EP4661965A1 - Compositions comprising kidney-tropic aavs and methods of use thereof - Google Patents

Abstract

Recombinant adeno-associated virus (AAV) vectors are leading gene delivery platforms, and several AAV-mediated therapies have recently been approved for clinical use. Disclosed herein are compositions comprising AAV capsid proteins demonstrating improved tropism and improved transduction efficiency for kidney cells and kidney-related cells, and methods of using AAV particles and AAV vectors comprising these AAV capsid proteins to efficiently deliver a gene of interest or a transgene to target cells or tissues and to treat a subject in need thereof.

Description

COMPOSITIONS COMPRISING KIDNEY-TROPIC AAVS AND METHODS OF USE THEREOF

I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/502,917 filed 17 May 2023 and to U.S. Provisional Patent Application No. 63/492,224 filed 25 March 2023, each of which is incorporated by reference herein in its entirety.

II. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01HL089221, UH3AR075336, and U01AI170064 awarded by the National Institute of Health. The government has certain rights in the invention.

III. REFERENCE TO THE SEQUENCE LISTING

[0003] The Sequence Listing submitted 25 March 2024 as an XML file named “23-2075-WO- Sequence_Listing”, created on 25 March 2024 and having a size of 7,706 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

IV. FIELD

[0004] The present disclosure generally relates to kidney -targeting gene delivery vehicles and related molecules.

V. BACKGROUND OF THE INVENTION

[0005] Chronic kidney disease (CKD) is estimated to affect 8-16% of the population worldwide and has increased by 31.7% over the last ten years. (Hill NR, et al. (2016) PLoS One. 1 l(7):e0158765; Evans M, et al. (2022) Adv. Ther. 39(l):33-43). Ultimately, CKD progresses to end-stage renal disease, where dialysis or kidney transplantation are the only viable options for renal replacement therapy. (Schrezenmeier E, et al. (2021) Genet Med. 23(7): 1219-1224; Tonelli M, et al. (2011) Am. J. Transplant. 11(10):2093-2109). Maintenance dialysis therapy requires multiple prolonged sessions per week and is characterized by poor patient survival. Kidney transplant has developed into a successful long-term therapy, but the field remains limited by donor organ scarcity and the need for lifelong immunosuppression. (Malek SK, et al. (2011) Transpl. Int. 24(5):419-424; Keith DS, et al. (2016) Clin. J. Am. Soc. Nephrol. 11(4):684-693). Many kidney diseases such as cystinuria, polycystic kidney disease, and cystinosis amongst others have underlying genetic etiologies that may be amenable by gene therapy or genome editing, underscoring the crucial unmet need for an effective and safe kidney-targeting gene delivery vehicle. (Peek JL, et al. (2023) Nat. Rev. Nephrol. 19(7):451- 462; Rubin J D, et al. (2020) Mol. Diagnosis Ther. 24(4):375-396; Peek JL, et al. (2022) Curr Opin Nephrol Hypertens. 31(2): 175-179; Hildebrandt F. (2010) Lancet. 375(9722): 1287- 1295). Accordingly, there is a need to develop an effective and safe kidney -targeting gene delivery vehicle.

VI. BRIEF SUMMARY OF THE INVENTION

[0006] Disclosed herein is an AAV capsid protein. Disclosed herein is an AAV capsid protein having one or more substitutions in variable region IV (VR-IV).

[0007] Disclosed herein is an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.

[0008] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01.

[0009] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q.

[0010] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:31, SEQ ID NO:35, or SEQ ID NO:43. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:53 - SEQ ID N0:61.

[0011] Disclosed herein is an AAV capsid protein, wherein the capsid protein comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is an AAV capsid protein, wherein the capsid protein comprises one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. Disclosed herein is an AAV capsid comprising an adeno -associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q. Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in SEQ ID NO:31 or SEQ ID NO:35.

[0012] Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61.

[0013] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed AAV capsid protein. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV).

[0014] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.

[0015] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising a sequence having one or more substitutions relative to the sequence set forth in SEQ ID NO:01.

[0016] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 03. [0017] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01.

[0018] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:31, SEQ ID NO:35, or SEQ ID NO:43. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61.

[0019] Disclosed herein is an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01. [0020] Disclosed herein is an AAV vector comprising a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein. Disclosed herein is an AAV vector comprising a transgene or a heterologous nucleic acid for a therapeutic protein and/or a therapeutic RNA. Disclosed herein is an AAV vector comprising a transgene or a heterologous nucleic acid for treating a subject having a kidney disease and/or kidney disorder. Disclosed herein is an AAV particle comprising an AAV capsid comprising a disclosed AAV capsid protein. Disclosed herein is an AAV particle comprising (i) an AAV capsid comprising at least one disclosed AAV capsid protein and (ii) a vector genome. Disclosed herein is an AAV particle for use in a disclosed method. Disclosed herein is an AAV particle for use in a disclosed method of delivering a payload or a disclosed method of treating a subject.

[0021] Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector in a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 03. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. [0022] Disclosed herein is a method of generating AAV particles, the method comprising delivering to one or more cells a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein; culturing the one or more cells; and harvesting the AAV particles from the one or more producer cells.

[0023] Disclosed herein is a method of generating AAV particles, the method comprising delivering to one or more cells three plasmids, wherein the first plasmid is a helper plasmid, wherein the second plasmid is RepCap plasmid, wherein the second plasmid comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, and wherein the third plasmid is the cis-plasmid or transfer plasmid; culturing the one or more cells; and harvesting the AAV particles from the one or more cells.

[0024] Disclosed herein is a method of generating AAV capsid proteins, the method comprising performing multiple rounds of evolution in one or more kidney or kidney -related models from one or more species.

[0025] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells with a disclosed AAV particle; and expressing the encoded payload. Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.

[0026] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells in a subject in need thereof with a disclosed AAV particle; and expressing the encoded payload. Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells in a subject in need thereof with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.

[0027] Disclosed herein is a method of treating a subject, the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector. Disclosed herein is a method of treating a subject, the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector. Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle comprising a payload. Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subj ect with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid. Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid, wherein the contacting step allows for expression of the encoded payload, the encoded gene of interest, or the encoded transgene in the one or more cells.

VII. BRIEF DESCRIPTION OF THE FIGURES

[0028] FIG. 1A - FIG. IE show multidimensional evolution strategy across different kidney model systems in vivo and ex vivo yields AAV.k variants. FIG. 1A is a schematic of AAV capsid library evolution in mice and pigs via intravenous dosing, on human kidney organoids, and ex vivo non-human primate kidneys following perfusion of the arterial and ureteral route. FIG. IB shows next-generation (NGS) sequencing of AAV capsid library evolved from mouse kidney. Black dots represent individual 7-mer amino acid sequences with the brown dot representing AAV9. FIG. 1C shows NGS of AAV capsid library evolved from pig kidney cortex (blue) and medulla (orange). Zoom of top right quadrant of NGS plot demonstrating enrichment of specific 7-mer amino acid sequences within the pig kidney cortex with AAV9 highlighted in brown. FIG. ID shows NGS of AAV capsid library evolved from human kidney organoids. Black dots represent individual 7-mer amino acid sequences with the brown dot representing AAV9. FIG. IE shows NGS of AAV capsid library evolved from ex vivo perfusion of non-human primate kidney via the arterial route (blue) and ureteral route (orange). Zoom of top right quadrant of NGS plot demonstrating enrichment of specific 7-mer amino acid sequences within ex vivo perfused non-human primate kidneys. AAV9 highlighted in brown. In all plots, the X-axis represents the read depth of each sequence found within the library amplified from the specified tissue with the Y-axis representing the fold change enrichment vs the starting parental library.

[0029] FIG. 2A - FIG. 21 show that AAV.kl3 and AAV.k20 transduced mouse kidneys more efficiently thank AAV9 following system administration. FIG. 2A shows that 8-week-old C57/B6 mice were injected intravenously with AAV9, AAV.kl3, or AAV.k20 packaging a self-complementary cassette encoding for mCherry driven by a chicken -beta actin hybrid (Cbh) promoter at a dose of 5el3 vg/kg. 30 days post-injection, organs were harvested. FIG. 2B show representative images of native mCherry fluorescence in mouse kidney for mock, AAV9, AAV.kl3, or AAV.k20. Scale bar = 1 mm. FIG. 2C shows representative images of native mCherry fluorescence and lotus tetragonolobus lectin (LTL) staining of proximal tubules for AAV9, AAV.kl3, or AAV.k20. Scale bar = 70 pm. FIG. 2D shows representative images for immunofluorescence labeling of SI and S2 segments of proximal tubules in mouse kidneys. Scale bar = 100 pm. FIG. 2E shows representative images for immunofluorescence of nephrin in mouse kidneys. Scale bar = 130 pm. FIG. 2F shows representative images for immunofluorescence of collecting ducts labeled by dolichos biflorus agglutinin (DBA) in mouse kidneys. Scale bar = 130 pm. FIG. 2G shows vector genome copy numbers per pg DNA for kidney were calculated by normalizing mCherry copy numbers to the total pg DNA input for qPCR quantification and plotted as log vg/pg DNA, where each dot represents an individual mouse. FIG. 2H shows vector genome copy numbers per pg DNA for liver were calculated by normalizing mCherry copy numbers to the total pg DNA input for qPCR quantification and plotted as log vg/pg DNA, where each dot represents an individual mouse. FIG. 21 shows the quantification of native mCherry fluorescence intensity normalized to LTL signal in kidney for AAV.kl3 compared to AAV9 (p < 0.0063) and AAV.k20 compared to AAV9 (p < 0.0001) and fold change is listed above significance. Statistical significance was determined by one-way ANOVA with Tukey’s post-test for mCherry quantification analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns is not significant.

[0030] FIG. 3A - FIG. 3H show that AAV.kl3 and AAV.k20 transduced 2D and 3D human kidney organoids more efficiently than AAV9. FIG. 3A shows representative images for immunofluorescence labeling of mCherry and proximal tubules with Lrp2 in 2D human kidney organoids transduced at lelO vg/well with AAV9, AAV.kl3, or AAV.k20 packaging a self- complementary cassette encoding for mCherry driven by a chicken-beta actin hybrid (Cbh) promoter. Scale bar is 100 pm. FIG. 3B shows a schematic of 3D human kidney organoids that were differentiated and transduced with lei 1 vg/well or lel2 vg/well of AAV at Day 14. Five (5) days post-transduction, organoids were harvested for cryo-sectioning and DNA/RNA analysis. FIG. 3C shows an assay analyzing the uptake of selected AAVs in human kidney organoids at lei 1 vg/well (left) or lel2 vg/well (right). Vector genome copy number per pg DNA for organoids for both transduction assays were calculated by normalizing mCherry copy numbers to the total pg DNA input for qPCR quantification and plotted as log vg/pg DNA. Each dot is representative of a single organoid. FIG. 3D shows an assay analyzing gene expression of selected AAVs in human kidney organoids at lei 1 vg/well (left) or lel2 vg/well (right). mRNA levels for mCherry were normalized to the house keeping gene RPL13A from a RT-qPCR quantification. FIG. 3E provides representative images for immunofluorescence labeling of mCherry in human kidney organoids transduced at lei 1 vg/well with AAV9, AAV.kl3, or AAV.k20. Scale bar = 100 pm. FIG. 3F shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signal in human kidney organoids transduced with AAV9, AAV.kl3, or AAV.k20 at lei 1 vg/well. The comparison of AAV.kl3 to AAV9 was significant (p < 0.0445) as was the comparison of AAV.k20 to AAV9 (p < 0.0175). The calculated fold change in transduction between capsids are listed above significance. FIG. 3G shows quantification of mCherry immunofluorescence intensity normalized to DAPI signal in human kidney organoids transduced with AAV9, AAV.kl3, or AAV.k20 at lel2 vg/well. The comparison of AAV.kl3 to AAV9 was not significant (ns) but the comparison of AAV.k20 to AAV9 was significant (p < 0.0053). The calculated fold change in transduction between capsids are listed above significance. FIG. 3H provides representative images for immunofluorescence labeling of mCherry and proximal tubules with LTL in 3D human kidney organoids transduced at lel2 vg/well with AAV9, AAV.kl3, or AAV.k20 packaging a self-complementary cassette encoding for mCherry driven by a chicken-beta actin hybrid (Cbh) promoter. Scale bar is 130 pm. Bottom row of dashed boxes are zoomed-in areas focusing on localization of mCherry in proximal tubules. Scale bar is 30 pm. Statistical significance was determined by one-way ANOVA with Dunnett’s post-test for mCherry quantification analysis comparison to AAV9. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns is not significant.

