that retrograde renal pelvis (RP) injection can substantially enhance AAV vector- mediated renal gene transfer by an order of magnitude when select AAV capsids are used including AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, and AAVShHIO, while such enhancement is not attained by RP injection of other common serotype capsids or systemic intravenous (IV) vector administration. AAVKP1 was then tested, as the prototype of these efficient capsids in both mice and rhesus macaques, and it was found that the enhancement is primarily attributed to substantially augmented transduction in the proximal renal tubules that are difficult to transduce with the common serotype capsids. It was also shown that RP injection overcomes pre-existing humoral immunity, allowing effective renal transduction in anti-AAV neutralizing antibody-baring animals, while non-renal tissue transduction could be completely avoided by neutralization of AAV vector particles spilled over from the injected kidney. Thus, RP injection of the select AAV capsids harboring this particular renal transduction-enhancing attribute is a promising approach to effectively treat renal diseases with no restriction imposed by pre-existing immunity against AAV and likely allows redosing if necessary.

[0158] Adeno-associated virus (AAV) is a promising vector for in vivo gene therapy that can attain a high level of transduction in various organs. To date, eight commercial AAV vector gene therapy products have been approved by authorizing agencies in the United States, European countries and Japan. With the demonstration of the safety and efficacy of the first two local injection products, Glybera for intramuscular injection to treat lipoprotein lipase deficiency and Luxturna for subretinal injection to treat Leber congenital amaurosis type 2, the first intravenous injection product, Zolgensma for the treatment of spinal muscular atrophy, was approved by FDA in 2019. Since then three additional intravenous injection products have been approved by either or both of FDA and EMA for the treatment of hemophilia A, hemophilia B and Duchenne muscular dystrophy. The approval of intravenous AAV gene therapy products and the demonstration of their safety and efficacy have significantly expanded the utility of AAV vector-mediated gene therapy in the clinic. Moreover, an intracranial injection AAV vector product has been approved by EMA for the treatment of aromatic L-amino acid decarboxylase (AADC) deficiency.

[0159] As for the gene therapy for kidney diseases, the success of AAV vector-mediated gene therapy in humans, together with the recent remarkable progress in the understanding of genetic etiology of kidney diseases, has brought a significant hope to the patients who suffer from genetic kidney diseases that account for a significant fraction of both pediatric and adult patients with chronic kidney disease (CKD) (Hildebrandt, Lancet 375(9722): 1287-95, 2010, Lata et al., Ann Intern Med. 168(2): 100-109, 2018, Groopman et al. N Engl J Med. 380(2): 142-151 , 2019). However, in contrast to the remarkable progress in the intravenous approaches to target the liver, central nervous system and skeletal muscles, it remains difficult to achieve renal transduction at a high level similar to the level that could be attained in other organs by systemic AAV vector administration even at a high dose (Zincarelli et al., Mol Ther. 16(6): 1073- 80, 2008). The intravenous administration of AAV vectors based on common serotypes can only achieve mesangial cell and interstitial cell transduction in the kidney, and the current AAV vector-mediated renal gene delivery methods are not capable of effectively transducing clinically relevant cell types including tubular epithelial cells and podocytes at satisfactory levels for their therapeutic use (Ikeda et al., J Am Soc Nephrol (9):2287-229 2018, Lang et al., Nat Commun 10, 3415, 2019). Although recent advancement of capsid engineering technology generated many AAV vectors with novel phenotypes (Wang et al., Nat Rev Drug Discov. 18(5):358-37 2019, Li and Samulski, Nat Rev Genet. 21(4):2.55-272, 2020), only less than a handful of novel capsids have been characterized in the context of renal gene transfer demonstrating some encouraging results to a limited degree (Ikeda et al., J Am Soc Nephrol. 29(9):2287-2297, 2018, Gonzalez et al., Nat Commun 13, 5947, 2022). Thus, the kidney has been viewed as an organ that is not susceptible to AAV vector transduction and gene therapy for kidney disease lags behind other gene therapies.

