Kidney-targeting aav capsids and methods of use thereof

Abstract

Methods are disclosed for delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject, comprising locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein. Methods are also disclosed for treating a kidney disorder in a subject, comprising locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject.

Description

[0001] KIDNEY-TARGETING AAV CAPSIDS AND METHODS OF USE THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATION

[0003] This claims the benefit of U.S. Provisional Application No. 63 / 462,492, filed April 27, 2023, incorporated herein by reference.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under P51OD011092 awarded by The National Institutes of Health. The government has certain rights in the invention.

[0006] FIELD OF THE DISCLOSURE

[0007] The related 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.

[0008] SEQUENCE LISTING

[0009] The Sequence Listing is submitted as an XML file in the form of the file named “Sequence.xml” (12,188 bytes), which was created on April 24, 2024, which is incorporated by reference herein.

[0010] BACKGROUND

[0011] 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, six 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. Two additional intravenous injection products have been approved by either or both of FDA and EMA for the treatment of hemophilia A and hemophilia B and another intravenous AAV vector product for the treatment of Duchenne muscular dystrophy is expected to be approved by FDA in the very near future.

[0012] 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. SUMMARY OF THE DISCLOSURE

[0013] Methods are disclosed for delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject, comprising locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein.

[0014] Methods are also disclosed for treating a kidney disorder in a subject, comprising locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject.

[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] FIGS. 1A-1B. AAV Barcode-Seq identified robust AAV capsids that outperform AAV9 for renal transduction following renal vein (RV) administration. (A) Male C57BL / 6J mice were injected with AAV-CAG-Barcode (BC) library that contained a total of 47 different AAV capsids at a dose of 2xl013vg / kg (intravenous, IV) or 3xlOnvg / mouse (RV) (n=3 per group for IV, n=4 per group for RV). Kidneys were harvested 6 weeks post-injection and relative transduction efficiency of each AAV capsid was determined by AAV DNA / RNA Barcode-Seq. (B) Relative renal transduction efficiency of common AAV serotype capsids and AAVAnc80 (light gray bars) and the AAV capsids showing remarkably enhanced renal transduction than AAV9 by RV injection (dark gray bars). Mean ± s.e.m. For the full list of AAV vectors, see FIGS. 2A-2B.

[0018] FIGS. 2A-2B. Relative transduction efficiency of AAV capsids in the kidney following intravenous (IV) or renal vein (RV)administration. Male C57BL / 6J mice were injected with AAV-CAG- BC library at a dose of 2x1013 vg / kg (IV) or 3x1011 vg / mouse (RV) (n=3 per group for IV, n=4 per group for RV). Kidneys were harvested 6 weeks post-injection and relative transduction efficiency of each AAV capsid was determined by AAV DNA / RNA Barcode-Seq. Mean ± s.e.m. AAV capsids that show significantly higher renal transduction than AAV9 determined by vector genome RNA transcript levels are included in FIG. 1 and annotated by asterisks (*). Hash (#) indicates AAV capsids that show more than 5 times higher vector genome DNA levels than AAV9 while exhibiting discordantly low vector RNA transcript levels.

[0019] FIGS. 3A-3C. Renal transduction of AAVKP1 was remarkably enhanced by renal vein (RV) injection. (A) Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato at a dose of 3xl0nvg / mouse (n=4 per group). Kidneys were harvested 2 weeks post-injection. (B) Quantification of vector genomes in the kidney by qPCR. Vector genome copy numbers were expressed as double-stranded vector genome copy numbers per diploid genomic equivalent. Mean + s.e.m. An interaction of the following two independent variables, AAV capsids and routes of administration, was statistically confirmed by two-way ANOVA followed by Tukey’s post hoc test. ****, adjusted p<0,0001; ns, not significant. (C) Representative images of the native tdTomato fluorescence signals in the kidney. The kidney transduced with AAVKP1 via renal vein injection (bottom right panel) exhibited much more tdTomato signals compared to the kidney transduced with AAVKP1 via tail vein injection (top right panel) or the kidneys transduced with AAV9 via tail vein or renal vein injection (left two panels). Although the tdTomato signals were primarily confined to the glomeruli following tail vein or renal vein injection of AAV9 or tail vein injection of AAV-KP1, renal vein injection of AAVKP1 led to significantly enhanced transduction in renal tubules in the cortex. Scale bar, 1 mm.

[0020] FIGS. 4A-4B. AAVKP1 transduced proximal tubule cells, but not podocytes, following renal vein (RV) injection. Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato at a dose of 3x10" vg / mouse (n=4 per group). Kidneys were harvested 2 weeks post-injection. (A) Representative images of native tdTomato and LTL (proximal tubules) are shown. The graph at the bottom shows a quantitative assessment of tdTomato-positive area in proximal renal tubules. Mean ± s.e.m. An interaction between AAV capsids and routes of administration was confirmed by two-way ANOVA followed by Tukey’s post hoc test. ****, adjusted p<0,0001 ; ns, not significant. (B) Representative images of native tdTomato and WT1 (podocyte nuclei), showing no transduction in podocytes. Scale bars, 50 pm. LTL, tetragonolobus lectin; WT1, Wilms tumor 1.

[0021] FIGS. 5A-5B. Renal transduction with AAVKP1 was not enhanced by ischemia. Eight-week- old C57BL / 6J male mice were injected with AAVKPl-CAG-tdTomato at a dose of 3xl0nvg / mouse. Kidneys were harvested 2 weeks post-injection. Representative images of native tdTomato in the kidney are shown. (A) AAVKP1 was administered intravenously following renal ischemia for 15 min. Transduction was compared between the kidney that has undergone 15-min ischemia (15 min) and the contralateral kidney that has undergone no ischemia (0 min), showing no difference. (B) AAVKP1 was administered via renal vein with various ischemic time periods (0 min, 5 min, 10 min and 15 min). An equivalent degree of enhanced transduction was observed in the cortex across these conditions, indicating that ischemia does not affect kidney transduction. Scale bar, 1 mm.

[0022] FIGS. 6A-6B. Renal vein (RV) 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 tail vein (A) or the renal vein (B). For the tail vein injection, microspheres were circulated for 30 min. There was no circulation time for RV injection. Kidneys were harvested following PBS perfusion. Representative images of microspheres (green) and CD31 (magenta, endothelial cells) in the kidney are shown. Arrows in (A) point to microsphere accumulation in the renal interstitium. Scale bar, 50 pm.