[0031] FIG. 4A - FIG. 4D show that AAV.kl3 and AAV.k20 transduced transplanted pig kidneys more efficiently than AAV9. FIG. 4A (left) shows a schematic of a nephrectomy was performed in a 50 kg pig. Briefly, the kidney graft was flushed with cold preservation solution and stored on ice. During the cold storage period, 5.9el2 vg of AAV.kl3-scCbh-mCherry was administered for 2 hours via the renal artery and 5.9el2 vg was administered via the ureteral route. Following static cold storage, kidneys were transplanted back into the same pig and kidneys were recovered 14 days post-transplant. Immunohistochemistry (H4C) of mCherry was performed on biopsies of pig kidney tissue, where positive signal is stained brown. FIG. 4A (right) shows representative H4C images with proximal tubules, distal tubules and glomeruli labelled. FIG. 4B (left) shows that a nephrectomy was performed in a 48.9 kg pig, and that the ex vivo machine perfusion was initiated for 2 hours. Then, 6. Iel2 vg of AAV.k20-scCbh- mCherry was perfused for 2 hours via the arterial route and 6.1el2 vg was administered retrograde via the ureteral route. F oilowing machine perfusion, kidneys were transplanted back into the same pig and kidneys were recovered 9 days post-transplant. FIG. 4B (right) shows representative IHC images have proximal tubules, distal tubules and glomeruli labelled. FIG. 4C (left) shows that a nephrectomy was performed in a 42 kg pig, and that the ex vivo machine perfusion was initiated. In this experiment, 7.5el2 vg of AAV.k20-scCbh-mCherry was administered for 2 hours via the ureteral route. Following machine perfusion, kidneys were transplanted into the same pig and kidneys were recovered 9 days post-transplant. FIG. 4C (right) shows representative IHC images have proximal tubules, distal tubules and glomeruli labelled. FIG. 4D (left) shows that a nephrectomy was performed in pigs weighing between 40-50 kg, and that the machine perfusion was initiated for 2 hours. A total of 7el2 vg of AAV9, AAV.kl3, or AAV.k20 packaging scCbh-mCherry was administered for 2 hours via the ureteral route. Following machine perfusion, kidneys were transplanted into the same pig and kidneys were recovered 9 days post-transplant. FIG. 4D (right) shows representative IHC images from AAV9, AAV.kl3, and AAV.k20 follow transplantation and recovery of the kidneys.

[0032] FIG. 5A shows the NGS of AAV capsid library evolved from pig kidney cortex (blue) and medulla (orange) was analyzed for unique sequences, compared, and plotted as a Venn diagram. Pig kidney cortex had 12,304 unique sequences and pig kidney medulla had 10,068 unique sequences. There were 1138 overlapping sequences. FIG. 5B shows that consensus motif analysis of the top 100 enriched AAV for pig cortex and medulla evolution, for residues 452 - 458 (VP1 numbering). FIG. 5C shows the amplicon-EZ sequencing of AAV capsid library evolved from nonhuman primate kidney perfusion via arterial (blue) and ureteral (orange) was analyzed for unique sequences, compared, and plotted as a Venn diagram. The arterial route had 4,552 unique sequences while the ureteral route had 4,711 unique sequences. There were 5,913 overlapping sequences. FIG. 5D shows the consensus motif analysis of the top 100 enriched AAV for ex vivo NHP arterial and ureteral evolution, for residues 452 - 458 (VP1 numbering).

[0033] FIG. 6A shows the analysis of amino acid prevalence for the parental and evolved libraries at each individual position in VR-IV (452-458). Libraries are plotted against amino acids, where the least and most enriched residues are blue and red, respectively, while the 50th percentile is white. FIG. 6B shows the analysis of amino acid prevalence for all positions in VR-IV (452-458) of parental and each evolved library. Positions in VR-IV (452-458) are plotted against amino acids for parental and each evolved library, where the least and most enriched residues are blue and red, respectively, while the 50th percentile is white. [0034] FIG. 7 shows the comparison of multiple recombinant AAV9, AAV.kl3, and AAV.k20 production yields for adherent and suspension systems. Purified yields are plotted as vector genomes per liter of media. Each symbol and color combination represents a different transgene cassette used for the production. Blue triangle represents a self-complementary Cbh- mCherry cassette, orange circle represents a self-complementary Cbh-PDLl cassette, and a purple square represents a single-stranded CBA-luciferase cassette. Statistical significance was determined by one-way ANOVA with Tukey’s post-test for analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns is not significant.

[0035] FIG. 8 shows additional representative images for immunofluorescence labeling of mCherry and collecting ducts with DBA in mouse kidneys. Scale bar = 130 pm.

[0036] FIG. 9A shows RNA was extracted from undifferentiated iPSCs and human kidney organoids across different experimental conditions. RT-qPCR was performed for OCT4 mRNA levels, a marker expressed in stem cells, and was normalized to human beta-actin. FIG. 9B shows additional representative images for immunofluorescence labeling of mCherry and proximal tubules with LTL in human kidney organoids transduced at lel2 vg/well with AAV9, AAV.kl3, or AAV.k20 packaging a self-complementary Cbh-mCherry. Scale bar = 130 pm. [0037] FIG. 10A - FIG. 10B show TH1 cells transduced at MOI with respective AAV capsid packaging self-complementary Cbh-mCherry cassette @ MOI of 500K. Cells seeded at 2E5 cells/well in 24 well plate and imaged at 18 hours post-transduction and 4 days post transduction.

[0038] FIG. 11A - FIG. 11B shows cells that were seeded at 2E5 cells/well in 24 well plate and a luciferase assay was performed 24 hours post-transduction. Cells were transduced at MOI with respective AAV capsid packaging single-stranded CB A-Luciferase cassette @ MOI of 500K.

[0039] FIG. 12 shows that kidney organoids were transduced at lei 1 vg/well with respective AAV capsid packaging a single- stranded CBA-Luciferase cassette. AAV.k20 outperformed transduction in human kidney organoids compared to AAV.

[0040] FIG. 13A - FIG. 13C showed the ability of AAV.k20 to effectively transduce nonhuman primate kidneys. Here, FIG. 13A shows the location of the 10 biopsies taken from the left and right kidneys. FIG. 13B the transduction efficiency of AAV.k20 following in situ delivery. FIG. 13C shows the distribution of AAV.k20 viral genomes.

[0041] FIG. 14A - FIG. 14F show biodistribution of various AAV9, AAV.kl3, and AAV.k20 following IV injection in mice (FIG. 14A - FIG. 14C) accompanied by a luciferase assay for each of these AAVs (FIG. 14D - FIG. 14F). [0042] FIG. 15A - FIG. 15F show the ability of AAV.k20 to effectively transduce nonhuman primate kidneys. Here, FIG. 15A shows the sites of 10 biopsies of the transduced kidney. FIG. 15B - FIG. 15C show the biodistribution of the AAV.k20 viral genomes, the transduction efficiency of AAV.k20 following in situ delivery. FIG. 15D - FIG. 15E show mCherry expression as assessed by qPCR. FIG. 15F is a western blot for vinculin (a housekeeping gene) and mCherry (the transgene of interests), demonstrating that the varying degree of mCherry correlated with mCherry RNA levels for those biopsies.

VIII. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present disclosure describes nucleic acid molecules, viral vectors, viral capsid proteins, viral particles, plasmids, cells, kits, pharmaceutical formulations, and compositions thereof and methods of using the disclosed compositions. It is to be understood that the inventive aspects of which are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0044] All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

A. DEFINITIONS

[0045] Before the present compounds, compositions, articles, systems, devices, vectors, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, example methods and materials are now described.

[0046] This disclosure describes inventive concepts with reference to specific examples. However, the intent is to cover all modifications, equivalents, and alternatives of the inventive concepts that are consistent with this disclosure. [0047] As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0048] The phrase “consisting essentially of’ limits the scope of a claim to the recited components in a composition or the recited steps in a method as well as those that do not materially affect the basic and novel characteristic or characteristics of the claimed composition or claimed method. The phrase “consisting of’ excludes any component, step, or element that is not recited in the claim. The phrase “comprising” is synonymous with “including”, “containing”, or “characterized by”, and is inclusive or open-ended. “Comprising” does not exclude additional, unrecited components or steps. -

[0049] In an aspect, when referring to any numerical value, the term “about” means a value falling within a range that is ± 10% of the stated value.

[0050] Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0051] In an aspect, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In an aspect, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value unless otherwise stated or otherwise evident from the context.

[0052] In an aspect, the term “in vitro” refers to events or experiments that occur in an artificial environment, e.g., in a petri dish, test tube, cell culture, etc., rather than within a multicellular organism. In an aspect, the term “in vivo” refers to events or experiments that occur within a multicellular organism. [0053] In an aspect, the term “comparable” in the context of a particular value and a reference value means that the particular value is consistent with the reference value, or that the deviation from the reference value (above or below) is at most 10%.

[0054] References in the specification and concluding claims to parts by weight of a particular element or component in a composition denotes the weight relationship between the element or component and any other elements or components in the composition or article for which a part by weight is expressed. Thus, in a compound containing 2 parts by weight component X and 5 parts by weight component Y, X and Y are present at a weight ratio of 2:5, and are present in such ratio regardless of whether additional components are contained in the compound.

[0055] In an aspect, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. In an aspect, a disclosed method can optionally comprise one or more additional steps, such as, for example, repeating an administering step or altering an administering step.

[0056] In an aspect, the term “subject” refers to the target of administration. In an aspect, a subject can be a human being. The term “subject” includes domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, fruit fly, etc.). Thus, the subject of the herein disclosed methods can be a vertebrate, such as a mammal, a fish, a bird, a reptile, or an amphibian. Alternatively, the subject of the herein disclosed methods can be a human, non-human primate, horse, pig, rabbit, dog, sheep, goat, cow, cat, guinea pig, or rodent. The term does not denote a particular age or sex, and thus, adult and child subjects, as well as fetuses, whether male or female, are intended to be covered. In an aspect, a subject can be a human patient. In an aspect, a subject can have a disease, a disorder, an infection, a symptom, and/or a complication, be suspected of having a disease, a disease, a disorder, an infection, a symptom, and/or a complication, or be at risk of developing a disease, a disorder, an infection, a symptom, and/or a complication. For example, a subject can have risk factors for developing a disease, a disorder, an infection, a symptom, and/or a complication. A subject can be at risk due to genetic predisposition, employment type (e.g., a health care worker, a miner), attendance at a specific location (e.g., school), attendance at social events (e.g., sporting events, concerns, religious services, political rallies and events, social justice rallies, marches, and events, etc.), by use of public transportation or public services, exposure to natural and man-made disasters (e.g., Chernobyl, 9/11 attacks, etc.). [0057] In an aspect, a subject can have one or more kidney diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0058] In an aspect, a subject can have a genetic disorder. In an aspect, a subject can have a missing, deficient, and/or mutant protein or enzyme can be encoded by NPHSL NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COI.4A4, PKD1, PKD2, PKHD1. DZIP1L, or any combination thereof.