[0160] In addition to the above-described target organ-specific challenge in AAV vector-mediated renal gene delivery, the high prevalence of pre-existing anti-AAV neutralizing antibodies in humans (Boutin et al. Hum. Gene Ther. 21(6):704-12, 2010) has remained as the major universal limitation to be overcome in AAV-mediated gene therapy. Up to 70% of adult human population is positive for anti-AAV capsid antibodies developed by natural infection that make the therapy ineffective; therefore, individuals with high levels of anti-AAV antibodies have so far been excluded from the therapy-eligible groups and may continue not to benefit from the therapy. In addition, redosing for maintaining therapeutic effects is currently not possible due to the robust humoral immune responses of AAV vectors that occur upon the first vector administration and are difficult to prevent or attenuate. Several strategies have been developed that include the use of less antigenic AAV capsids generated by genetic engineering (Grimm et al., J Virol. 82(12):5887- 91, 2008, Tse et al., Proc Natl Acad Sci U S A. 114(24):E4812-E4821, 2017), pharmacological intervention with immunosuppressants (Meliani et al., Nat Commun. 9(l):4098, 2018) or removal of immunoglobulins from the bloodstream by plasmapheresis and immunoadsorption (Salas et al., Blood Adv. 3(17) :2632-2641 , 2019) or intravenous administration of an IgG-cleaving enzyme (Elmore et al., JCI Insight 5(19) :e 13988, 2020, Leborgne et al., Nat Med. 26(7): 1096-1101, 2020); however, their clinical efficacies have yet to be determined.

[0161] Renal pelvis (RP) injection was exploited as an approach to overcome these two challenges in gene delivery to the kidney, that is, inefficient renal gene transfer by traditional approaches using the common AAV serotype capsids and pre-existing humoral immunity both of which significantly limit the efficacy of renal gene therapy. The rationale for the RP approach is twofold: (1) As opposed to systemic vector administration that dilutes the vector in the central compartment, local AAV vector administration has been presumed to have a potential to enhance target tissue transduction by increasing local vector concentrations, and (2) Although the urinary tract is not generally considered as an immunoprivileged area, the immunoglobulin concentration in the urine is > 1000 times less than that in the plasma (Burdon, Immunology 21 (2):363-8, 1971). Therefore, it is not unreasonable to presume that RP injection can potentially evade preexisting humoral immunity. It has been shown that AAV vectors locally administered to immune -privileged organs or spaces such as eyes and the central nervous system could mediate effective vector transduction in target organs even in the presence of circulating anti-AAV neutralizing antibodies (Barker et al., J Gene Med. 11 (6):486-97, 2009, Shinohara et al., Mol Neurobiol. 56(6):4203-421, 2019). Thus, the RP approach is likely to overcome the challenges at least to some degree; however, the potential efficacy of AAV vector- mediated renal transduction by RP in the presence or absence of pre-exiting anti-AAV neutralizing antibodies has not been assessed before. In the present study, renal transduction efficiency of 47 AAV capsids was comprehensively assessed following RP injection in C57BL / 6J mice using our AAV Barcode-Seq technology (Adachi et al., Nat Commun. 5:3075, 2014). The 47 capsids included common AAV serotypes and genetically engineered capsids as detailed below. As a result, nine AAV capsids (AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, and AAVShHIO) were identified with which gene delivery to the kidney can be remarkably enhanced. Among the above nine AAV capsids, the first six capsids, AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, and AAV2.7m8, can lead to substantially higher transduction in the kidney than that of the benchmark AAV9 capsid by an order of magnitude by RP injection compared to IV injection. Subsequent single capsid validation analyses using AAVKP1 and AAV9 capsids revealed that RP injection of AAVKP1, but not AAV9, enables effective gene transfer to proximal renal tubules, which is not possible by IV injection of cither capsid. In addition, although RP-injcctcd AAV9 capsid effectively transduced the liver, the liver transduction of RP-injected AAVKP1 was substantially attenuated, demonstrating the high renal specificity of the RP approach using AAVKP1. The efficacy of the RP approach was also determined using AAVKP1 in non-human primates. To this end, a real-time fluorescence image-guided balloon catheter-based retrograde transureteral renal pelvis administration method was developed and applied the method to inject AAVKP1 vector into the kidney unilaterally in two rhesus macaques, one showing anti-AAVKPl neutralizing antibody negative and the other showing marginally negative (1:7.5). As a result, robust renal tubular transduction of AAVKP1 was attained, with minimal vector dissemination to the liver in these two rhesus macaques. This retrograde transureteral renal pelvis injection method was applied to two anti-AAVKPl neutralizing antibody -positive rhesus macaque and demonstrated effective renal tubule transduction at a comparable level despite the presence of anti- AAV neutralizing antibodies and nearly complete block of vector genome dissemination to the liver. These observations provide the basis for effective AAV vector-mediated renal gene delivery applicable to a broader range of patients population, emphasizing the potential advantage of renal pelvis administration for clinical translation.