[0023] FIGS. 7A-7D. Limited extra-renal dissemination of AAVKP1 following renal vein (RV) injection prevented off-target liver transduction. Eight-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato at a dose of 3xlOnvg / mouse (A, B) or IxlO13vg / kg (C, D) (n=4 per group). (A) Vector genomes in the liver were quantified by qPCR 2 weeks post-injection. Vector genome copy numbers were expressed as double-stranded vector genome copy numbers per diploid genomic equivalent. An interaction between AAV capsids and routes of administration was confirmed by two-way ANOVA followed by Tukey’s post hoc test. (B) Representative images of native tdTomato and DAPI in the liver. The tdTomato signals in the liver of mice injected with AAVKP1 via renal vein (bottom right panel) were markedly reduced compared to the intense liver signals observed in mice injected with AAVKP1 via tail vein (top right panel) or in mice injected with AAV9 via either tail vein or renal vein injection (left two panels). Scale bar, 100 pm. (C) Vector genomes in the kidney were quantified by qPCR 10 min after RV injection. An unpaired t-test was used for statistical assessment. (D) Blood vector concentration-time curves following RV injection. Area under the curve was calculated and statistical significance was determined by unpaired t-test. ****, adjusted p<0,0001; ***, adjusted p<0,001; ns, not significant. Mean ± s.e.m.

[0024] FIGS. 8A-8E. AAVKP1 transduced proximal renal tubules in non-human primate following renal artery (RA) injection. A 14-month-old male rhesus macaque was injected with AAVKPl-CAG- tdTomato via the renal artery at a dose of 8.2xl012vg / animal. Transduction efficiencies in the kidney and liver were assessed 3 weeks post-injection. (A) A fluoroscopic image of transcatheter renal artery injection. Contrast was injected to visualize the injection path. (B) A representative image of native tdTomato in the kidney. Strong and widespread transduction was observed in the cortex. Scale bar, 1 mm. (C) Representative images of native tdTomato and LTL (proximal renal tubules). (D) A representative image of native tdTomato and WT1 (podocytes). No podocyte transduction was observed. (E) A representative image of native tdTomato in the liver. Scale bars in C-E, 100 pm. LTL, tetragonolobus lectin; WT1, Wilms tumor 1.

[0025] SEQUENCES

[0026] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and one letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0027] SEQ ID NOs: 1-6 are exemplary amino acid sequences of an AAV capsid protein. SEQ ID NOs: 7-8 are the nucleic acid sequences of primers.

[0028] DETAILED DESCRIPTION OF SEVERAL ASPECTS

[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 vectors by IV and RV injections. Six AAV vectors (AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, AAV7m8) were identified that transduce the kidney more efficiently than AAV9 by RV injection. 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. It was documented that the renal transduction profile is substantially different between healthy and CKD kidneys.

[0031] Methods are disclosed for delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject. In further aspects, methods are disclosed for treating a kidney disorder in a subject, that includes locally delivering an AAV vector encoding a therapeutic protein to cells of kidney.

[0032] Terms

[0033] 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.

[0034] 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.

[0035] The term “administration” refers to providing or giving a subject an agent by any effective route. Exemplary routes of administration include, but are 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. 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.

[0036] 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.

[0037] 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.

[0038] TABLE A

[0039] Original Residue Conservative Substitutions

[0040] Ala ser

[0041] Arg lys

[0042] Asn gin; his

[0043] Asp glu

[0044] Cys ser

[0045] Gin asn

[0046] Glu asp

[0047] Gly pro

[0048] His asn; gin lie leu; val

[0049] Leu ile; val

[0050] Lys arg; gin; glu

[0051] Met leu; ile

[0052] Phe met; leu; tyr

[0053] Ser thr

[0054] Thr ser

[0055] Trp tyr

[0056] Tyr trp; phe

[0057] Val ile; leu 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.

[0058] “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 niL / min / 1.73 m2for three consecutive months (see 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.

[0059] 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:

[0060] Stage 1: Slightly diminished function; kidney damage with normal or relatively high GFR (>90 mL / min / 1.73 m2). Kidney damage includes pathological abnormalities or markers of damage, including abnormalities in blood or urine test or imaging studies.

[0061] Stage 2. Mild reduction in GFR (60-89 mL / min / 1.73 m2) with kidney damage.

[0062] Stage 3. Moderate reduction in GFR (30-59 mL / min / 1.73 m2).

[0063] Stage 4. Severe reduction in GFR (15-29 mL / min / 1.73 m2). Preparation for renal replacement therapy.

[0064] Stage 5. Established kidney failure (GFR < 15 mL / min / 1.73 m2, 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).

[0065] 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 l., 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.)

[0066] 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).

[0067] 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%.

[0068] 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.

[0069] 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.

[0070] 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 (z.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.

[0071] A “heterologous” sequence is a sequence that is not normally (in the wild-type sequence) found adj cent 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.

[0072] 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, i.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.

[0073] 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, 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.

[0074] The term “mammal” includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects.

[0075] 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.”

[0076] 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.

[0077] 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.

[0078] 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. Nat . 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)).

[0079] 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.

[0080] 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).

[0081] 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.

[0082] 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.

[0083] 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.

[0084] The term “polypeptide fragment” refers to a portion of a polypeptide which exhibits at least one useful epitope. The term “functional fragments of a polypeptide” refers to all fragments of a polypeptide that retain an activity of the polypeptide. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell. An “epitope” is a region of a polypeptide capable of binding an immunoglobulin generated in response to contact with an antigen.

[0085] The term “substantially purified polypeptide” as used herein refers to a polypeptide which is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In one aspect, the polypeptide is at least 50%, for example at least 80% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In another aspect, the polypeptide is at least 90% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated. In yet another aspect, the polypeptide is at least 95% free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.

[0086] “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.

[0087] 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 promoter-dependent 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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 episomes.

[0093] 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.

[0094] A “transgene” refers to an exogenous gene supplied by a vector or a virus, such as an AAV.

[0095] Overview

[0096] Clause 1 : A method of delivering a composition comprising an adeno-associated virus (AAV) to one or more kidney cell types in a subject, comprising: locally delivering to the kidney of the subject an adeno- associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, thereby delivering the composition comprising the AAV to the one or more kidney cell types in the subject.