[0059] In an aspect, “effective amount” and “amount effective” can refer to an amount that is sufficient to achieve the desired result such as, for example, the treatment and/or prevention of a disease, a disorder, an infection, a symptom, and/or a complication, or a suspected disease, disorder, infection, symptom, and/or complication (related to, for example, to a kidney disease and/or kidney disorder). In an aspect, an effective amount can alleviate and/or improve one or more symptoms and/or complications associated with a kidney disease and/or kidney disorder. In an aspect, a “therapeutically effective amount” refers to an amount (i.e., vector genome / body weight or vg/kg) that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms, but is generally insufficient to cause adverse side effects. In an aspect, “therapeutically effective amount” means an amount of a disclosed composition that (i) treats the particular disease, disorder, and/or infection, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, and/or disorder, or (iii) delays the onset of one or more symptoms of the particular disease, condition, and/or disorder described herein. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed; the duration of the treatment; drugs used in combination or coincidental with a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions employed, and other like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of a disclosed composition and/or a pharmaceutical preparation comprising one or more disclosed composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, then the effective daily dose can be divided into multiple doses for purposes of administration. Consequently, a single dose of a disclosed compositions and/or a pharmaceutical preparation comprising one or more disclosed compositions, or methods can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. In further various aspects, a preparation can be administered in a “prophylactically effective amount”; that is, an amount effective for prevention of a disease, a disorder, an infection, a symptom, and/or a complication. [0060] In an aspect, “control” refers a standard or reference condition, against which results are compared. In an aspect, a control is used at the same time as a test variable or subject to provide a comparison. In an aspect, a control is a historical control that has been performed previously, a result or amount that has been previously known, or an otherwise existing record. A control can be a positive or negative control.

[0061] In an aspect, the term “diagnosed” means having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a disease, a disorder, an infection, a symptom, and/or a complication (related to, for example, a kidney disease and/or kidney disorder that can be diagnosed or treated by one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods. For example, “suspected of having” can mean having been subjected to a physical examination by a person of skill, for example, a physician, and found to have a condition (e.g., a kidney disease and/or kidney disorder) that can likely be treated by one or more of the disclosed capsid proteins, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods.

[0062] The words “treat” or “treating” or “treatment” refer to therapeutic or medical treatment wherein the object is to slow down (lessen), ameliorate, and/or diminish an undesired physiological change, disease, pathological condition, or disorder in a subject. In an aspect, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Treatment may not necessarily result in the complete clearance of an infection but may reduce or minimize complications, the side effects, and/or the progression of a disease, a disorder, an infection, a symptom, and/or a complication. The success or otherwise of treatment can be monitored by physical examination of the subject as well as cytopathological, DNA, and/or mRNA detection techniques. The words “treat” or “treating” or “treatment” include palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., a human), and includes: (i) preventing the undesired physiological change, disease, pathological condition, or disorder from occurring in a subject that can be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the physiological change, disease, pathological condition, or disorder, i.e., arresting its development; or (iii) relieving the physiological change, disease, pathological condition, or disorder, i.e., causing regression of the disease. For example, in an aspect, treating an infection can reduce the severity of an established infection in a subject by l%-100% as compared to a control (such as, for example, a subject not having the disease, the disorder, the infection, the symptom, and/or the complication. In an aspect, treating can refer to a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease, disorder, infection, symptom, and/or complication. In an aspect, treating can refer to 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction of one or more symptoms. It is understood that treatment does not necessarily refer to a cure or complete ablation or eradication of the disease, disorder, infection, symptom, and/or complication. However, in an aspect, treatment can refer to a cure or complete ablation or eradication of the disease, disorder, infection, symptom, and/or complication. [0063] Methods and techniques to monitor a subject’s response to a disclosed method can comprise qualitative (or subjective) means as well as quantitative (or objective) means. In an aspect, qualitative means (or subjective means) can comprise a subject’s own perspective. For example, a subject can report how he/she is feeling, whether he/she has experienced improvements and/or setbacks, whether he/she has experienced an amelioration or an intensification of one or more symptoms, or a combination thereof. In an aspect, quantitative means (or objective means) can comprise methods and techniques that include, but are not limited to, the following: (i) fluid analysis (e.g., tests of a subject’s fluids including but not limited to aqueous humor and vitreous humor, bile, blood, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal drainage and phlegm), peritoneal fluid, pleural fluid, saliva, sebum (skin oil), semen, sweat, synovial fluid, tears, vaginal secretion, vomit, and urine), (ii) imaging (e.g., ordinary x-rays, ultrasonography, radioisotope (nuclear) scanning, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and angiography), (iii) endoscopy (e.g., laryngoscopy, bronchoscopy, esophagoscopy, gastroscopy, GI endoscopy, coloscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy), (iv) analysis of organ activity (e.g., electrocardiography (ECG), electroencephalography (EEG), and pulse oximetry), (v) biopsy (e.g., removal of tissue samples for microscopic evaluation), and (vi) genetic testing.

[0064] A “patient” refers to a subject afflicted with a disease, disorder, infection, symptom, and/or complication (e.g., a kidney disease and/or kidney disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having a disease, disorder, infection, symptom, and/or complication (relating to, for example, a kidney disease and/or kidney disorder). In an aspect, a patient can refer to a subject that has been diagnosed with or is suspected of having an established disease, disorder, infection, symptom, and/or complication and is seeking treatment or receiving treatment.

[0065] In an aspect, the term “prevent” or “preventing” or “prevention” refers to precluding, averting, obviating, forestalling, stopping, or hindering something from happening, especially by advance action. It is understood that where reduce, inhibit, or prevent are used herein, unless specifically indicated otherwise, the use of the other two words is also expressly disclosed. In an aspect, preventing a disease, disorder, infection, symptom, and/or complication is intended. The words “prevent” and “preventing” and “prevention” also refer to prophylactic or preventative measures for protecting or precluding a subject (e.g., having a kidney disease and/or kidney disorder) not having a given infection related complication from progressing to that complication. Individuals in which prevention is required include those who have an infection.

[0066] In an aspect, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, the following: retrograde ureteral infusion, renal arterial administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, inter utero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent. Administration can be performed by one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol can be employed with a kidney (or part thereof) obtained for a subj ect. In an aspect, a kidney can be obtained from a donor subject and can be subjected to an ex vivo perfusion protocol prior to implantation into a subject in need thereof. In an aspect, a kidney can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion protocol, and can be returned to the subj ect in need thereof.

[0067] In an aspect, the term “ex vivo” can refer generally to activities that take place outside an organism or subject such as experimentation, modification, differentiation, manipulation, and/or measurement done in or on living tissue in an artificial environment outside the organism. In an aspect, ex vivo experimentation, ex vivo modification, ex vivo differentiation, ex vivo manipulation, and/or ex vivo measurement can occur with a minimum alteration of the natural conditions. In an aspect, “ex vivo” can comprise living cells, tissues, or organs (e.g., kidney or kidney -related) taken from a subject in need thereof or a donor subject and cultured and/or maintained and/or perfused in a laboratory apparatus, usually under sterile conditions, and typically for a limited duration of time (e.g., a few hours or up to about 24 hours, up to about 48 hours, up to about 72 hours, up to about 96 hours, up to about 120 hours, up to about 144 hours, up to about 168 hours, or more depending on the circumstances and/or the desired characteristics. In an aspect, tissues, cells, or organs can be collected, frozen, and later thawed for ex vivo treatment. [0068] In an aspect, the term “perfuse” or “perfusion” can refer to the act of forcing a fluid to flow through the lumen of a hollow structure, e.g., forcing a fluid to flow from an artery or other blood vessel supplying a vascular bed of a tissue through the vascular bed of the tissue. In an aspect, a disclosed perfusate can be a bodily fluid, preservation solution, or any other fluid suitable for perfusion of tissue, grafts, or organs. A disclosed bodily fluid can be blood, lymph, plasma, serum, cerebral spinal fluid, urine or any other bodily fluid. A disclosed preservation solution can be any organ preservation solution, including, but not limited to, a solution, a saline, or machine perfusion solution. In an aspect, a disclosed fluid can comprise a disclosed AAV vector.

[0069] In an aspect, “modifying the method” can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration, or by changing the duration of time of administration or between administrations to a subject.

[0070] In an aspect, “concurrently” means (1) simultaneously in time, or (2) at different times during the course of a common treatment schedule.

[0071] The term “contacting” can refer to bringing one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed AAV vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, or any combination thereof with a target area or intended target area (e.g., one or more parts and/or regions of a kidney) in such a manner that the one or more of the disclosed capsid proteins, the disclosed AAV particles, the disclosed AAV vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, or any combination thereof can exert an effect on the intended target or targeted area either directly or indirectly.

[0072] In an aspect, “determining” can refer to measuring or ascertaining the presence and severity of a disease, disorder, infection, symptom, and/or complication (e.g., relating to a kidney disease and/or kidney disorder). Methods and techniques used to determining the presence and/or severity of a disease, disorder, infection, symptom, and/or complication are typically known to the medical arts. For example, the art is familiar with the ways to identify and/or diagnose the presence, severity, or both of a disease, disorder, infection, symptom, and/or complication. [0073] In an aspect, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. In an aspect, a pharmaceutical carrier employed can be a solid, liquid, or gas. In an aspect, examples of solid carriers can include lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, and stearic acid. In an aspect, examples of liquid carriers can include sugar syrup, peanut oil, olive oil, and water. In an aspect, examples of gaseous carriers can include carbon dioxide and nitrogen. In preparing a disclosed composition for oral dosage form, any convenient pharmaceutical media can be employed. For example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like can be used to form oral liquid preparations such as suspensions, elixirs and solutions; while carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like can be used to form oral solid preparations such as powders, capsules and tablets. Because of their ease of administration, tablets and capsules are the preferred oral dosage units whereby solid pharmaceutical carriers are employed. Optionally, tablets can be coated by standard aqueous or nonaqueous techniques. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid and the like. It can also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents, such as aluminum monostearate and gelatin, which delay absorption. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters) and poly(anhydrides). Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacterial -retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable media just prior to use. Suitable inert carriers can include sugars such as lactose. Desirably, at least 95% by weight of the particles of the active ingredient have an effective particle size in the range of 0.01 to 10 micrometers.

[0074] In an aspect, the term “derivative” or “variant” refers to a compound having a structure derived from the structure of a parental compound (such as, e.g., a polypeptide having the sequence set forth in any of SEQ ID NO:24 - SEQ ID NO:43 or a nucleic acid having the sequence set forth in any of SEQ ID NO: 171 - SEQ ID NO: 190) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds.

[0075] In an aspect, disclosed AAV genomes or AAV vectors, when present in a suitable producer cell and in the presence of AAV Rep and Cap proteins, can replicate and package into AAV viral particles, particularly infectious viral particles.

[0076] In an aspect, “genome particles (gp),” “genome equivalents,” or “genome copies” can refer to a viral titer or the number of virions containing the AAV DNA genome, regardless of infectivity or functionality.

[0077] In an aspect, “adeno- associated virus” or “AAV” refers to a viral particle consisting of at least one AAV capsid protein VP1, VP2, and/or VP3, preferably all three capsid proteins, and an encapsidated polynucleotide AAV genome or AAV vector. A disclosed AAV can typically be a recombinant AAV. An AAV can be a non-naturally occurring AAV. The AAV can comprise one or more heterologous polynucleotides, i.e., polynucleotides other than wildtype AAV polynucleotides, such as transgenes. An example of a transgene is a therapeutic gene.