[0162] Results

[0163] AAV Barcode-Seq identified AAV capsids that outperform AAV9 in renal transduction by retrograde renal pelvis (RP) injection.

[0164] It was first investigated which AAV capsids transduce the kidney most efficiently by RP injection. To this end, AAV Barcode-Seq (Adachi et al., Nat Commun. 5:3075, 2014) was employed. A total of 47 different AAV vector-mediated renal transduction profiles in mice were determined following RP administration. In this approach, AAV capsids package AAV-CAG-BC vector genomes whose barcodes (BCs) are unique to each capsid (Figure 1A). The AAV-CAG-BC genomes carry the ubiquitous CAG promoter that drives barcode expression as mRNA, which enables assessment of relative transduction efficiency at both DNA and RNA levels (DNA / RNA Barcode-Seq). An AAV barcode library was produced that contains 47 AAV capsids (11 serotypes and 36 mutants, Table 1) and injected the library into 8-week- old C57BL / 6J male mice by RP (3xlOuvg / mouse, n=3) according to the protocol reported in the literature (Konkalmatt et al., JCI Insight l(8):e85888, 2016).

[0165] Table 1. AAV vectors included in the Barcode library.

[0166] Vectors Reference Vectors Reference

[0167] AAV 1 Earley AAVbb.2 Gao

[0168] AAV 1 9mtlOO See below AAVDJ Grimm

[0169] AAVl_9mt30 See below AAVHN1 See below

[0170] AAVl_9mt76 See below AAVHN2 See below

[0171] AAV2 Earley AAVHN3 See below

[0172] AAV2G9 Shen AAVhu.l l Gao

[0173] AAV2i8 Asokan AAVhu.13 Gao

[0174] AAV2retro Tervo AAVhu.37 Gao

[0175] AAV2R585E Adachi AAVKP1 Pekrun

[0176] AAV2R585E9_2 Adachi AAVKP2 Pekrun

[0177] AAV3 Earley AAVKP3 Pekrun

[0178] AAV4 Earley AAVLK03 Lisowski

[0179] AAV5 Earley AAVNP40 Paulk

[0180] AAV6 Earley AAVNP59 Paulk

[0181] AAV7 Earley AAVPHP.B Deverman

[0182] AAV8 Earley AAVPHP.eB Chan

[0183] AAV9 Earley AAVPHP.S Chan

[0184] AAV9AA272 See below AAVpol Bello

[0185] AAV9AA22 See below AAVrh.8 Gao

[0186] AAV9W22A See below AAVrh.10 Gao

[0187] AAV 10 Earley AAVrh.20 Gao

[0188] AAV 11 Earley AAVrh.43 Gao

[0189] AAV2.7m8 Dalkara AAVShHIO Klimczak

[0190] AAVAnc80 Zinn

[0191] References:

[0192] Earley, L. F. et al., . J Virol 91 (2017). doi.org: 10.1128 / JVI.01980-16.

[0193] Shen, S. et al. J Biol Chem 288, 28814-28823 (2013).

[0194] Asokan, A. et al. Nat Biotechnol 28, 79-82 (2010). doi.org: 10.1038 / nbt.l599

[0195] Tervo, D. G. et al. Neuron 92, 372-382 (2016). doi.org:10.1016 / j. neuron.2016.09.021

[0196] Adachi, K et al. Nat Commun 5, 3075 (2014), doi.org:10.1038 / ncomms4075 Dalkara, D. et al. Sci Transl Med 5, 189ral76 (2013). doi.org: 10.1126 / scitranslmed.3005708 Zinn, E. et al.. Cell Rep 12, 1056-1068 (2015). doi.org:10.1016 / j.celrep.2015.07.019 Gao, G. et al. J Virol 78, 6381-6388 (2004). doi.org: 10.1128 / JVI.78.12.6381-6388.2004 Grimm, D. et al.. J Virol 82, 5887-5911 (2008). doi.org: 10.1128 / JVI.00254-08 Pekrun, K. et al. JCI insight 4 (2019). doi.org: 10.1172 / jci.insight.131610 Lisowski, L. et al. Nature 506, 382-386 (2014). doi.org: 10.1038 / naturel 2875 Paulk, N. K. et al Mol Ther 26, 289-303 (2018). doi.org:10.1016 / j.ymthe.2017.09.021 Deverman, B. E. et al. Nat Biotechnol 34, 204-209 (2016). doi.org: 10.1038 / nbt.3440 Chan, K. Y. et al. Nat Neurosci 20, 1172-1179 (2017). doi.org: 10.1038 / nn.4593 Bello, A. et al. Gene therapy 16, 1320-1328 (2009). doi.org: 10.1038 / gt.2009.82 Klimczak, R. R., et al. PLoS One 4, e7467 (2009). doi.org:10.1371 / journal.pone.0007467