[0097] Clause 2: The method of clause 1, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

[0098] Clause 3: The method of clause 1, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0099] Clause 4: The method of any one of clauses 1-3, wherein locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

[0100] Clause 5: The method of any one of clauses 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 of the subject.

[0101] Clause 6: The method of any one of clauses 1-5, wherein the AAV transduces the one or more kidney cell types of the subject with an efficiency about 5 times to about 200 times greater than that of AAV9.

[0102] Clause 7: The method of any one of clauses 1-6, wherein the genome comprises a nucleic acid molecule encoding a therapeutic protein.

[0103] Clause 8: A method of treating a kidney disorder in a subject, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject.

[0104] Clause 9: The method of clause 8, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

[0105] Clause 10: The method of clause 8, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0106] Clause 11: The method of any one of clauses 8-10, wherein the locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

[0107] Clause 12: The method of any one of clauses 8-11, 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.

[0108] Clause 13: The method of any one of clauses 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.

[0109] Clause 14: The method of any one of clauses 8-13, wherein the kidney disorder is a glomerular disease, a tubulointerstitial disease, a renal vascular disease, a genetic kidney disease, am 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.

[0110] Clause 15: A composition comprising an adeno-associated virus (AAV) for use in delivering a therapeutic protein to one or more kidney cell types in a subject, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome comprises a nucleic acid molecule encoding the therapeutic protein.

[0111] Clause 16: The composition for use of clause 15, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

[0112] Clause 17: The composition for use of clause 15, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

[0113] Clause 18: The composition for use of any one of clauses 15-17, wherein locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

[0114] Clause 19: The composition for use of any one of clauses 15-18, 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 of the subject.

[0115] Clause 20: The composition for use of any one of clauses 15-19, wherein the AAV transduces the one or more kidney cell types of the subject with an efficiency about 5 times to about 200 times greater than that of AAV9.

[0116] Clause 21 : The composition for use of any one of clauses 15-20, wherein the subject has a kidney disorder.

[0117] Clause 22: The composition for use of any one of clauses 15-21, wherein the kidney disorder is a glomerular disease, a tubulointerstitial disease, a renal vascular disease, a genetic kidney disease, am 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.

[0118] AAV Vectors and Their Use

[0119] 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.

[0120] 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.

[0121] 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 (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.

[0122] 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 are 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.

[0123] 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 (VP1, 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 icosahedral symmetry. VP1, VP2 and VP3 are 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 .

[0124] In some aspects, a recombinant adeno-associated virus (rAAV) is generated having a capsid of interest. In AAV, the capsid includes VP1, 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 EFloc promoter, the GAPDH promoter, the PGK promoter, the RSV promoter, and the [I-actin promoter.

[0125] 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 proteins under the control of inducible promoters. Still other stable host cells may be generated by one of skill in the art.

[0126] The minigene, rep sequences, cap 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, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 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.

[0127] 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 [3-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 (PDGFR[3) 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.

[0128] 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.

[0129] 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 (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et 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. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used.

[0130] The vectors disclosed herein can contain nucleic acid sequences encoding an intact AAV capsid which may be from a single AAV serotype.

[0131] 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, or AAV2.7m8 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, or AAV2.7m8 capsid protein.

[0132] 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.

[0133] In some aspects, the 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 Wilms 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, P-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 glomerulopath (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, djenkol bean poisoning (djenkolism), 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 l[3-associated kidney disease, hepatorenal syndrome, herbal supplements and kidney disease, high altitude renal syndrome, high blood pressure and kidney disease, HIV-associated immune complex kidney disease (HIVICK), HIV-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-related 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-methylenedioxymethamphetamine) and kidney failure, meatal stenosis, medullary cystic kidney disease (urolodulin-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 (Exner 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.

[0134] 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, Bartter 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.

[0135] 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, b0 +amino acid transporter, nephrocystin 1, CLCN5, polycystin-1, polycystin-2, BBS. When the proteins of interest is too large to 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 (PDGFRfl) 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.

[0136] In some aspects, the AAV vector can further comprise a polynucleotide the express functional RNA molecules 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 overexpressed 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 molecules 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 are 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.

[0137] 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 identify 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, or AAV2.7m8 capsid proteins, and are shown below:

[0138] AAV KPI

[0139] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPGNGLDKGEP VNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLE PLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDTDSAADPQPLGEPPA APSGLGTGTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHL YKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVK EVTQNEGTKTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAM GRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSG TTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLV NPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNL QSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIK NTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNT EGVYSEPRPIGTRYLTRNL (SEQ ID NO: 1)

[0140] AAVKP2

[0141] MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGE PVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVL EPLGLVEEGAKTAPGKKRPVEHSPVEPDSSSGIGKTGQQPAKKRLNFGQTGDADSVPDPQPLGQPP AAPSGLGTNTMATGSGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNH LYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQV KEVTQNEGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQA VGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTS GTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSL VNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANN LQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMI KNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVD TNGVYSEPRPIGTRYLTRPL (SEQ ID NO: 2)

[0142] AAVKP3

[0143] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPL GLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAP SGVGPNTMAAGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYK QISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEV TQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTN QSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGP AMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSN TAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPV PADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVY SEPRPIGTRYLTRNL (SEQ ID NO: 3)

[0144] AAV-DJ

[0145] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPL GLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPIGEPPAAP SGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKE VTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGR SSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTTN TQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPG PAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGN RQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTP VPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGV YSEPRPIGTRYLTRNL (SEQ ID NO: 4)

[0146] AAV2G9

[0147] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSVAGPSNMAVQGRNWLPGPCYRQQRVSKTSADNNNSEFAWTGATKYHLNGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQ AATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVP ANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVY SEPRPIGTRYLTRNL (SEQ ID NO: 5)

[0148] AAV2_7m8

[0149] MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPV NEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPL GLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAP SGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYK QISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVT QNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRS SFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQS RLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPA MASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNLA LGETTRPARQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPP QILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDF TVDTNGVYSEPRPIGTRYLTRNL (SEQ ID NO: 6).

[0150] The disclosed AAV can be delivered to the kidney of a subject by any method. These include direct parenchymal injection, renal vein injection, and renal artery injection.

[0151] 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'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

[0152] 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 non-protected counterparts.

[0153] Exemplary excipients that can 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.