[0078] In an aspect, a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect on the cell or tissue in which it is present, or on a mammal in which the gene is expressed. Examples of beneficial effects include amelioration of a sign or symptom of a condition or disease, prevention or inhibition of a condition or disease, or conferral of a desired characteristic. Therapeutic genes include, but are not limited to, genes that correct a genetic deficiency in a cell or mammal. In an aspect, a therapeutic gene can be NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I. MX IB, SMARCAL1, C0Q2, PDSS2, MTTL1, SCARB2, FN1, CO/.4A5, CO/.4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP IL.

[0079] In an aspect, “transfer plasmid” or “pTransfer” contains the viral genome. The pTransfer further comprises two ITRs, a transgene, gene of interest, heterologous nucleic acid, and/or payload, a promoter, and one or more cis-regulatory elements (e.g., Lox sites, WPRE, poly A, etc.).

[0080] In an aspect, an “AAV inverted terminal repeat (ITR)” sequence” or “ITR” can comprise an approximately 145 -nucleotide sequence that is present at both termini of the native single- stranded AAV genome.

[0081] In an aspect, a “transgene” is a polynucleotide encoding a gene that is delivered to a cell by a disclosed AAV vector.

[0082] In an aspect, a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product after being transcribed, and sometimes also translated. The term “gene” or “coding sequence” refers to a nucleotide sequence in vitro or in vivo that encodes a gene product. In some instances, the gene consists or consists essentially of coding sequence, that is, sequence that encodes the gene product. In other instances, the gene comprises additional, non-coding, sequence that permits, facilitates or directs the cellular expression machinery to express the encoded product. Such sequences can include, but are not limited to promoters, enhancers, transcriptional termination and/or poly(A) addition signals, and elements that affect transcript processing and/or stability. A gene may or may not include regions preceding and following the coding region, e.g., 5’ untranslated (5’ UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0083] A “gene product” is a molecule resulting from expression of a particular gene or fragment thereof. Gene products can include, for example, a polypeptide, an aptamer, an interfering RNA, an mRNA, and the like. A “gene product” can be a polypeptide, peptide, protein or interfering RNA including short interfering RNA (siRNA), miRNA or small hairpin RNA (shRNA). In an aspect, a disclosed gene product can be a therapeutic gene product, e.g., a therapeutic protein or a therapeutic RNA (e.g., an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA).

[0084] AAV can be replication competent or replication incompetent. By “replication competent” is meant that the virus or viral particle is infectious and capable of replication in a suitable infected cell. In an aspect, the disclosed AAV can be replication-incompetent. [0085] In an aspect, the term “viral vector” refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral particle. The viral vector can contain a nucleic acid (e.g., a transgene, a gene of interest, and/or a payload) encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle can be utilized for the purpose of transferring nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0086] In an aspect, an “AAV virus” or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein such as VP1 (typically by all of the capsid proteins of a wild- type AAV) and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as a “recombinant AAV vector particle” or simply a “AAV vector”. Thus, production of an AAV particle necessarily includes production of an AAV vector, as such a vector is contained within an AAV particle.

[0087] In an aspect, “viral capsid polypeptide” refers to the proteinaceous shell or coat of a viral particle. A viral capsid polypeptide permits packaging or assembly of the capsid polypeptide into a viral particle that is competent for delivery of nucleic acid to the host cell. Capsids function to encapsidate, protect, transport, and release into a host cell a viral genome. Capsids are generally comprised of oligomeric structural subunits of a polypeptide of the viral capsid polypeptides.

[0088] In an aspect, “encapsidated” means enclosed within a viral capsid. As an example, the AAV genome comprises three overlapping sequences which encode capsid proteins, VP1, VP2 and VP3, which start from one promoter, p40. The AAV capsid is composed of a mixture of VP1, VP2, and VP3 totaling 60 monomers arranged in icosahedral symmetry in a ratio of 1 : 1 : 10.

[0089] In an aspect, “packaging” refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle.

[0090] In an aspect, “payload” refers to a nucleic acid that is encapsidated within a viral vector, e.g., an AAV vector. A payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA. In an aspect, a “payload” refers to one or more polynucleotides or polynucleotide regions encoded by or within a viral genome or an expression product of such polynucleotide or polynucleotide region, e.g., a transgene, a polynucleotide encoding a polypeptide or multi-polypeptide or a modulatory nucleic acid or regulatory nucleic acid. In an aspect, a disclosed payload can comprise any nucleic acid that is useful for modulating the expression in a target cell transduced or contacted with the AAV particle carrying the payload. In an aspect, modulation can be by supplementation of the payload in a target cell or tissue. In an aspect, modulation can be gene replacement of the payload in a target cell or tissue. In an aspect, modulation can be by inhibition using a modulatory nucleic acid of the payload in a target cell or tissue. In an aspect, a disclosed payload can comprise a combination of coding and non-coding nucleic acid sequences, and can be codon-optimized. In an aspect, a payload can comprise one or more regulatable elements. In an aspect, a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload. In an aspect, a disclosed payload can encode a gene therapy product. A gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In an aspect, a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated. In an aspect, a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product.

[0091] In an aspect, the term “polypeptide” refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide can be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a nonpolypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide.” Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology or synthesized through chemical means such as conventional solid phase peptide synthesis, etc. In an aspect, the term “polypeptide sequence” or “amino acid sequence” can refer to the polypeptide material itself and/or to the sequence information (i.e., the succession of letters or three letter codes used as abbreviations for amino acid names) that biochemically characterizes a polypeptide. A polypeptide sequence presented herein is presented in an N-terminal to C-terminal direction unless otherwise indicated.

[0092] A variant amino acid or DNA sequence can be at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose, e.g., that are freely available on the world wide web (e.g., BLASTp or BLASTn with default settings). Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites permitting ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are well established. Any cysteine residue not involved in maintaining the proper conformation of a polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to a polypeptide to improve its stability or facilitate oligomerization.

[0093] In an aspect, “polynucleotide sequence” can refer to the polynucleotide material itself and/or to the sequence information (i.e., the succession of letters used as abbreviations for bases) that biochemically characterizes a specific nucleic acid. A polynucleotide sequence presented herein is presented in a 5’ to 3’ direction unless otherwise indicated.

[0094] In an aspect, the term “corresponding to,” when used in reference to an amino acid or polynucleotide sequence means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has structural properties, functional properties, or both that are similar relative to an amino acid or polynucleotide sequence in a similar location in another polypeptide or polynucleotide molecule. Homologues of a given polypeptide in different species “correspond to” each other, as do regions or domains of homologous polypeptides from different species. Similarly, capsid polypeptides of different serotypes of viral vectors, including but not limited to adeno-associated virus (AAV) vectors, “correspond to” each other, as do regions of such polypeptides, defined, for example by alignment of their amino acid sequences. While other alignment parameters can be used to define such regions, for the avoidance of doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) using default parameters.

[0095] In an aspect, “promoter” or “promoters” are known to the art. Depending on the level and tissue-specific expression desired, a variety of promoter elements can be used. A promoter can be tissue-specific or ubiquitous and can be constitutive or inducible, depending on the pattern of the gene expression desired. A promoter can be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0096] “Tissue-specific promoters” are known to the art and include, but are not limited to, neuron-specific promoters, kidney specific promoters, muscle-specific promoters, liverspecific promoters, skeletal muscle-specific promoters, and heart-specific promoters.

[0097] In an aspect, a “ubiquitous/constitutive promoter” refer to a promoter that allows for continual transcription of its associated gene. A ubiquitous/constitutive promoter is always active and can be used to express genes in a wide range of cells and tissues, including, but not limited to, the liver, kidney, skeletal muscle, cardiac muscle, smooth muscle, diaphragm muscle, brain, spinal cord, endothelial cells, intestinal cells, pulmonary cells (e.g., smooth muscle or epithelium), peritoneal epithelial cells, and fibroblasts. Ubiquitous/constitutive promoters include, but are not limited to, a CMV major immediate-early enhancer/ chicken beta-actin promoter, a cytomegalovirus (CMV) major immediate-early promoter, an Elongation Factor 1-a (EFla) promoter, a simian vacuolating virus 40 (SV40) promoter, an AmpR promoter, a PyK promoter, a human ubiquitin C gene (Ubc) promoter, a MFG promoter, a human beta actin promoter, a CAG promoter, a EGR1 promoter, a FerH promoter, a FerL promoter, a GRP78 promoter, a GRP94 promoter, a HSP70 promoter, a [3-kin promoter, a murine phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, a ROSA promoter, human Ubiquitin B promoter, a Rous sarcoma virus promoter, or any other natural or synthetic ubiquitous/constitutive promoters.

[0098] In an aspect, an “inducible promoter” refers to a promoter that can be regulated by positive or negative control. Factors that can regulate an inducible promoter include, but are not limited to, chemical agents (e.g., the metallothionein promoter or a hormone inducible promoter), temperature, and light.

[0099] As used herein, an “isolated” biological component (such as a nucleic acid molecule, protein, or virus) has been substantially separated or purified away from other biological components (e.g., other chromosomal and extra-chromosomal DNA and RNA, proteins and/or organelles). Nucleic acids, proteins, and/or viruses that have been “isolated” include nucleic acids, proteins, and viruses purified by standard purification methods. The term also embraces nucleic acids, proteins, and viruses prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids or proteins. The term “isolated” (or purified) does not require absolute purity; rather, it is intended as a relative term. Thus, for example, an isolated or purified nucleic acid, protein, virus, or other active compound is one that is isolated in whole or in part from associated nucleic acids, proteins, and other contaminants. In an aspect, the term “substantially purified” refers to a nucleic acid, protein, virus or other active compound that has been isolated from a cell, cell culture medium, or other crude preparation and subjected to fractionation to remove various components of the initial preparation, such as proteins, cellular debris, and other components.

[0100] “Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as “substantially identical” or “essentially similar” when they are optimally aligned. For example, sequence similarity oridentity can be determined by searching against databases such as FASTA, BLAST, etc., but hits should be retrieved and aligned pairwise to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences can have “substantial sequence identity” if the percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more, preferably 90%, 95%, 98%, 99% or more. Such sequences are also referred to as “variants” herein, e.g., other variants of AAV capsid proteins. It should be understood that sequence with substantial sequence identity do not necessarily have the same length and may differ in length. For example, sequences that have the same nucleotide sequence but of which one has additional nucleotides on the 3’- and/or 5 ’-side are 100% identical.

[0101] A “codon-optimized” nucleic acid refers to a nucleic acid sequence that has been altered such that the codons are optimal for expression in a particular system (such as a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or in a particular mammalian species (such as human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

[0102] Disclosed are the components to be used to prepare one or more of the disclosed capsid proteins, the disclosed vectors, the disclosed nucleic acid molecules, the disclosed compositions thereof, the disclosed pharmaceutical formulations, and/or the disclosed methods used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds cannot be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules including the compounds are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the compositions of the invention. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific aspects or combination of aspects of the methods of the invention.

B. COMPOSITIONS

1. AAV CAPSID PROTEINS

[0103] Disclosed herein is an AAV capsid protein. Disclosed herein is an AAV capsid protein having one or more substitutions in variable region IV (VR-IV).

[0104] Disclosed herein is an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.

[0105] In an aspect, a disclosed AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:24 - SEQ ID NO:43.

[0106] In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:04. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:05. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:06. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:07. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:08. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:09. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 10. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 11. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 12. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 13. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 14. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 15. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 16. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 17. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 18. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO: 19. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:20. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:21. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:22. In an aspect, positions 452-458 of the AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:23.