[0197] AAVl_9mtl00, AAVl_9mt30, AAVl_9mt76 - AAVl-derived capsid mutants containing portions of the AAV9 capsid sequence, see US Patent 11,866,462, incorporated herein by reference. AAVl_9mtl00, AAVl_9mt30 and AAVl_9mt76 are AAV 1.9-9919911, AAV1.9- 1191999 and AAV 1.9-9119919, respectively (AAV 1.9-9919911, AAV 1.9- 1191999 and AAV 1.9-9119919 are the names used in the US Patent 11866462). AAV9AA272 - an AAV9 capsid mutant, AAV9N272A, described in US Patent US11,459,558, incorporated herein by reference. AAV9AA22 - an AAV9 capsid mutant, AAV9W22A / W23A, is disclosed in PCT Publication No. WO 2018 / 119330A2, incorporated herein by reference. AAV9W22A - an AAV9 capsid mutant, AAV9W22A, is also disclosed in PCT Publication WO2018119330A2, incorporated herein by reference. AAVHN1, AAVHN2, and AAVHN3 - AAV9 capsid mutants with a peptide insertion at amino acid position 589, are disclosed in US Patent No. 11,459,558, incorporated herein by reference.

[0198] Briefly, the renal blood flow and urinary flow was blocked and the renal pelvis was slowly filled with the AAV vector solution over one minute followed by 14-min dwelling time. Six weeks after injection, relative transduction efficiency of each AAV vector in the kidney compared to AAV9 was determined by AAV DNA / RNA Barcode-Seq. As shown in Figure IB (transduction efficiency of all AAV capsids in Figure 8), it was found that (a) by RP injection, nine AAV capsids, AAVKP1, AAVKP2, AAVKP3 (Pekrun et aL, JCI Insight (22):el31610, 2019), AAVDJ (Grimm et al., J Virol. 82( 12) :5887-911, 2008), AAV2G9 (Shen, Horowitz et al., J Biol Chem. 288(40):28814-23, 2013), AAV2.7m8 (Dalkara et al. , Sci Transl Med. 5(189): 189ra76, 2013), AAV3 (Rutledge et al., J Virol. 72( l):309- 19, 1998), AAVLK03 (Lisowski et al., Nature 506(7488): 382-6, 2014) and AAVShHIO (Klimczak et al., PLoS One 4(10):e7467, 2009) showed significantly higher renal transduction than AAV9 at the vector genome DNA level, and (b) AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, and AAV2.7m8 showed significantly higher renal transduction than AAV9 at the vector genome transcripts level while AAV3, AAVLK03 and AAVShHIO did not. There was DNA / RNA correlation for the first six AAV capsids, but the other three capsids showed significantly higher renal transduction than AAV9 at DNA level, but their mRNA expression levels were discordantly lower than AAV9.

[0199] Augmented renal transduction with the nine AAV capsids was not observed when they were administrated intravenously, highlighting the importance of local vector administration to exploit this peculiar attribute of the nine capsids for enhanced renal gene delivery.

[0200] RP injection of AAVKP1 enables effective transduction in the proximal renal tubules.

[0201] To validate the AAV Barcode-Seq data and determine the cell type tropism of each AAV capsid, a single capsid validation study was performed. For this, AAVKP1 was selected as the prototype of the abovedescribed 9 capsids and used AAV9 as the benchmark, and AAVKPl-CAG-tdTomato and AAV9-CAG- tdTomato vectors were produced. Each of the vectors was then injected into 8-wcck-old C57BL / 6J male mice via RP at a dose of 3xlOuvg / mouse (n=4 per group, Figure 2A). Two weeks after injection, vector genome copy numbers were determined by DNA qPCR and renal transduction was assessed by immunofluorescence microscopy. Quantification of vector genome in the kidney showed that, while vector genome copy numbers were comparable between the AAV9 and AAVKP1 -injected mice when the vectors were administered by IV, there were >20 times more vector genomes in the AAVKP1 -injected mice than the AAV9-injected mice (Figure 2B). In consistent with the vector genome copy number data, the histological assessment also showed substantially enhanced transduction in the kidney with AAVKP1 compared to AAV9 following RP injection (Figure 2C). Interestingly, enhanced transduction of AAVKP1 was mainly observed in the cortex. Detailed analysis by co-staining of proximal renal tubule cells revealed that AAVKP1 -mediated enhanced renal transduction was primarily attributed to proximal tubule transduction, showing 6.5% transduction in the proximal renal tubule cells by RP injection of AAVKP1 (Figure 3A). Costaining of podocytes was also performed, demonstrating no transduction in podocytes with AAV9 or AAVKP1 via RP (Figure 3B).