[0154] 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. %. I f 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. In one example, an aqueous virion-stabilizing formulation comprises a carbohydrate, such as sorbitol, at a concentration of between 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.

[0155] 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 deacetylate 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.

[0156] 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 IO16of the AAV virions, such as 108to IO14AAV 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.

[0157] 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 1O1SAAV virions in a single dose, or two, four, five, six or more doses that collectively result in delivery of, e.g., IO3to 1016AAV virions. One of skill in the art can readily determine an appropriate number of doses to administer.

[0158] 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 101 1, 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. 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.

[0159] EXAMPLES

[0160] 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 was preformed using AAV Barcode-Seq. Although intravenous (IV) systemic administration and local renal vein (RV) administration of AAV vectors show no remarkable administration method-dependent differences in renal transduction profiles in the majority of the 47 AAV capsids, six AAV capsids exhibited remarkable enhancement of renal transduction when delivered by RV injection. Among the six AAV capsids, i.e., AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9 and AAV2.7m8, 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 IV and RV injections, RV 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 local intravascular vector administration 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.

[0161] Example 1

[0162] Barcode-Seq identified AAV capsids that outperform AAV9 in renal transduction by local injection

[0163] It was first investigated which AAV capsids transduce the kidney most efficiently by IV and RV injections in mice. The RV route for local intravascular injection because it has been commonly used in mouse studies. To this end, AAV Barcode-Seq (Adachi et al., Nat Commun 5:3075, 2014) was employed, and a total of 47 different AAV capsid-mediated renal transduction profiles were determined in mice following IV or RV administration. In this approach, AAV capsids package AAV-CAG-BC vector genomes whose barcodes (BCs) are unique to each capsids (FIG. 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 contained 47 AAV capsids (11 naturally occurring serotypes and 36 capsid-engineered mutants, Table 1) and injected into 8-week-old C57BL / 6J male mice by IV at a dose of 2xl013vg / kg (n=3) or by RV at a dose of 3x10’1vg / mouse (n=4). RV injection was performed with the blockage of the local renal blood flow for 15 min based on the methods reported previously (Rocca et al., Gene therapy 21: 618- 628, 2014; Konkalmatt et al., JCI insight 1, 2016) to increase the vector exposure. Six weeks after injection, relative transduction efficiency of each AAV capsid in the kidney compared to AAV9 was determined by AAV DNA / RNA Barcode-Seq. As shown in FIG. IB (transduction efficiency of all AAV capsids are shown in FIGS. 2A-2B), it was found that (a) AAV9 capsid transduced the kidney most efficiently by IV; (b) by RV injection, the following six AAV capsids, AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9 and AAV2.7m8, showed remarkably higher renal transduction than AAV9 with all but AAV2.7m8 showing more than 10 fold enhancement; and (c) AAV3, AAVLK03 and AAVShHIO showed significantly higher renal gene transfer than AAV9 at a vector genome DNA level but the vector genomes delivered by these two capsids were transcriptionally less active than those delivered by AAV9, showing a discordance of AAV vector genome DNA and vector genome RNA transcript levels. Since this phenotype of AAVLK03 was previously reported in the liver (Gonzalez-Sandoval et al., bioRxiv, 2022) and the discordance between vector genome DNA and vector genome transcript levels was also observed with AAV3, AAVLK03 and AAVshHIO in IV injection (FIGS. 2A-2B), this phenomenon is most likely not related to the method of vector administration. Importantly, systemic and local vector administrations showed distinct profiles in renal transduction and the renal transduction enhancing effect of the six AAV capsids that were identified was observed only when they were locally administered, highlighting the importance of careful consideration of administration route -dependent attributes of AAV capsids in designing gene therapy approaches for the treatment of kidney diseases.

[0164] Table 1. AAV vectors included in the Barcode library.

[0165] Vectors Reference* Vectors Reference*

[0166] AAV1 Earley AAVbb.2 Gao

[0167] AAVl_9mtl00 Nakai AAVDJ Grimm

[0168] AAVl_9mt30 Nakai AAVHN1 Nakai

[0169] AAVl_9mt76 Nakai AAVHN2 Nakai

[0170] AAV2 Earley AAVHN3 Nakai

[0171] AAV2G9 Shen AAVhu.ll Gao

[0172] AAV2i8 Asokan3AAVhu.13 Gao AAV2retro Tervo AAVhu.37 Gao

[0173] AAV2R585E Adachi AAVKP1 Pekrun

[0174] AAV2R585E9_2 Adachi AAVKP2 Pekrun

[0175] AAV3 Earley1AAVKP3 Pekrun

[0176] AAV4 Earley AAVLK03 Lisowski

[0177] AAV5 Earley AAVNP40 Paulk

[0178] AAV6 Earley AAVNP59 Paulk

[0179] AAV7 Earley AAVPHP.B Deverman

[0180] AAV8 Earley AAVPHP.eB Chan

[0181] AAV9 Earley31AAVPHP.S Chan

[0182] AAV9AA272 Nakai AAVpol Bello

[0183] AAV9AA22 Nakai AAVrh.8 Gao

[0184] AAV9W22A Nakai AAVrh.10 Gao

[0185] AAV10 Earley AAVrh.20 Gao

[0186] AAV 11 Earley AAVrh.43 Gao

[0187] AAV2.7m8 Dalkara AAVShHIO Klimczak

[0188] AAVAnc80 Zinn

[0189] *References:

[0190] Earley, L. F. et al., . J Virol 91 (2017). doi.org:10.1128 / JVI.01980-16

[0191] Shen, S. et al. J Biol Chem 288, 28814-28823 (2013)

[0192] Asokan, A. et al. Nat Biotechnol 28, 79-82 (2010). doi.org: 10.1038 / nbt.1599

[0193] Tervo, D. G. et al. Neuron 92, 372-382 (2016). doi.org: 10.1016 / j. neuron.2016.09.021

[0194] Adachi, K et al. Nat Commun 5, 3075 (2014), doi.org: 10.1038 / ncomms4075

[0195] Dalkara, D. et al. Sci Transl Med 5, 189ral76 (2013). doi.org: 10.1126 / scitranslmed.3005708

[0196] Zinn, E. et al.. CellRep 12, 1056-1068 (2015). doi.org:10.1016 / j.celrep.2015.07.019

[0197] Gao, G. et al. J Virol 78, 6381-6388 (2004). doi.org:10.1128 / JVL78.12.6381-6388.2004