[0107] In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:24. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:25. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:26. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:27. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:28. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:29. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:30. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:31. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:32. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:33. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:34. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:35. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:36. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:37. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:38. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:39. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:40. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:41. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:42. In an aspect, a disclosed AAV capsid protein can comprise the amino acid sequence of SEQ ID NO:43.

[0108] In an aspect, a disclosed AAV capsid protein can be a variant of a parental wild-type capsid protein. In an aspect, a disclosed parental wild-type capsid protein can be a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO. In an aspect, a disclosed parental wild-type capsid protein can be VP1 of AAV9. In an aspect, a disclosed parental wild-type capsid protein can be VP2 of AAV9. In an aspect, a disclosed parental wild-type capsid protein can be VP3 of AAV9.

[0109] In an aspect, a disclosed AAV capsid protein can improve gene transfer and/or expression in one or more region(s) or part(s) of kidney when compared to a disclosed parental wild-type capsid protein. In an aspect, gene transfer and/or expression can be improved at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times. In an aspect, a disclosed region or part of kidney can be adrenal glands, cortex, medulla, renal column, pyramid, renal pelvis, major calyx, minor calyx, papillae, or ureter. In an aspect, a disclosed region or part of kidney can be the proximal tubule in the cortex.

[0110] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01.

[0111] In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO: 8873. In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO:23. In an aspect of a disclosed AAV capsid protein, positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO:44 - SEQ ID NO:52.

[0112] In an aspect of a disclosed AAV capsid protein, positions 452-458 of the AAV capsid can comprise a truncated substitution such as AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0113] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q. In an aspect, the one or more disclosed amino acid substitutions can comprise substitutions other than GVSLGGG.

[0114] In an aspect of a disclosed AAV capsid protein, each of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 1 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 2 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 3 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 4 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 5 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 6 or more of positions 452 - 458 can have an amino acid substitution. In an aspect of a disclosed AAV capsid protein, 7 or more of positions 452 - 458 can have an amino acid substitution.

[0115] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:31, SEQ ID NO:35, or SEQ ID NO:43. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61. In an aspect, a disclosed AAV capsid protein is not SEQ ID NO:57.

[0116] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having one or more substitutions relative to the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72. SEQ ID NO:77, SEQ ID NO:82. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising a sequence having one or more substitutions relative to the sequence set forth in SEQ ID NO: 87, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137. For example, in an aspect, a disclosed wild-type sequence (as presented below in Table 1) has one or more substitutions in one or more regions of the protein.

TABLE 1 - SEQUENCES FOR WILD-TYPE OR NON-SUBSTITUTED AAV CAPSID PROTEINS

[0117] In an aspect, the disclosed one or more substitutions can comprise 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, or 10 substitutions. In an aspect, the disclosed one or more substitutions can comprise 7 substitutions.

[0118] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:65 or SEQ ID NO:66. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:70 or SEQ ID NO:71. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:75 or SEQ ID NO:76. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 80 or SEQ ID NO:81. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:85 or SEQ ID NO:86. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:90 or SEQ ID NO:91. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:95 or SEQ ID NO:96. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 111. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 125 or SEQ ID NO: 126. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 131. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 135 or SEQ ID NO: 136. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 140 or SEQ ID NO: 141. For example, in an aspect, a disclosed AAV capsid variant can comprise the sequence set forth below in Table 2.

TABLE 2 - SEQUENCES FOR AAV CAPSID PROTEINS

[0119] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:62 but for one or more substitutions in the region of SEQ ID NO:63 or SEQ ID NO:64. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 67 but for one or more substitutions in the region of SEQ ID NO:68 or SEQ ID NO:69. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:72 but for one or more substitutions in the region of SEQ ID NO:73 or SEQ ID NO:74. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:77 but for one or more substitutions in the region of SEQ ID NO:78 or SEQ ID NO:79. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:82 but for one or more substitutions in the region of SEQ ID NO:83 or SEQ ID NO:84. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:87 but for one or more substitutions in the region of SEQ ID NO:88 or SEQ ID NO:89. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:92 but for one or more substitutions in the region of SEQ ID NO:93 or SEQ ID NO:94. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:97 but for one or more substitutions in the region of SEQ ID NO:98 or SEQ ID NO:99. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 102 but for one or more substitutions in the region of SEQ ID NO: 103 or SEQ ID NO: 104. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 107 but for one or more substitutions in the region of SEQ ID NO:108 or SEQ ID NO: 109. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 112 but for one or more substitutions in the region of SEQ ID NO: 113 or SEQ ID NO: 114. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 117 but for one or more substitutions in the region of SEQ ID NO: 118 or SEQ ID NO: 119. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 122 but for one or more substitutions in the region of SEQ ID NO: 123 or SEQ ID NO: 124. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 127 but for one or more substitutions in the region of SEQ ID NO: 128 or SEQ ID NO: 129. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 132 but for one or more substitutions in the region of SEQ ID NO: 133 or SEQ ID NO: 134. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 137 but for one or more substitutions in the region of SEQ ID NO: 138 or SEQ ID NO: 139.

[0120] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:62 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 67 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:72 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:77 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:82 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:87 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:92 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:97 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 102 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 107 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 112 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 117 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 122 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 127 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 132 with one or more substitutions in variable region IV (VR-IV). Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO: 137 with one or more substitutions in variable region IV (VR-IV).

[0121] In an aspect, a disclosed AAV capsid protein can be used to improve and/or enhance gene transfer to one or more kidney cells, kidney-derived cell types, and/or kidney -related cell types when compared to the wild-type AAV capsid protein. In an aspect, a disclosed AAV capsid protein can be used to effect widespread transduction of one or more kidney cells, kidney-derived cell types, and/or kidney-related cell types.

[0122] In an aspect, a disclosed AAV capsid protein can be used to transduce one or more kidney cells, kidney-derived cell types, and/or kidney-related cell types more efficiently than that of the wild-type AAV capsid protein. In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0123] In an aspect, a disclosed AAV capsid protein can be used to improve and/or enhance gene transfer to any region or part of the kidney. In an aspect, a disclosed AAV capsid protein can demonstrate an improved correlation in dose-response (e.g., thereby improving efficiency). [0124] In an aspect, a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO:01. In an aspect, a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137.

[0125] In an aspect, disclosed kidney cells, kidney -derived cell types, and/or kidney-related cell types can comprise kidney epithelial cells and/or kidney endothelial cell types. In an aspect, kidney cells, kidney-derived cell types, and/or kidney-related cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof. In an aspect, a disclosed region or part of the kidney can comprise the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

[0126] In an aspect, a disclosed AAV capsid protein can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed AAV capsid protein can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0127] In an aspect, a disclosed AAV capsid protein can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subj ect’s kidney disease and/or kidney disorder. In an aspect, a disclosed AAV capsid protein can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed AAV capsid protein can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed AAV capsid protein can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0128] In an aspect, a disclosed kidney disease or disorder comprises Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof. [0129] In an aspect, a disclosed AAV capsid protein can be used to improve kidney function in the subject. In an aspect, a disclosed AAV capsid protein can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed AAV capsid protein can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed AAV capsid protein can be used to reduce the risk of developing a inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed AAV capsid protein can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney-related cell types.

[0130] In an aspect, a disclosed AAV capsid protein can be used to treat a subject in need thereof. In an aspect, a disclosed AAV capsid protein can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, a subject in need thereof can have one or more kidneys diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof. In an aspect, a disclosed AAV capsid protein can be incorporated into a disclosed AAV capsid. In an aspect, a disclosed AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed kidney cells, kidney-derived cell types, and/or kidney -related cell types). In an aspect, a disclosed AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types and/or one or more tissue types. In an aspect, a disclosed AAV capsid variant can efficiently transduce one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types. In an aspect, a disclosed AAV capsid protein can outperform the parental wild-type AAV in one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types.

[0131] In an aspect, a disclosed AAV capsid protein can be used in a method of reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed AAV capsid protein can be used in a method of improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV capsid protein can be used in a method of reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV capsid protein can be used in a method of reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV capsid protein can be used in a method of enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed AAV capsid protein can be used in a method of extending and/or improving the life expectancy of a subj ect. [0132] Disclosed herein is an AAV capsid comprising a disclosed AAV capsid protein. Disclosed herein is an AAV capsid protein, wherein the capsid protein comprises the sequence set forth in SEQ ID NO:03. Disclosed herein is an AAV capsid protein, wherein the capsid protein comprises a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is an AAV capsid protein, wherein the capsid protein comprises one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. In an aspect of a disclosed AAV capsid, positions 452 - 458 of the AAV capsid protein can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect of a disclosed AAV capsid, positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect of a disclosed AAV capsid, positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect of a disclosed AAV capsid, positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect of a disclosed AAV capsid, positions 452 - 458 of the AAV capsid protein can comprise the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO:23.

[0133] In an aspect, positions 452-458 of the AAV capsid can comprise a truncated substitution such as AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0134] Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q. Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in SEQ ID NO:31 or SEQ ID NO:35.

[0135] Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61. In an aspect, a disclosed AAV capsid protein is not SEQ ID NO:57. In an aspect of a disclosed AAV capsid, the one or more amino acid substitutions in the AAV capsid protein can comprise substitutions other than GVSLGGG (SEQ ID NO: 50). [0136] Disclosed herein is a library of AAV capsid proteins. Disclosed herein is a library of AAV capsid proteins having one or more substitutions in variable region IV (VR-IV). Disclosed herein is a library of AAV capsid proteins comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a library of AAV capsid proteins comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is a library of AAV capsid proteins comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. Disclosed herein is a library of AAV capsid proteins, wherein positions 452 - 458 relative to SEQ ID NO:01 can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23. Disclosed herein is a library of AAV capsid proteins, wherein positions 452 - 458 relative to SEQ ID NO:01 can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO: 8873. Disclosed herein is a library of AAV capsid proteins, wherein positions 452 - 458 relative to SEQ ID NO:01 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23. Disclosed herein is a library of AAV capsid proteins, wherein positions 452 - 458 relative to SEQ ID NO:01 can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO:8873. Disclosed herein is a library of AAV capsid proteins, wherein positions 452 - 458 relative to SEQ ID NO:01 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 191 - SEQ ID NO:8873. Disclosed herein is a library of AAV capsid proteins, wherein the capsid proteins comprise the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43 or SEQ ID NO:53 - SEQ ID N0:61.

2. NUCLEIC ACID MOLECULES

[0137] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed AAV capsid protein. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein having one or more substitutions in variable region IV (VR-IV).

[0138] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01. In an aspect, a disclosed encoded AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:24 - SEQ ID NO:43.

[0139] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising a sequence having one or more substitutions relative to the sequence set forth in SEQ ID NO:01.

[0140] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO: 03.

[0141] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO: 8873. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 191 - SEQ ID NO:8873.