[0202] Ischemia did not enhance renal transduction of AAVKP1.

[0203] Since RP injection was performed with the 15-min blockade of the local arterial and venous blood flow, it was investigated if ischemia influences renal transduction. To this end, RP injection of AAVKPl- CAG-tdTomato (3xl0nvg / mouse) was performed with variable ischemic time (0 min, 5 min, 10 min and 15 min) (Figure 9). While there was no apparent difference in renal transduction between the 5-, 10-, and 15- minute groups, RP injection without blockade of renal blood flow resulted in substantially lower transduction than other groups, showing no proximal tubule transduction. These observations indicate that, for effective renal gene delivery by RP, transient blockade of renal blood flow is important, but there is no benefit of extended blockade for more than 5 min and 5 min blockade appears to be sufficient to maximize renal transduction by RP. However, as described below, effective renal tubule transduction could be attained by RP without the transient blockade of renal blood flow in non-human primates. Augmented renal interstitial accumulation of vector particles is a potential mechanism of the enhanced renal transduction by RP injection.

[0204] To investigate the distribution and extravasation of AAV vector particles in the kidney following AAV vector injection by RP, fluorescent microspheres of 25 nm in diameter were employed to mimic AAV vector particles. This size of the microspheres was chosen as it is close to the diameter of AAV virions. After 30-min circulation, intravenously administered microspheres mainly accumulated in the mesangial area of the glomeruli and showed limited extravasation to the interstitial space, consistent with the previous findings that nanoparticles accumulate in the mesangial area in size-dependent manner (Choi et al., Proc Natl Acad Sci USA. 108(16) :6656-61 , 2011, Guo et al., Nat Comrnun. 8( 1 ) : 87, 2017). In contrast, microspheres administered via RP accumulated in the renal interstitial space effectively (Figure 10A). It is important to note that interstitial accumulation was not achieved in the absence of the blockade of renal artery and vein (Figure 10B). These observations suggest that peritubular capillary wall poses a significant barrier for AAV to transduce tubular epithelial cells following IV injection, but RP injection bypasses this barrier, leading to efficient proximal tubule transduction. Lack of transduction enhancement by RP injection without local blockade of the blood flow also supports the importance of interstitial accumulation of AAV particles for proximal tubule transduction.

[0205] Limited extra-renal dissemination of AAVKP1 following RP injection suppressed off-target liver transduction

[0206] Several studies have reported off-target organ transduction especially in the liver following local injection of AAV9 (Meyer et al., Mol Ther. 23(31:477-87, 2015, Quirin et al., Mol Ther Methods Clin Dev. 8:8-20, 2018, Rubin et al., Hum Gene Ther. 30( 12): 1559-1571, 2019, Mathiesen et al., Mol Ther Methods Clin Dev. 19:447-458, 2020). Therefore, the off-target liver transduction following RP injection of AAV9 and AAVKP1 was next assessed. Quantification of vector genome in the liver two weeks after RP injection revealed that RP injection of AAVKP1 significantly suppressed the off-target liver transduction. On the other hand, off-target liver transduction was not prevented by RP injection of AAV9 (Figure 4A). Immunofluorescence study also confirmed substantially suppressed off-target liver transduction by RP injection of AAVKP1 (Figure 4B). Based on these observations, it was hypothesized that difference in pharmacokinetics following RP injection leads to the contrasting renal and hepatic transduction between AAV9 and AAVKP1. Quantification of vector genome 10 min after local injection showed >10 times more vector genomes in AAVKP1 -injected kidney than AAV9-injected kidney following RP injection (Figure 4C), indicating that injected AAVKP1 stays in the kidney more efficiently than AAV9. It is important to note that transduction with AAV vector does not occur within 10 min following vector exposure in vivo (Kotchey et al., Mol Ther. 19(6): 1079-89, 2011); therefore, it is unlikely that the higher concentration of AAVKP1 than AAV9 in the injected kidney is the consequence of higher transduction of the AAVKP1- injcctcd kidney. Rather, the accumulated AAVKP1 vector genomes represent AAVKP1 vector particles trapped by the mesangial interstitial space and the renal interstitium. Consistent with this inference, blood concentration of AAVKP1 was more than 50 times less than that of AAV9 throughout the study time points (0 min, 10 min, 30 min, 1 h, 4 h and 8 h after RP injection, Figure 4D), indicating that substantially more AAV vector particles are trapped in the kidney when A AVKP1 capsid is used compared to AAV9. These findings support our hypothesis that less AAVKP1 vector spill-over by RP injection due to the sequestration of AAV vector particles within the kidney contributes to the efficient renal transduction while minimizing off-target organ transduction. On the other hand, our study also indicates that AAV9 is not the most appropriate AAV capsid for RP injection for the purpose of renal gene delivery with limited off-target effects.