[0198] Grimm, D. et al.. J Virol 82, 5887-5911 (2008). doi.org: 10.1128 / JVI.00254-08

[0199] Pekrun, K. et al. JCI insight 4 (2019). doi.org: 10.1172 / jci.insight.l31610

[0200] Lisowski, L. et al. Nature 506, 382-386 (2014). doi.org: 10.1038 / naturel 2875

[0201] Paulk, N. K. et al Mol Ther 26, 289-303 (2018). doi.org:10.1016 / j.ymthe.2017.09.021

[0202] Deverman, B. E. et al. Nat Biotechnol 34, 204-209 (2016). doi.org: 10.1038 / nbt.3440

[0203] Chan, K. Y. et al. Nat Neurosci 20, 1172-1179 (2017). doi.org: 10.1038 / nn.4593

[0204] Bello, A. et al. Gene therapy 16, 1320-1328 (2009). doi.org:10.1038 / gt.2009.82

[0205] Klimczak, R. R., et al. PLoS One 4, e7467 (2009). doi.org:10.1371 / journal.pone.0007467

[0206] Groopman, E. E. et al. N Engl J Med 380, 142-151 (2019). https: / / doi.org: 10.1056 / NEJMoal 806891

[0207] Earley, L. F. et al. J Virol 91 (2017). https: / / doi.org: 10.1128 / JVI.01980-16

[0208] Nakai: AAVl_9mtl00, AAVl_9mt30, AAVl_9mt76 - AAV1 -derived capsid mutants containing portions of the AAV9 capsid sequence (PCT Publication No. WO2017192750A1). AAVl_9mtlOO, AAVl_9mt30 and AAVl_9mt76 are AA VI.9-9919911, AAV1.9- 1191999 and AAV1.9-9119919, respectively (Note: AAV1.9-9919911, AAV1.9- 1191999 and AA VI.9-9119919 are the nomenclature used in the figures of PCT Publication No. WO2017192750A1). AAV9AA272 - an AAV9 capsid mutant, AAV9N272A, disclosed in US Patent No. 11459558B2. AAV9AA22 - an AAV9 capsid mutant, AAV9W22A / W23A, disclosed in PCT Publication No. WO2018119330A2. AAV9W22A - an AAV9 capsid mutant, AAV9W22A, described in PCT Publication No. WO2018119330A2. AAVHN1, AAVHN2, AAVHN3 - AAV9 capsid mutants with a peptide insertion at amino acid position 589 described in US Patent No. 11,459,558.

[0209] Example 2

[0210] Renal vein injection of AAVKP1 enabled effective proximal tubule cell transduction in mice

[0211] To validate the AAV Barcode-Seq data and characterize the cell type tropism, the AAV-CAG- tdTomato genome was individually packaged with AAV9 and AAVKP1 capsids to produce AAV9-CAG- tdTomato and AAVKPl-CAG-tdTomato vectors. Each of these two vectors was injected into 8-week-old C57BL / 6J male mice by IV or RV at a dose of 3x10'1vg / mouse (n=4 per group, FIG. 3A). AAVKP1 (Pekrun et al., JCI insight 4, 2019) was selected from the 6 capsids showing the enhancement as it was one of the most efficient capsids for kidney transduction by RV based on the AAV Barcode-Seq data. Two weeks after injection, vector genome copy numbers were determined by DNA qPCR and renal transduction was assessed by immunofluorescence microscopy. Quantification of vector genomes in the kidney showed that, while vector genome copy numbers were comparable between the AAV9 and AAVKP1 -injected mice in the IV group, there were >30 times more vector genomes in the AAVKP1 -injected mice than the AAV9- injected mice (FIG. 3B). Vector genome copy numbers in the AAV9-injected animals were not different between the IV and RV groups, demonstrating that there is no advantage of local administration over systemic administration when AAV9 capsid is used. In consistent with the vector genome copy number data, the histological assessment also showed substantially enhanced transduction in the kidney with AAVKP1 following RV injection compared to the IV injection (FIG. 3C). 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 11% transduction in the proximal renal tubule cells by RV injection of AAVKP1, whereas AAVKP1 only transduced mesangial cells in the glomeruli by IV injection (FIG. 4A). On the other hand, cell tropism of AAV9 remained the same regardless of administration routes, showing mainly mesangial cell transduction. We also performed co-staining of podocytes, demonstrating no transduction in podocytes with AAV9 or AAVKP1 regardless of the administration routes (FIG. 4B).

[0212] Example 3

[0213] Ischemia did not enhance renal transduction of AAVKP1

[0214] Since RV injection was performed with the 15 min-blockage of the local arterial and venous flow, it was investigated if ischemia influences renal transduction. Intravenous injection of AAVKP1-CAG- tdTomato (3xlOnvg / mouse) into 8-week-old C57BL / 6J male mice following 15 min-renal ischemia did not alter the transduction, showing only mesangial cell transduction (FIG. 5A). This indicates that ischemia is not the cause of enhanced renal transduction by RV. RV injection of AAVKPl-CAG-tdTomato (3x1011vg / mouse) was also performed with varying ischemic time periods from < 1 min up to 14 min (FIG. 5B). There was no apparent difference in transduction efficiency by RV between the following four ischemic time periods (<1 min, 5 min, 10 min and 15min). Please note that testing RV in no ischemic time period condition is not possible because renal blood flow needs to be stopped when the agent is injected via RV to the kidney. In the shortest ischemic time period condition for RV injection (i.e., 0 min in FIGS. 5B), the renal blood flow was stopped for no more than 1 min.

[0215] Example 4

[0216] Augmented renal interstitial accumulation of vector particles is a potential mechanism of the enhanced renal transduction by local vector administration

[0217] When AAV vectors are administered intravascularly, vascular endothelial cells pose a significant physical and functional barrier for in vivo AAV vector transduction in the organs whose endothelium is devoid of fenestrae (i.e., continuous endothelium) such as the brain, heart and skeletal muscles (Kotchey et al., Mol Ther 19:1079-1089, 2011). In the kidney, cortical peritubular capillaries and glomerular endothelial cells are both fenestrated by holes of approximately 60 to 80 nm in diameter with the former having a diaphragm and the latter being devoid of it (Satchell et al., Am J Physiol Renal Physiol 296:F947-956, 2009) while the other blood vessels are lined with continuous endothelium devoid of fenestrae. Based on these microanatomical structure of the kidney, the mesangial interstitial space is readily reached by intravascularly injected AAV vector particles through the fenestrae in the glomerular endothelial cells. The peritubular interstitium in the cortex is also presumed to be reached by intravascularly injected AAV vector particles relatively easily due to the fenestrated nature of the peritubular capillaries, although not as easily as the mesangial interstitial space due to the presence of a diaphragm in the fenestrae.