[0142] In an aspect, the sequence set forth in SEQ ID NO: 04 can be encoded by the sequence set forth in SEQ ID NO: 151. In an aspect, the sequence set forth in SEQ ID NO:05 can be encoded by the sequence set forth in SEQ ID NO: 152. In an aspect, the sequence set forth in SEQ ID NO:06 can be encoded by the sequence set forth in SEQ ID NO: 153. In an aspect, the sequence set forth in SEQ ID NO:07 can be encoded by the sequence set forth in SEQ ID NO: 154. In an aspect, the sequence set forth in SEQ ID NO:08 can be encoded by the sequence set forth in SEQ ID NO: 155. In an aspect, the sequence set forth in SEQ ID NO:09 can be encoded by the sequence set forth in SEQ ID NO: 156. In an aspect, the sequence set forth in SEQ ID NO: 10 can be encoded by the sequence set forth in SEQ ID NO: 157. In an aspect, the sequence set forth in SEQ ID NO: 11 can be encoded by the sequence set forth in SEQ ID NO: 158. In an aspect, the sequence set forth in SEQ ID NO: 12 can be encoded by the sequence set forth in SEQ ID NO: 159. In an aspect, the sequence set forth in SEQ ID NO: 13 can be encoded by the sequence set forth in SEQ ID NO: 160. In an aspect, the sequence set forth in SEQ ID NO: 14 can be encoded by the sequence set forth in SEQ ID NO: 161. In an aspect, the sequence set forth in SEQ ID NO: 15 can be encoded by the sequence set forth in SEQ ID NO: 162. In an aspect, the sequence set forth in SEQ ID NO: 16 can be encoded by the sequence set forth in SEQ ID NO: 163. In an aspect, the sequence set forth in SEQ ID NO: 17 can be encoded by the sequence set forth in SEQ ID NO: 164. In an aspect, the sequence set forth in SEQ ID NO: 18 can be encoded by the sequence set forth in SEQ ID NO: 165. In an aspect, the sequence set forth in SEQ ID NO: 19 can be encoded by the sequence set forth in SEQ ID NO: 166. In an aspect, the sequence set forth in SEQ ID NO:20 can be encoded by the sequence set forth in SEQ ID NO: 167. In an aspect, the sequence set forth in SEQ ID NO:21 can be encoded by the sequence set forth in SEQ ID NO: 168. In an aspect, the sequence set forth in SEQ ID NO:22 can be encoded by the sequence set forth in SEQ ID NO: 169. In an aspect, the sequence set forth in SEQ ID NO:23 can be encoded by the sequence set forth in SEQ ID NO: 170. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452 - 458 can comprise the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO:23.

[0143] In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein positions 452-458 of the AAV capsid can comprise a truncated substitution such as AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0144] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising a sequence having one or more substitutions relative to the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 82, SEQ ID NO: 87, SEQ ID NO: 92, SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137. For example, in an aspect, a disclosed wildtype sequence (Table 1) has one or more substitutions in one or more regions of the protein. In an aspect, the disclosed one or more substitutions can comprise 1 substitution, 2 substitutions, 3 substitutions, 4 substitutions, 5 substitutions, 6 substitutions, 7 substitutions, 8 substitutions, 9 substitutions, or 10 substitutions. In an aspect, the disclosed one or more substitutions can comprise 7 substitutions.

[0145] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:65 or SEQ ID NO:66. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:70 or SEQ ID NOVI. Disclosed herein is an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:75 or SEQ ID NO:76. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:80 or SEQ ID NO:81. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:85 or SEQ ID NO:86. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NOVO or SEQ ID NOVI. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:95 or SEQ ID NO:96. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 105 or SEQ ID NO: 106. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 111. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 115 or SEQ ID NO: 116. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 120 or SEQ ID NO: 121. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 125 or SEQ ID NO: 126. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 130 or SEQ ID NO: 131. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 135 or SEQ ID NO: 136. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 140 or SEQ ID NO: 141. For example, in an aspect, a disclosed encoded AAV capsid variant can comprise the sequence set forth in Table 2.

[0146] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:62 but for one or more substitutions in the region of SEQ ID NO:63 or SEQ ID NO:64. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:67 but for one or more substitutions in the region of SEQ ID NO:68 or SEQ ID NO:69. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:72 but for one or more substitutions in the region of SEQ ID NO:73 or SEQ ID NO:74. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:77 but for one or more substitutions in the region of SEQ ID NO:78 or SEQ ID NO:79. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:82 but for one or more substitutions in the region of SEQ ID NO:83 or SEQ ID NO:84. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:87 but for one or more substitutions in the region of SEQ ID NO:88 or SEQ ID NO: 89. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:92 but for one or more substitutions in the region of SEQ ID NO:93 or SEQ ID NO:94. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:97 but for one or more substitutions in the region of SEQ ID NO:98 or SEQ ID NO:99. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 102 but for one or more substitutions in the region of SEQ ID NO: 103 or SEQ ID NO: 104. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 107 but for one or more substitutions in the region of SEQ ID NO: 108 or SEQ ID NO: 109. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 112 but for one or more substitutions in the region of SEQ ID NO: 113 or SEQ ID NO: 114. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 117 but for one or more substitutions in the region of SEQ ID NO: 118 or SEQ ID NO: 119. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 122 but for one or more substitutions in the region of SEQ ID NO: 123 or SEQ ID NO: 124. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 127 but for one or more substitutions in the region of SEQ ID NO: 128 or SEQ ID NO: 129. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 132 but for one or more substitutions in the region of SEQ ID NO: 133 or SEQ ID NO: 134. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 137 but for one or more substitutions in the region of SEQ ID NO: 138 or SEQ ID NO: 139.

[0147] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137, with one or more substitutions in variable region IV (VR-IV).

[0148] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q. In an aspect of a disclosed nucleic acid molecule, the one or more amino acid substitutions can comprise substitutions other than GVSLGGG (SEQ ID NO:50).

[0149] In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein each of positions 452 - 458 can have an amino acid substitution. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein, wherein 1 or more of positions 452 - 458 can have an amino acid substitution, or 2 or more of positions 452 - 458 can have an amino acid substitution, or 3 or more of positions 452 - 458 can have an amino acid substitution, 4 or more of positions 452 - 458 can have an amino acid substitution, or 5 or more of positions 452 - 458 can have an amino acid substitution, or 6 or more of positions 452 - 458 can have an amino acid substitution, or 7 or more of positions 452 - 458 can have an amino acid substitution.

[0150] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:31, SEQ ID NO:35, or SEQ ID NO:43. Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:53 - SEQ ID N0:61.

[0151] Disclosed herein is a nucleic acid molecule comprising the sequence set forth in SEQ ID NO:02, wherein the nucleotides at positions 1354-1374 comprise 7 amino acids, wherein 1 or more of the 7 amino acids has been substituted. Disclosed herein is a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 171 - SEQ ID NO: 190. Disclosed herein is a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 171 - SEQ ID NO: 190, wherein the encoded AAV capsid protein can improved and/or enhanced gene transfer to one or more kidney cells or kidney-derived cell types when compared to the wild-type capsid protein. Disclosed herein is a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 171 - SEQ ID NO: 190, wherein the encoded AAV capsid protein can demonstrate improved and/or enhanced gene transfer to any region or part of the kidney. Disclosed herein is a nucleic acid molecule comprising the sequence set forth in any one of SEQ ID NO: 171 - SEQ ID NO: 190, wherein the encoded AAV capsid protein can demonstrate an improved correlation in dose-response.

[0152] In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein that can be used to improve and/or enhance gene transfer to one or more kidney cells, kidney- derived cell types, and/or kidney -related cell types when compared to the wild-type AAV capsid protein. In an aspect, a disclosed nucleic acid molecule can be used to effect widespread transduction of one or more kidney cells, kidney-derived cell types, and/or kidney -related cell types.

[0153] In an aspect, a disclosed nucleic acid molecule can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than that of a nucleic acid molecule encoding the wild-type capsid protein. In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0154] In an aspect, a disclosed nucleic acid molecule can be used to improve and/or enhance gene transfer to any region or part of the kidney. In an aspect, a disclosed nucleic acid molecule can demonstrate an improved correlation in dose-response (e.g., thereby improving efficiency). [0155] For example, in an aspect, a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO:01. In an aspect, a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137.

[0156] In an aspect, disclosed kidney cells or kidney-derived cell types can comprise kidney epithelial cells and/or kidney endothelial cell types. In an aspect, disclosed kidney cells or kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0157] In an aspect, a disclosed region or part of the kidney can comprise the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

TABLE 3 - AMINO ACID AND NUCLEOTIDE SEQUENCES FOR SUBSTITUTIONS

[0158] In an aspect, a disclosed nucleic acid molecule can comprise the nucleotide sequence for one or more regulatory elements. For example, a disclosed regulatory element can comprise a promoter operably linked to a disclosed nucleic acid molecule, wherein the promoter drives the expression of a disclosed capsid protein, a disclosed encoded polypeptide, a disclosed encoded therapeutic agent, or both.

[0159] In an aspect, a disclosed nucleic acid molecule can comprise the sequence for at least one of polypeptide, a glycopeptide, a polysaccharide, a glycolipid, a lipid, or a nucleic acid polymer, or a combination thereof. In an aspect, a disclosed nucleic acid molecule can comprise the sequence for at least one therapeutic agent. In an aspect, a disclosed therapeutic agent can be an oligonucleotide therapeutic agent. In an aspect, a disclosed oligonucleotide therapeutic agent can be a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptidenucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed nucleic acid-based molecule can comprise one or more modifications at any position applicable. In an aspect, a disclosed therapeutic agent can comprise a CRISPR-based endonuclease (e.g., Cas9). In an aspect, a disclosed CRISPR-based endonuclease can be derived from a CRISPR/Cas type I, type II, or type III system.

[0160] In an aspect, a disclosed nucleic acid molecule can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed nucleic acid molecule can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0161] In an aspect, a disclosed nucleic acid molecule can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s kidney disease and/or kidney disorder. In an aspect, a disclosed nucleic acid molecule can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed nucleic acid molecule can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed nucleic acid molecule can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0162] In an aspect, a disclosed kidney disease or disorder comprises Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0163] In an aspect, a disclosed nucleic acid molecule can be used to improve kidney function in the subject. In an aspect, a disclosed nucleic acid molecule can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed nucleic acid molecule can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed nucleic acid molecule can be used to reduce the risk of developing inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed nucleic acid molecule can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney -related cell types.

[0164] In an aspect, a disclosed nucleic acid molecule can be used to treat a subject in need thereof. In an aspect, a disclosed nucleic acid molecule can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, a subject in need thereof can have one or more kidneys diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof. [0165] In an aspect, a gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In an aspect, a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated. In an aspect, a disclosed payload nucleic acid can encode atransgene having a beneficial or desirable gene product.

[0166] In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein can be incorporated into a disclosed AAV capsid. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types). In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types and/or one or more tissue types. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid variant that can efficiently transduce one or more disclosed kidney cells, kidney-derived cell types, and/or kidney -related cell types. In an aspect, a disclosed nucleic acid molecule can encode an AAV capsid protein that can outperform the parental wild-type AAV in one or more disclosed kidney cells, kidney-derived cell types, and/or kidney -related cell types.

In an aspect, a disclosed nucleic acid molecule can be formulated for administration via one or more routes. Such routes are well known to those skilled in the art and include, but are not limited to, the following: retrograde ureteral infusion, renal arterial administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, inter utero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration of a disclosed nucleic acid molecule can be continuous or intermittent. In an aspect, a disclosed nucleic acid molecule can be administered via one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol employing a disclosed nucleic acid molecule can be employed with a kidney (or part thereof) obtained for a subject. In an aspect, a kidney can be obtained from a donor subject and can be subjected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule prior to implantation into a subject in need thereof. In an aspect, a kidney can be obtained from the subj ect in need thereof, can be subj ected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, and can be returned to the subject in need thereof. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule can be applied to other relevant tissues in the subject in need thereof.

[0167] In an aspect, a disclosed nucleic acid molecule can be used in a method of reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed nucleic acid molecule can be used in a method of improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed nucleic acid molecule can be used in a method of reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed nucleic acid molecule can be used in a method of reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, disclosed nucleic acid molecule can be used in a method of enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed nucleic acid molecule can be used to extend and/or improve the life expectancy of a subject.