[0207] Establishment of real-time image-guided retrograde transureteral renal pelvis injection procedure.

[0208] To translate our findings into a more clinically relevant setting, a non-surgical renal pelvic injection procedure was established for AAV vector administration in non-human primates. A real-time image-guided retrograde transureteral renal pelvis injection procedure was successfully established that comprises the following steps: (1) Under general anesthesia, insert a cystoscope to the bladder through the urethra and identify the ureteral orifices; (2) Advance a guidewire from the cystoscope via the ureter (Figure 5A) to the renal pelvis of the treatment side; (3) Remove the scope and advance a balloon catheter over the wire to the proximal aspect of the ureter; (4) Inflate the balloon of the catheter at the proximal aspect of the ureter adjacent to the ureteropelvic junction or at the ureteropelvic junction to achieve an adequate ureteral occlusion (Figure 5B); (5) Measure the volume of the renal pelvis by injecting a contrast agent under a fluoroscopy with the ureter occluded by the balloon catheter; (6) Ensure a proper positioning of the catheter and occlusion of the ureter (Figure 5C); (7) Inject about 0.4 mL / kg (ranging from 0.29 mL / kg to 0.45 mL / kg) body weight of a test agent (e.g., AAV vector solution) followed by air (which is equal to catheter dead space + up to 250% of the pelvic space, usually 0.1 -0.3 mL / kg) injection to retrogradely deliver an AAV vector solution to the renal parenchyma with a raised pressure (approximately 30 to 70 crnHiO or approximately 22 to 52 mmHg) inside the renal pelvis; (8) Maintain the consistent balloon occlusion during the dwelling time of the AAV vector solution in the kidney; (9) Change the animal position to the lateral decubitus (with the injected kidney side down) after 5 min of the dwelling time and wait for another 5 min with ureter occlusion maintained with the inflated balloon; (10) After a total of 10 min of the dwelling time, change the animal position to the supine position and deflate the balloon; (11) Assure the catheter position by injecting a contrast agent; and (12) Remove the balloon catheter. AAV vector solutions were administered into animals in PBS / 5% sorbitol and were not mixed with a contrast agent.

[0209] This procedure was performed on 5 rhesus macaques with no major clinical complications or procedural failure (Tabic 2). There was a modest elevation of liver enzymes one day post-injection in all animals (Table 3), but it was resolved within a week. It was not clear whether the elevation was related to AAV vector itself or procedure-related, but due to the transient and modest nature of the elevation, it was likely to be related to the anesthesia and / or the procedure. It was also confirmed that there is no histological damage in the kidney following the procedure (Figures 5D and 5E).

[0210] Retrograde transureteral renal pelvis injection of AAVKP1 vector transduced proximal renal tubule with high efficiency and specificity in rhesus macaques.

[0211] To assess the efficacy of the retrograde transureteral renal pelvis injection in renal transduction in non-human primates (NHPs), AAVKPl-CAG-tdTomato vector was injected into 3-year-old female rhesus macaque (Animal #2 in Table 2) and 8-year-old female rhesus macaque (Animal #3 in Table 2) at 1.3xl013vg / animal (2.8xl012vg / kg) and 1.7xl013vg / animal (2.8xl012vg / kg), respectively. Renal transduction and off-target liver transduction were assessed three weeks after injection. Results of Animal #2 were shown in Figure 6. Efficient cortical transduction, especially, high rate of proximal tubule transduction was observed (Figure 6A and 6B). On the other hand, glomerular cells including podocytes were not transduced (Figure 6C). Consistent with mice data, off-target liver transduction was minimal (Figure 6D). Similar results were obtained from Animal #3.