[0218] To investigate the distribution and extravasation of AAV vector particles in the kidney following AAV vector injection by IV and RV, 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 (FIG. 6A), consistent with the previous findings that nanoparticles accumulate in the mesangial area in size-dependent manner (Choi et aL, Proc Natl Acad Sci U SA 108:6656-6661, 2011; Guo et al., Nat Commun 8:878, 2017). This observation is in line with knowledge of the above described anatomical structure of the kidney and the observed effective transduction of mesangial cells with AAV vectors. On the other hand, limited extravasation of microspheres from the peritubular capillary to the interstitial space was observed (indicated by arrows in FIG. 6A). Next, microspheres were administered via RV to assess their extravasation and accumulation in the kidney. Interestingly, the RV administration resulted in efficient interstitial accumulation of the microspheres (FIG. 6B) mainly in the renal cortex. These observations suggest that peritubular capillary endothelium poses a significant barrier for AAV vector to transduce tubular epithelial cells following IV injection even with the presence of fenestration in the endothelium, while RV injection allows AAV vector to pass through this barrier, leading to efficient proximal tubule transduction. In addition, this observation indicates that renal tubular basement membrane is not a significant barrier for AAV vector transduction allowing effective entry of AAV particles from the renal interstitium into renal tubular cells from the basolateral surface.

[0219] Example 5 Limited extra-renal dissemination of AAVKP1 following local intravascular injection suppressed off- target liver transduction

[0220] Several studies have reported off-target organ transduction especially in the liver following local injection of AAV9 (Rubin et ah, Human gene therapy 30:1559-1571, 2019; Meyer et ah, Mol Ther 23:477- 487, 2015; Matheisen et ah, Mol Ther Methods Clin Dev 19:447-458, 2020; Quirin et al., Mol Ther Methods Clin Dev 8: 8-20, 2018). Therefore, off-target liver transduction was assessed following RV injection of AAV9 and AAVKP1. Quantification of vector genomes in the liver two weeks after IV and RV injection revealed that both AAV9 and AAVKP1 are equally liver tropic by IV injection, while RV injection of AAVKP1 reduced the off-target liver transduction by 14-fold compared to IV injection. On the other hand, off-target liver transduction was not prevented by RV injection of AAV9 (FIG. 7A). Immunofluorescence study also confirmed substantially suppressed off-target liver transduction by RV injection of AAVKP1 compared to AAV9 (FIG. 7B). Based on these observations, it was hypothesized that the difference in pharmacokinetics following local injection leads to the contrasting renal and hepatic transduction between AAV9 and AAVKP1. Quantification of vector genomes 10 min after local injection showed >10 times more vector genomes in AAVKP1 -injected kidney than the AAV9-injected kidney following RV injection (FIG. 7C), 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: 1079-1089, 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- injected 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 RV injection, FIG. 7D), indicating that substantially more AAV vector particles are trapped in the kidney when AAVKP1 capsid is used compared to AAV9. These findings support the hypothesis that less AAVKP1 vector spillover by RV 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 local vector delivery with limited off-target effects. Example 6

[0221] Local intravascular administration of AAVKP1 enabled effective proximal tubule cell transduction in non-human primates

[0222] To translate the findings into a clinically relevant setting, local intravascular injection of AAVKP1 was performed in a non-human primate. In place of the RV approach, a balloon catheter-mediated renal artery (RA) injection method was performed with transient blockage of renal blood flow. RA injection was chosen as it is a more physiological approach than retrograde RV injection. In the procedure, a balloon catheter was inserted via the femoral artery and vein, and the catheter tips were placed at the renal artery and vein (FIG. 8A). After arterial balloon occlusion, AAVKPl-CAG-tdTomato was infused via the left renal artery into a 14-month-old male rhesus macaque at 8.2xl012vg / animal (2.5xl012vg / kg) followed by venous occlusion with 10-min dwelling time. Renal transduction and off-target liver transduction were assessed three weeks after injection. The results demonstrated that effective proximal renal tubular transduction was attained with minimal liver transduction (FIGS. 8B, 8C and 8E). In contrast to the transduction profiles observed in mice, glomerular transduction was not observed (FIG. 8D). The transduced cells in the kidney were exclusively LTL-positive cells, showing high specificity to proximal renal tubules. These results demonstrate that AAVKP1 is an effective AAV capsid for renal gene transfer in non-human primate by local intravascular administration.

[0223] Thus, a small subset of AAV capsids were identified that exhibit significantly enhanced gene transfer to the kidney by local intravascular vector administration. By using the AAV capsids showing this peculiar phenotype, clinically relevant kidney cells were transduced; i.e., proximal renal tubules, at the level that was not previously attainable. According to the previous studies assessing renal transduction of common AAV serotypes by IV (Ikeda et al., JASN 29:2287-2297, 2018; Lang et al., Nat Commun 10:3415, 2019; Schievenbursch et al., Mol Ther 18:1302-1309, 2010) and RV (Rocca et al., Gene therapy 21:618-628, 2014) injections, it has been generally considered that AAV9 is one of the most efficient capsids regardless of administration routes. However, our unbiased, head-to-head comparison study that sought to determine renal transduction efficiency with various AAV capsids covering both common serotypes and engineered capsids has demonstrated that effective AAV capsids are not necessarily the same between systemic IV injection and local intravascular injection, and has shown that remarkable enhancement of renal transduction could be attained by local intravascular injection but it could be attained only when a particular subset of AAV capsids were used. In the study, we have identified the following 6 AAV capsids: AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9 and AAV2.7m8, as those that outperform AAV9 only when administered by RV injection. AAV9 is the serotype that shows the most robust in vivo transduction in various organs following IV injections and our AAV Barcode-Seq experiment has also reconfirmed it in the context of renal gene transfer. AAVKP (Pekrun et al., JCI insight 4, 2019) and AAVDJ (Grimm et al., J Virol 82:5887-5911, 2008) were generated by capsid shuffling and in vitro screening in pancreatic islets and hepatoma cells, respectively. AAV2G9 was generated by engraftment of a galactose binding domain of AAV9 into AAV2 capsid (Shen et al., J Biol Chem 288:28814-28823, 2013). AAV2.7m8 was generated by in vivo screening in photoreceptors of mice retinas (Dalkara et al., Sci Transl Med 5: 189ral76, 2013). Therefore, their high performance in renal gene transfer by local intravascular administration was unexpected and not predictable, exemplifying the utility of high-throughput AAV capsid phenotype characterization such as AAV Barcode-Seq. Nonetheless, our discovery that there are a small subset of AAV capsids that can substantially enhance renal transduction by local vector administration represents a breakthrough in renal gene transfer and opens a new avenue to the development of much more effective approaches for gene therapy for kidney diseases.