3. CELLS

[0168] Disclosed herein is a cell comprising a disclosed nucleic acid molecule. Disclosed herein is a cell comprising a disclosed AAV having a capsid comprising a capsid protein. Disclosed herein is a cell comprising a disclosed capsid having a disclosed variant. Disclosed herein is a cell comprising a disclosed viral vector. Disclosed herein is a cell comprising an AAV vector particle. Disclosed herein are producer cells capable of generating AAV having a disclosed AAV capsid variant. Disclosed herein are cells used to perform and/or effect a disclosed method. Disclosed herein are cells used to perform and/or effect a disclosed method of directed evolution of the AAV capsid protein. Disclosed herein are cells used to perform and/or effect a disclosed method of generating AAV particles. Disclosed herein are cells used to perform and/or effect a disclosed method of delivering a payload. Disclosed herein are cells used to perform and/or effect a disclosed method of treating a subject.

4. VECTORS AND PARTICLES

[0169] Disclosed herein is a vector comprising a disclosed nucleic acid molecule. Disclosed herein is a vector comprising a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein.

[0170] Disclosed herein is an AAV vector comprising a gene of interest and a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01. In an aspect of a disclosed AAV vector, an encoded AAV capsid protein can comprise an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:24 - SEQ ID NO:43.

[0171] Disclosed herein is an AAV vector comprising a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein. Disclosed herein is an AAV vector comprising at least one heterologous nucleic acid. Disclosed herein is an AAV vector comprising a vector genome. In an aspect, a disclosed vector genome can comprise a first inverted terminal repeat (ITR) and a second ITR. In an aspect, a disclosed vector genome can comprise a nucleic acid sequence encoding a transgene or a payload between the first ITR and the second ITR. Disclosed herein is an AAV vector comprising at least one heterologous nucleic acid and at least one inverted terminal repeat (ITR). In an aspect, for example, the at least one ITR can be AAV2 ITR.

[0172] Disclosed herein is an AAV vector comprising a heterologous nucleic acid for a therapeutic protein and/or a therapeutic RNA. Disclosed herein is an AAV vector comprising a heterologous nucleic acid for treating a subject having a kidney disease and/or kidney disorder. Disclosed herein is an AAV vector comprising a nucleic acid sequence encoding a transgene. Disclosed herein is an AAV vector comprising a nucleic acid sequence encoding a transgene for treating a subject having a kidney disease and/or kidney disorder.

[0173] Disclosed herein is an AAV particle comprising an AAV capsid comprising a disclosed AAV capsid protein. Disclosed herein is an AAV particle comprising (i) an AAV capsid comprising at least one disclosed AAV capsid protein and (ii) a vector genome.

[0174] Disclosed herein is an AAV particle for use in a disclosed method. Disclosed herein is an AAV particle for use in a disclosed method of delivering a payload or a disclosed method of treating a subject.

[0175] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03.

[0176] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. In an aspect, positions 452 - 458 of the AAV capsid protein can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect, positions 452 - 458 of the AAV capsid protein can comprise the sequence set forth in any one of SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect, positions 452 - 458 of the AAV capsid protein can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23. In an aspect, positions 452 - 458 of the AAV capsid protein can comprise a sequence that is at least 85% identical to any one of SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect, positions 452 - 458 of the AAV capsid protein can comprise the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO:23. In an aspect, positions 452-458 of the AAV capsid can comprise a truncated substitution such as AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0177] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q. [0178] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:31 or SEQ ID NO:35. In an aspect of a disclosed AAV capsid, the one or more amino acid substitutions in the AAV capsid protein can comprise substitutions other than GVSLGGG (SEQ ID NO:50).

[0179] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having one or more substitutions relative to the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO: 92. SEQ ID NO: 97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, or SEQ ID NO: 137.

[0180] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:65 or SEQ ID NO:66, SEQ ID NO:70 or SEQ ID NO:71, SEQ ID NO:75 or SEQ ID NO:76, SEQ ID NO:80 or SEQ ID NO:81, SEQ ID NO:85 or SEQ ID NO:86, SEQ ID NOVO or SEQ ID NO:91, SEQ ID NO:95 or SEQ ID NO:96, SEQ ID NO: 100 or SEQ ID NO: 101, SEQ ID NO: 105 or SEQ ID NO: 106, SEQ ID NO: 110 or SEQ ID NO: 111, SEQ ID NO: 115 or SEQ ID NO: 116, SEQ ID NO: 120 or SEQ ID NO: 121, SEQ ID NO: 125 or SEQ ID NO: 126, SEQ ID NO: 130 or SEQ ID NO: 131. SEQ ID NO : 135 or SEQ ID NO : 136, or SEQ ID NO : 140 or SEQ ID NO : 141.

[0181] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:62 but for one or more substitutions in the region of SEQ ID NO:63 or SEQ ID NO:64. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:67 but for one or more substitutions in the region of SEQ ID NO:68 or SEQ ID NO:69. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:72 but for one or more substitutions in the region of SEQ ID NO:73 or SEQ ID NO:74. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:77 but for one or more substitutions in the region of SEQ ID NO:78 or SEQ ID NO:79. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:82 but for one or more substitutions in the region of SEQ ID NO:83 or SEQ ID NO:84. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 87 but for one or more substitutions in the region of SEQ ID NO:88 or SEQ ID NO:89. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:92 but for one or more substitutions in the region of SEQ ID NO:93 or SEQ ID NO:94. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO:97 but for one or more substitutions in the region of SEQ ID NO:98 or SEQ ID NO:99. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 102 but for one or more substitutions in the region of SEQ ID NO: 103 or SEQ ID NO: 104. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 107 but for one or more substitutions in the region of SEQ ID NO: 108 or SEQ ID NO: 109. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 112 but for one or more substitutions in the region of SEQ ID NO: 113 or SEQ ID NO: 114. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 117 but for one or more substitutions in the region of SEQ ID NO: 118 or SEQ ID NO: 119. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 122 but for one or more substitutions in the region of SEQ ID NO: 123 or SEQ ID NO: 124. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 127 but for one or more substitutions in the region of SEQ ID NO: 128 or SEQ ID NO: 129. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 132 but for one or more substitutions in the region of SEQ ID NO: 133 or SEQ ID NO: 134. In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having the sequence set forth in SEQ ID NO: 137 but for one or more substitutions in the region of SEQ ID NO: 138 or SEQ ID NO: 139.

[0182] In an aspect of a disclosed AAV particle, the AAV capsid comprises an AAV capsid protein having a sequence having at least 85% identity to the sequence set forth in SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:92. SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, or SEQ ID NO: 137, with one or more substitutions in variable region IV (VR-IV). [0183] In an aspect, a disclosed nucleic acid sequence encoding a transgene or heterologous nucleic acid can be operably linked to one or more transcription regulatory elements. In an aspect, the one or more transcription regulatory elements can increase the transcription and/or expression of the transgene or heterologous nucleic acid.

[0184] In an aspect, a disclosed nucleic acid sequence encoding a transgene or heterologous nucleic acid (i.e., gene of interest) can be operably linked to a promoter. In an aspect, a disclosed transcription regulatory element can comprise a ubiquitous promoter operably linked to a disclosed transgene or heterologous nucleic acid, wherein the ubiquitous promoter drives the expression of a disclosed transgene or heterologous nucleic acid. In an aspect, a disclosed transcription regulatory element can comprise a ubiquitous promoter operably linked to a disclosed transgene or heterologous nucleic acid, wherein the tissue specific promoter drives the expression of a disclosed transgene or heterologous nucleic acid. In an aspect, a tissuespecific promoter can be a kidney-specific promoter and/or a kidney-specific enhancer. In an aspect, a disclosed transcription regulatory element can comprise a kidney-specific promoter and/or a kidney-specific enhancer.

[0185] In an aspect, a disclosed kidney-specific promoter can comprise a GOT promoter, an SGLT2 promoter, a PEPCK promoter, a KAP promoter, a KAP promoter including an A GT intron, a THP promoter, an A QP-2 promoter, a promoter of the B 1 subunit of vacuolar proton ATPase, a Hox-B7 promoter, a Ksp-cadherin promoter, a PAX-8 promoter, a promoter, a 11- beta-HSD 2 promoter, a renin promoter, a nephrin promoter, a podocin promoter, a tenascin-C promoter, a Osr-2 promoter, or any combination thereof. In an aspect, a disclosed kidneyspecific promoters can comprise a human homologue of a GOT promoter, an SGLT2 promoter, a PEPCK promoter, a KAP promoter, a KAP promoter including an A GT intron, a THP promoter, an A QP-2 promoter, a promoter of the Bl subunit of vacuolar proton ATPase, a Hox-B7 promoter, a Ksp-cadherin promoter, a PAX-8 promoter, a promoter, a l l -beta-HSD 2 promoter, a renin promoter, a nephrin promoter, a podocin promoter, a tenascin-C promoter, a Osr-2 promoter, or any combination thereof. In an aspect, a disclosed kidney-specific promoter can comprise an active fragment of GOT promoter, an SGLT2 promoter, a PEPCK promoter, a KAP promoter, a KAP promoter including an A GT intron, a THP promoter, an A QP-2 promoter, a promoter of the B 1 subunit of vacuolar proton ATPase, a Hox-B7 promoter, a Ksp- cadherin promoter, a PAX-8 promoter, a promoter, a 11 -beta-HSD 2 promoter, a renin promoter, a nephrin promoter, a podocin promoter, a tenascin-C promoter, a Osr-2 promoter, or any combination thereof. [0186] In an aspect, a disclosed transcriptional regulatory element can comprise a podocytespecific transcriptional regulatory element. In an aspect, a disclosed transcription regulatory element can comprise a minimal NPHS1 promoter and/or a minimal NPHS2 promoter.

[0187] In an aspect, a disclosed payload can encode a therapeutic RNA or a therapeutic protein. In an aspect, a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein. In an aspect, the therapeutic RNA is a circular RNA (cirRNA). In an aspect, a disclosed therapeutic RNA can be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA. In an aspect, a disclosed transgene or disclosed payload can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by NPHS1, NRHS2, PLCE1, CD2AR, LAMB2, NRHS2, ACTN4, TRRC6, WT1, I.MX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COI.4A4, PKD1, RKD2, PKHD1, DZIP1L, or any combination thereof. In an aspect, a disclosed transgene or heterologous nucleic acid can encode apolipoprotein LI, fibrocystin, myosin heavy chain 9, nephrocystin 1, poly cystin 1, poly cystin 2, or any combination thereof. In an aspect, a disclosed transgene or disclosed heterologous nucleic acid can encode NRHSL NRHS2, PLCE1, CD2AR, LAMB2, NRHS2, ACTN4, TRRC6, WT1, I.MX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COI.4A4, PKD1, PKD2, RKHD DZIP1L, or any combination thereof.

TABLE 4 - INFORMATION FOR EXEMPLARY GENES OF INTEREST

[0188] In an aspect, a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule. In an aspect, a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).

[0189] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve and/or enhance gene transfer to one or more kidney cells or kidney-derived cell types when compared to an AAV particle having the wild-type capsid protein. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to effect widespread transduction of one or more kidney cells or kidney-derived cell types. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than that of an AAV particle or AAV vector having the wild-type capsid protein.

[0190] In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0191] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve and/or enhance gene transfer to any region or part of the kidney. In an aspect of a disclosed AAV particle or a disclosed AAV vector, improved and/or enhanced gene transfer to kidney cells or kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0192] In an aspect, a disclosed AAV particle or a disclosed AAV vector can demonstrate an improved correlation in dose-response (e.g., thereby improving efficiency).

[0193] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pretreatment level. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pretreatment quality of life. [0194] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s kidney disease and/or kidney disorder. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0195] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve kidney function in the subject. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to reduce the risk of developing inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney-related cell types.