[0212] Retrograde transureteral renal pelvis injection can overcome the challenge posed by preexisting humoral immunity.

[0213] It is known that local injection of an AAV vector to immune-privileged organs such as eyes and central nervous system can allow successful transduction in target cells in the presence of neutralizing antibodies (NAbs) in the blood circulation. Immunoglobulin concentration in cerebrospinal fluid is one thousand times less than that in the plasma (Begley et al., Curr Pharm Des. 14(16) : 1566-80, 2008). Accordingly, it has been shown that the presence of anti- AAV NAbs of up to 1: 128 in the blood does not block transduction in the CNS when AAV vector is administered intrathecally (Gray et al., Gene Ther. 20(4):450-9, 2013). As immunoglobulin concentration in the urine is also >1000 times less than that in the plasma (Burdon, Immunology 21(2):363-8, 1971), it was hypothesized that renal pelvis injection can evade pre-existing humoral immunity against AAV. To address this, AAVKPl-CAG-tdTomato vector was injected via RP into two female rhesus macaques (Animal #4, 15-year-old; and Animal #5, 3-year-old) harboring anti-AAV-KP1 NAbs at titers of 1 :25.6 (Animal #4) and 1 :20.1 (Animal #5), and renal and hepatic transduction was assessed three weeks post-injection. As shown in FIGS. 7A and 7E, robust transduction was observed in the cortex of the kidney despite the presence of pre-existing NAbs at the time of vector administration. Transduction pattern in the kidney of these two animals (Animals #4 and #5) was not different from that obtained from Animals #2 and #3 carrying no detectable or marginal positive (1:7.5) for anti-AAVKPl NAbs, showing efficient proximal renal tubule transduction and no glomerular transduction (Figures 7B, 7C, 7F and 7G). Importantly, off-target liver transduction was completely blocked in Animals #4 and #5 (Figures 7D and 7H), which could be explained by very rapid clearance of AAVKPl-CAG- tdTomato vector particles from the blood circulation in the presence of NAbs (Figure 71).

[0214] In the cortex of the injected kidneys, infiltration of mononuclear cells and tubulitis with no glomerular involvement were sometimes observed in the transduced area (Figures 11A and 1 IB). As the contralateral kidney (Figures 11 A and 1 IB) or the kidney injected with the vehicle only (Figure 11C, NHP1) did not show this pathological change, it was concluded that this is due to the immune response against persistently expressed tdTomato proteins in the kidney.

[0215] These observations are consistent with the previous study demonstrating that even low levels of neutralizing antibody (at a titer of 1:4) can prevent the transduction of AAV vector administered intravenously (Gray et al., Mol Ther. 19(6): 1058-69, 2011). In summary, it was successfully demonstrated that pre-existing neutralizing antibody attack could be effectively avoided by RP injection and efficient transduction in proximal renal tubules with high specificity in NHPs is possible by utilizing the RP injection approach with appropriate choice of AAV capsid. The RP injection is a versatile approach for renal gene transfer and significantly broaden the applications of AAV vector-mediated gene therapies that target the kidney.

[0216] Table 2. List of non-human primates (NHPs) in this study

[0217] Table 3 Liver and kidney biomarkers in NHPs treated with RP injection of AAV

[0218] NHP1 Pre-inj ection 1 day 1 week 3 weeks

[0219] BUN (mg / dL) 15.2 17.5 12.5 11.9

[0220] Cre (mg / dL) 0.6 0.8 0.6 0.5

[0221] ALT (IU / L) 24 132 49 23

[0222] AST (IU / L) 40 248 51 71 NHP3 Pre-inj ection 1 day 1 week 3 weeks

[0223] BUN (mg / dL) 11.1 19.6 10.9 11.3

[0224] Cre (mg / dL) 0.6 0.8 0.6 0.8

[0225] ALT (IU / L) 36 49 30 48

[0226] AST (IU / L) 33 101 21 28

[0227] In view of the many possible aspects to which the principles of our invention may be applied, it should be recognized that illustrated aspects are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A method of delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject with or without immunity against AAV, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein via renal pelvic injection, wherein the renal pelvic injection comprises direct renal pelvic injection or retrograde transureteral renal pelvic injection, thereby delivering the composition comprising the AAV to the one or more kidney cell types in the subject.

2. The method of claim 1 , wherein the AAV is AAVKP 1 , A AVKP2, AA VKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03 or AAVShHIO.