[0224] It was shown that AAVKP1 efficiently transduces proximal renal tubule cells by local vector administration. Proximal renal tubules account for a half of the tubular epithelial cells (Clark et al., Kidney Int 95:787-796, 2019) and is an important target cell type for both basic research (Chevalier et aL, Am J Physiol Renal Physiol 31LF145-161, 2016) and gene therapy (Hildebrandt et al., Lancet 375: 1287-1295, 2010). RV injection is not parenchymal injection, and injected solution infiltrates into the whole kidney, resulting in widely distributed proximal renal tubule transduction. In contrast to other organs such as brain where target cells tend to be consolidated in one region, this is critical for the renal gene transfer. It was demonstrated herein that the extended blockage of the renal arterial and venous flow performed in previous studies (Rocca et al., Gene therapy 21:618-628, 2014; Asico et al., Biochemical and biophysical research communications 497: 19-24, 2018; Konkalmatt et al., JCI insight 1, 2016) is not required for efficient renal transduction of AAVKP1. Minimizing the ischemic time only for completing RV injection is beneficial.

[0225] The results demonstrated that AAVKP1 has limited dissemination following local injection that leads to transduction in the kidney with high specificity. Recent clinical trials raised concerns about adverse effects related to dissemination of AAV vectors to off-target organs, such as hepatotoxicity, genotoxicity, thrombotic microangiopathy and dorsal root ganglia (DRG) toxicity (Li et al., Nat Rev Genet 21:255-272, 2020; Colella et al., Mol Ther Methods Clin Dev 8:87-104, 2018). Local administration of AAVKP1 can prevent or reduce these side effects of AAV vector-mediated gene therapy. It was demonstrated that the degree of vector spillover after local administration is capsid dependent. Locally administered AAV9 caused substantial spillover that resulted in off-target liver transduction comparable to systemic administration, indicating that AAV9 is not suitable for local injection.

[0226] Example 7 Materials and Methods

[0227] Animal experiments. All the animal experiments were performed according to the guideline for animal care at Oregon Health & Science University (OHSU). C57BL / 6J mice were purchased from the Jackson Laboratory (Strain ID is 664). For AAV Barcode-Seq analysis, 8-week-old C57BL / 6J male mice were injected with an AAV barcode library via the tail vein at a dose of 2xl013vg / kg (n=3), and via the renal vein (n=4) at a dose of 3xl0uvg / mouse. Six weeks after injection, mice were euthanized, and kidneys were harvested. For AAV9 / AAVKP1 characterization study, 8-week-old C57BL / 6J male mice were injected with AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato via tail vein or the renal vein at a dose of 3xl0n vg / mouse (n=4 per group). Two weeks after injection, mice were euthanized, and kidneys and livers were harvested for viral genome quantification and histological assessment by immunofluorescence microscopy. For a pharmacokinetic study, the whole blood samples were collected from the retro-orbital plexus at 0 min (RV only, when 15 min-dwelling was completed), 1 min (IV only), 10 min, 30 min, 1 h, 4 h and 8 h following administration of AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato into 8-week-old C57BL / 6J male mice at a dose of IxlO13vg / kg (n=4 per group). To quantify the viral genome in the kidney after local vector injection, injected kidneys were harvested 10 min after the completion of 15 min-dwelling time following renal vein injection of AAV9-CAG-tdTomato or AAVKPl-CAG-tdTomato into 8-week-old C57BL / 6J male mice at a dose of IxlO13vg / kg (n=4 per group).

[0228] Mouse surgical procedures. For renal vein injection (modified from the previous protocol9), mice were anesthetized by isoflurane inhalation and placed on heated surgical pad (8002062012, Stryker Medical) to maintain a constant body temperature. A medial abdominal incision was made and intestines were removed from the abdominal cavity to expose the left renal vasculature and the kidney. Removed intestines were kept moist throughout the surgery. A non-traumatic micro-serrefine clamp was placed on the renal artery and vein and 50 pl of AAV vector solution was injected using a 31 gauge needle (328468, BD Medical). Ischemic time required for injection is less than 1 min. Fifteen min after injection, the clamp was removed to observe the restoration of blood flow (verified by color change), and the incision was closed. To assess the effect of ischemia on renal transduction, a clamp was placed on the renal artery and vein for 15 min by making a flank incision and tail vein injection was performed after removal of the clamp. Renal vein injection was also performed with different lengths of ischemia (<1 min (RV injection), 5 min, 10 min and 15 min). In the < 1 min group, renal vein injection was performed with minimal length of ischemia required for injection.

[0229] AAV vectors and plasmids. AAV-CAG-BC library, AAV9-CAG-tdTomato and AAVKPl-CAG- tdTomato were produced in HEK293 cells (RRID: CVCL-6871, Agilent) by an adenovirus-free plasmid transfection method and purified by two rounds of cesium chloride density-gradient ultracentrifugation followed by dialysis as described previously (Earley et al., J Virol 91, 2017). AAV9 and AAVKP1 helper plasmids were provided by J. M. Wilson and M. A. Kay, respectively. pAAV-CAG-tdTomato was a gift from E. Boyden (59462, Addgene). For AAV barcode library production, we used pdsAAV-CAG-VBCx plasmids (where VBC is viral barcode and x is an integer identification number indicating each different viral barcode). pdsAAV-CAG-VBCx plasmids are double-stranded AAV vector plasmids and same as pdsAAV-U6-VBCx described previously (Earley et al., J Virol 91, 2017) except that human U6 small nuclear RNA promoter has been replaced by CAG promoter and a SV40 polyadenylation signal has been added.