3. The method of claim 1 or claim 2, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-9.

4. The method of claim 1 or claim 2, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-9.

5. The method of any one of claims 1 -4, wherein the AAV transduces at least one of mesangial cells, glomerular endothelial cells, parietal epithelial cells, podocytes, proximal tubule cells, Loop of Henle cells, distal tubule cells, collecting duct cells, fibroblasts, pericytes, or vascular smooth muscle cells.

6. The method of any one of claims 1-5, wherein the AAV transduces the one or more kidney cell types with an efficiency about 5 times to about 200 times greater than that of AAV9.

7. The method of any one of claims 1-6, wherein the genome comprises a nucleic acid molecule encoding a therapeutic protein.

8. A method of treating a kidney disorder in a subject with or without immunity against AAV, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAV capsid protein via renal pelvic injection, wherein the renal pelvic injection comprises direct renal pelvic injection or retrograde transureteral renal pelvic injection, and wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject.

9. The method of claim 8, wherein the AAV is AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV2.7m8, AAV3, AAVLK03, or AAVShHIO.

10. The method of claim 8 or claim 9, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-9.

11. The method of claim 8, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-9.

12. The method of any one of claims 8-11, wherein the renal pelvic injection comprises retrograde transurctcral renal pelvic injection.

13. The method of any one of claims 8-12, wherein the AAV transduces at least one of mesangial cells, glomerular endothelial cells, parietal epithelial cells, podocytes, proximal tubule cells, Loop of Henle cells, distal tubule cells, collecting duct cells, fibroblasts, pericytes, or vascular smooth muscle cells.

14. The method of any one of claims 8-12, wherein the AAV transduces the one or more kidney cell types with an efficiency about 5 times to about 200 times greater than that of AAV9.

15. The method of any one of claims 8-14, wherein the kidney disorder is a glomerular disease, a tubulointerstitial disease, a renal vascular disease, a genetic kidney disease, an autoimmune kidney disease, an infectious kidney disease, an inborn error of metabolism, a renal cancer, an acute kidney injury, chronic kidney disease, end stage kidney disease, a ciliopathy, polycystic kidney disease, nephronophthisis, Bardet- Biedl syndrome, Meckel-Gruber syndrome, a collagenopathy, Alport syndrome, congenital nephrotic syndrome, steroid resistant nephrotic syndrome, glomerulonephritis, a nephrolithiasis, cystinuria, Dent disease, renal tubular acidosis, Fanconi syndrome, hereditary interstitial kidney disease, diabetic nephropathy, thin basement membrane syndrome, nephrotic diabetes insipidus, hereditary tyrosinemia, cystinosis, Fabry disease, Gitelman syndrome, Bartter syndromes, Lowe syndrome, Liddle’s syndrome, or steroid-resistant nephrotic syndrome.

16. The method of any one of claims 1-15, wherein transureteral renal pelvic injection comprises: a) identifying a ureteral orifice of the subject using a cystoscope; b) advancing a guidewire from the cystoscope through the ureteral orifice and through a ureter of the subject to a renal pelvis of the subject; c) removing the cystoscope; d) advancing a balloon catheter comprising a balloon over the guide wire to the proximal aspectof the ureter of the subject; e) inflating the balloon of the balloon catheter to occlude the ureter; f) measuring the volume of the renal pelvis of the subject in the ureter occluded by the balloon catheter in step e); g) positioning a catheter and injecting the composition comprising the AAV vector through the catheter into the renal pelvis of the subject; and h) injecting a volume of the air through the catheter into the renal pelvis, thereby retrogradely delivering the composition comprising the AAV vector to renal parenchyma of the subject, under pressure.

17. The method of claim 16, wherein the volume of the renal pelvis of the subject is measured using a contrast agent and fluoroscopy.

18. The method of claim 16, wherein the volume of the renal pelvis of the subject is measured using computed tomography (CT), magnetic resonance imaging (MRI) or sonography using ultrasound.

19. The method of any one of claims 16-18, wherein the volume of the air injected through the catheter into the renal pelvis is about 0.05 mL / kg to about 0.4 mL / kg body weight of the subject.

20. The method of any one of claims 16-19, further comprising elevating the pressure to about 30-70 crnFbO during delivery of the composition comprising the AAV vector.

21. The method of any one of claims 16-20, wherein the balloon of the balloon catheter is inflated at a ureteropelvic junction of the subject.

22. The method of any one of claims 16-20, wherein the balloon of the balloon catheter is inflated at the proximal aspect of the ureter adjacent to a ureteropelvic junction of the subject.

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