[0230] AAV Barcode-Seq analysis. Total DNA was extracted from tissues using QIAamp MinElute Virus Spin Kit (57704, Qiagen) or KingFisher Cell and Tissue DNA kit (97030196, Fisher Scientific) following proteinase K (25530049, Invitrogen) treatment. Total RNA was extracted from tissues using TRIzol

[0231] (15596018, Invitrogen) followed by DNase treatment using TURBO DNA-free kit (AM1907, Invitrogen). 0.8 jjg of DNase-treated RNA was reverse transcribed with an RT-specific primer using High-Capacity cDNA Reverse Transcription Kit (4368813, Applied Biosystems) or SuperScript IV Reverse Transcriptase (18090200, Invitrogen) in a total volume of 20 pl. One pg DNA or 4 pl cDNA was used to PCR amplify VBC using platinum SuperFi II DNA Polymerase (12361010, Invitrogen). Information on primer sequence including frameshifting nucleotide, sample barcode was previously explained (. PCR products were mixed at an equimolar ratio and sequenced with either of the following settings at Massively Parallel Sequencing Shared Resource (MPSSR) at OHSU or Novogene (Sacramento, CA): 75-cycle single-end / 150-cycle single- end / 180-cycle single-end / 300-cycle paired-end on an Illumina NextSeq 500 / NovaSeq 6000 instruments. The Illumina sequencing data was analyzed. Four parameters in FastQC (Shen et al., J Biol Chem 288: 28814- 28823, 2013) per base sequence quality, per sequence quality scores, per base N content and sequence length distribution) were met in all the data sets we used in this study. The algorithm for data analysis was previously described (Adachi et al., Nat Commun 5:3075, 2014; Earley et al., J Virol 91, 2017). For RNA Barcode-seq, the sequence-dependent difference in the in vivo mRNA transcription and the reversetranscription PCR amplification efficiencies between VBCs was considered. In brief, we made two AAV9- CAG-VBCx libraries that contain all the AAV-CAG-VBCx genomes packaged with the same AAV9 capsid. These two libraries were produced independently from two independent pools of all the pdsAAV-CAG- VBCx plasmids mixed at an equimolar ratio. Each of the two AAV9-CAG-VBCx virus libraries was intravenously injected into 8-week-old C57BL / 6J male mice (n=3 each) to obtain a duplicated set of data each of which was obtained from 3 mice. The livers were harvested from the library-injected animals 6 weeks post-injection. Liver RNA was extracted and VBC RNA barcode transcripts were amplified by reverse transcription (RT)-PCR. The RT-PCR products were then subjected to the Barcode-Seq analysis, which provides output RNA barcode reads. The AAV-CAG-VBCx genomes were extracted from each of the two AAV9-CAG-VBCx virus library stocks and the VBC DNA barcodes were amplified by DNA-PCR, which provides input DNA barcode reads. The correction factors obtained by the ratio of output RNA barcode reads and input DNA barcode reads were used to cancel out the barcode sequence-dependent differences in the RNA barcode read count data.

[0232] AAV vector genome quantification. Total DNA was extracted from tissues as described in AAV Barcode-Seq analysis section. Blood DNA sample was prepared using Extract-N-Amp Blood PCR Kit (XNAB2R, Sigma) and diluted 100 times. Vector genome copy numbers were quantified by quantitative PCR (qPCR). In brief, 10-100 ng of tissue DNA or 1 pl of blood DNA sample was mixed with Power SYBR Green PCR Master Mix (43-676-59, Fisher Scientific) and 25 pmol primers in a 25 pl reaction volume and subjected to qPCR using Rotor-Gene Q (Qiagen). We amplified the tdTomato gene sequence for vector genome quantification using following primers: tdTomato forward (5’-ATGGACCTGTGATGCAGAAG- 3’, SEQ ID NO: 7) and tdTomato reverse (5’-TTCAGCTTCAGAGCCTGGTG-3’, SEQ ID NO: 8). Information on copy number standards and normalization was described previously (Adachi et al., Nat Commun 5:3075, 2014). Vector genome copy numbers were expressed as double-stranded vector genome copy numbers per diploid genomic equivalent. Statistics. Statistical analyses were performed using GraphPad Prism9 (version 9.5.0). Data are presented as mean + standard error of the mean. Comparison of two groups were performed using two-tailed unpaired t-test. For data sets with more than two groups (combination of AAV vectors and routes of administration), significance was determined by two-way ANOVA followed by Tukey’s post-hoc test. For pharmacokinetic study, area under the curve for each group was calculated using trapezoid rule, exa

[0233] 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, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, 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 capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

3. The method of claim 1, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

4. The method of claim 1, wherein locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

5. The method of claim 1, 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 of the subject.

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

7. The method of claim 1, wherein the genome comprises a nucleic acid molecule encoding a therapeutic protein.

8. A method of treating a kidney disorder in a subject, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome encodes a therapeutic agent that treats the kidney disorder in the subject.

9. The method of claim 8, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

10. The method of claim 8, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

11. The method of claim 8, wherein the locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

12. The method of claim 8, 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.

13. The method of claim 8 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.

14. The method of claim 8, 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- Bicdl syndrome, Mcckcl-Grubcr syndrome, a collagcnopathy, 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.

15. A composition comprising an adeno-associated virus (AAV) for use in delivering a therapeutic protein to one or more kidney cell types in a subject, comprising: locally delivering to the kidney of the subject an adeno-associated virus (AAV) comprising a genome and an AAVKP1, AAVKP2, AAVKP3, AAVDJ, AAV2G9, or AAV2.7m8 capsid protein, wherein the genome comprises a nucleic acid molecule encoding the therapeutic protein.

16. The composition for use of claim 15, wherein the capsid protein comprises an amino acid sequence at least 95% identical to one of SEQ ID NOs: 1-6.

17. The composition for use of claim 15, wherein the capsid protein comprises the amino acid sequence of any one of SEQ ID NOs: 1-6.

18. The composition for use of claim 15, wherein locally delivering to the kidney comprises direct parenchymal injection, renal vein injection, and / or renal artery injection.

19. The composition for use of claim 15, 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 of the subject.

20. The composition for use of claim 15, wherein the AAV transduces the one or more kidney cell types of the subject with an efficiency about 5 times to about 200 times greater than that of AAV9.

21. The composition for use of claim 15, wherein the subject has a kidney disorder.

22. The composition for use of claim 15, 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 inj ury, 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.

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