[0196] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to treat a subject in need thereof. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, a subject in need thereof can have one or more kidneys diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0197] In an aspect, a disclosed AAV particle or a disclosed AAV vector an AAV capsid protein can be incorporated into a disclosed AAV capsid. In an aspect, a disclosed AAV particle or a disclosed AAV vector an AAV capsid protein can demonstrate improved tropism for one or more cell types and/or one or more tissue types (such as, for example, one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types). In an aspect, a disclosed AAV particle or a disclosed AAV vector can exhibit improved transduction efficiency and/or properties when introduced to one or more cell types and/or one or more tissue types. In an aspect, a disclosed AAV particle or a disclosed AAV vector can efficiently transduce one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types. In an aspect, a disclosed AAV particle or a disclosed AAV vector can outperform the parental wild-type AAV in one or more disclosed kidney cells, kidney-derived cell types, and/or kidney -related cell types.

[0198] In an aspect, a disclosed AAV particle or a disclosed AAV vector can be AAV 1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrhlO, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, or AAVcy.7. In an aspect, a disclosed AAV vector can be bovine AAV, caprine AAV, canine AAV, equine AAV, ovine AAV, avian AAV, primate AAV, or non-primate AAV. In an aspect, a disclosed AAV vector can be AAV-DJ, AAV-HAE1, AAV-HAE2, AAVM41, AAV-1829, AAV2 Y/F, AAV2 T/V, AAV2i8, AAV2.5, AAV9.45, AAV9.61, AAV-B1, AAV- AS, AAV9.45A-String (e.g., AAV9.45-AS), AAV9.45Angiopep, AAV9.47-Angiopep, AAV9.47-AS, AAV-PHP.B, AAV- PHP.eB, AAV-PHP.S, AAV-F, AAVcc.47, or AAVcc.81.

[0199] In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be formulated for administration via one or more routes. Such routes are well known to those skilled in the art and include, but are not limited to, the following: retrograde ureteral infusion, renal arterial administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, inter utero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration of a disclosed AAV particle or a disclosed AAV vector can be continuous or intermittent. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered via one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol employing a disclosed AAV particle and/or a disclosed AAV vector can be employed with a kidney (or part thereof) obtained for a subject. In an aspect, a kidney can be obtained from a donor subj ect and can be subjected to an ex vivo perfusion protocol employing a disclosed AAV particle and/or a disclosed AAV vector prior to implantation into a subject in need thereof. In an aspect, a kidney can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion protocol employing a disclosed AAV particle and/or a disclosed AAV vector, and can be returned to the subject in need thereof. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed AAV particle and/or a disclosed AAV vector can be applied to other relevant tissues in the subject in need thereof.

[0200] In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used in a method of reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used in a method of improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used in a method of reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used in a method of reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used in a method of enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed AAV particle and/or a disclosed AAV vector can be used to extend and/or improve the life expectancy of a subject.

5. PHARMACEUTICAL FORMULATIONS

[0201] Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector in a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a disclosed nucleic acid molecule in a pharmaceutically acceptable carrier. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding a disclosed adeno- associated virus (AAV) capsid protein. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is a pharmaceutical formulation comprising a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. AAV particles, AAV vectors, AAV capsid proteins, and nucleic acid molecules are disclosed herein.

[0202] In an aspect, a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL to about 1 x 10¹⁴ DRP/mL. In an aspect, a disclosed pharmaceutical formulation can comprise about 1 x 10⁶ DRP/mL, 1 x 10⁷ DRP/mL, 1 x 10⁸ DRP/mL, 1 x 10⁹ DRP/mL, 1 x 10¹⁰ DRP/mL, 1 x 10¹¹ DRP/mL, 1 x 10¹² DRP/mL, 1 x 10¹³ DRP/mL, or 1 x 10¹⁴ DRP/mL.

[0203] In an aspect, a disclosed pharmaceutical formulation can be administered to a subject in need thereof. In an aspect, a disclosed pharmaceutical formulation can be formulated for administration via one or more routes. Such routes are well known to those skilled in the art and include, but are not limited to, the following: retrograde ureteral infusion, renal arterial administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, otic administration, inter utero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration of a disclosed pharmaceutical formulation can be continuous or intermittent. In an aspect, a disclosed pharmaceutical formulation can be administered via one or more ex vivo methods such as, for example, an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol can be employed with a kidney (or part thereof) obtained for a subject. In an aspect, a kidney can be obtained from a donor subject and can be subjected to an ex vivo perfusion protocol employing a disclosed pharmaceutical formulation prior to implantation into a subject in need thereof. In an aspect, a kidney can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion protocol, and can be returned to the subject in need thereof. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed pharmaceutical formulation can be applied to other relevant tissues in the subject in need thereof.

[0204] In an aspect, a disclosed pharmaceutical formulation can be used in a disclosed method. In an aspect, a disclosed pharmaceutical formulation can be used in a disclosed method of delivering a payload.

[0205] In an aspect, a disclosed pharmaceutical formulation can be used to improve and/or enhance gene transfer to one or more kidney cells or kidney-derived cell types when compared to an AAV particle having the wild-type capsid protein. In an aspect, a disclosed pharmaceutical formulation can be used to effect widespread transduction of one or more kidney cells or kidney -derived cell types. In an aspect, a disclosed pharmaceutical formulation can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than that of an AAV particle or AAV vector having the wild-type capsid protein. In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0206] In an aspect, a disclosed pharmaceutical formulation can be used to improve and/or enhance gene transfer to any region or part of the kidney. In an aspect of a disclosed pharmaceutical formulation, improved and/or enhanced gene transfer to kidney cells or kidney- derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof. In an aspect, a disclosed pharmaceutical formulation can demonstrate an improved correlation in dose-response (e.g., thereby improving efficiency).

[0207] In an aspect, a disclosed pharmaceutical formulation can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed pharmaceutical formulation can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0208] In an aspect, a disclosed pharmaceutical formulation can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s kidney disease and/or kidney disorder. In an aspect, a disclosed pharmaceutical formulation can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0209] In an aspect, a disclosed pharmaceutical formulation can be used to improve kidney function in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed pharmaceutical formulation can be used to reduce the risk of developing inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed pharmaceutical formulation can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney -related cell types.

[0210] In an aspect, a disclosed pharmaceutical formulation can be used to treat a subject in need thereof. In an aspect, a disclosed pharmaceutical formulation can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, a subject in need thereof can have one or more kidneys diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0211] In an aspect, a disclosed pharmaceutical formulation can have improved transduction efficiency and/or properties when introduced to one or more cell types and/or one or more tissue types. In an aspect, a disclosed pharmaceutical formulation can efficiently transduce one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types and/or can outperform the parental wild-type AAV in one or more disclosed kidney cells, kidney-derived cell types, and/or kidney-related cell types.

[0212] In an aspect, a disclosed pharmaceutical formulation can be used in a method of reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed pharmaceutical formulation can be used in a method of improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed pharmaceutical formulation can be used in a method of reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed pharmaceutical formulation can be used in a method of reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed pharmaceutical formulation can be used in a method of enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed pharmaceutical formulation can be used to extend and/or improve the life expectancy of a subject.

6. KITS

[0213] Disclosed herein is a kit comprising one or more disclosed compositions. In an aspect, a composition of a disclosed kit can comprise one or more disclosed nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed AAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs, or any combination thereof. In an aspect, a disclosed kit can comprise a combination of one or more active agents. In an aspect, a disclosed kit can comprise at least two components constituting the kit. Together, the components constitute a functional unit for a given purpose (such as, for example, performing directed evolution on AAV capsid proteins, generating AAV particles, or delivering a payload). Individual member components can be physically packaged together or separately. For example, a disclosed kit comprising an instruction for using the kit can be physically included with instructions for other individual member components. Instead, the instruction can be supplied as a separate member component, either in a paper form or an electronic form that can be supplied on computer readable memory device or downloaded from an internet website, or as recorded presentation. In an aspect, a disclosed kit for use in a disclosed method can comprise one or more containers holding a disclosed composition (i.e., disclosed nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed AAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs, or any combination thereof) and a label or package insert with instructions for use.

[0214] In an aspect, a disclosed kit can contain one or more additional agents (e.g., excipients, buffers, active agents, biologically active agents, pharmaceutically active agents, immunebased therapeutic agents, clinically approved agents, or a combination thereof). In an aspect, one or more active agents can treat, inhibit, and/or ameliorate one or more comorbidities in a subject. In an aspect, one or more active agents can treat, inhibit, and/or ameliorate a disease or a disorder (such as a kidney disease and/or kidney disorder), an infection, a symptom, a complication, or a combination thereof. In an aspect, suitable containers include, for example, bottles, vials, syringes, blister pack, etc. The containers can be formed from a variety of materials such as glass or plastic. The container can hold a disclosed composition (e.g., for example, disclosed nucleic acid molecules, disclosed plasmids, disclosed AAV capsid proteins, disclosed AAV particles, disclosed vectors, disclosed AAV vectors, disclosed pharmaceutical formulations, disclosed wild-type AAV capsid proteins, disclosed wild-type AAVs), or a disclosed pharmaceutical formulation and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert can indicate that a disclosed composition or a pharmaceutical formulation comprising a disclosed composition can be used for treating, preventing, inhibiting, and/or ameliorating a disease and/or a disorder (such as a kidney disease and/or kidney disorder), an infection, a symptom, a complication, or a combination thereof. A disclosed kit can comprise additional components necessary for administration such as, for example, other buffers, diluents, filters, needles, and syringes. The term “package insert” can refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications and/or warnings concerning the use of such therapeutic products. In an aspect, a disclosed kit can comprise some or all the components necessary to practice and/or perform one or more disclosed methods. In an aspect, a disclosed kit can be used in a method of delivering a payload. In an aspect, a disclosed kit can be used in a method of treating a subject.

[0215] In an aspect, a disclosed kit can be used in a method of reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed kit can be used in a method of improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed kit can be used in a method of reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed kit can be used in a method of reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed kit can be used in a method of enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subj ect in need thereof). In an aspect, a disclosed kit can be used in a method of extending and/or improving the life expectancy of a subject.

C. METHODS

1. METHODS OF GENERATING AND/OR MAKING AAV PARTICLES

[0216] Disclosed herein is a method of generating AAV particles, the method comprising delivering to one or more cells a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein; culturing the one or more cells; and harvesting the AAV particles from the one or more producer cells. Disclosed herein is a method of generating AAV particles, the method comprising delivering to one or more cells three plasmids, wherein the first plasmid is a helper plasmid, wherein the second plasmid is RepCap plasmid, wherein the second plasmid comprises a nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein, and wherein the third plasmid is the cis-plasmid or transfer plasmid; culturing the one or more cells; and harvesting the AAV particles from the one or more cells. Disclosed herein is a method for making an AAV particle, the method comprising providing one or more cells comprising a disclosed AAV vector and culturing the one or more cells under conditions allowing for forming an AAV particle.

[0217] In an aspect, a disclosed method can comprise expressing a disclosed encoded AAV capsid protein. In an aspect, a disclosed method can comprise culturing the one or more cells in a media. In an aspect, a disclosed method can comprise harvesting the AAV particles. In an aspect, a disclosed method can comprise purifying the harvested AAV particles. In an aspect, a disclosed method can comprise using the purified AAV particles in gene therapy. In an aspect, a disclosed method can comprise delivering to the one or more cells a helper plasmid. In an aspect, a disclosed method can comprise delivering to the one or more cells a cis-plasmid or a transfer plasmid encoding a gene of interest or a transgene. In an aspect, disclosed secreted AAV particles can comprise the gene of interest or the transgene. In an aspect, disclosed secreted AAV particles can comprise one or more base-editing components and/or one or more gRNAs.

[0218] In an aspect, a disclosed method can comprise harvesting from AAV particles and/or AAV particles by lysis of the host cells of the production culture or by harvest of the spent media from the production culture. Suitable methods of lysing cells are known to the skilled person and can include multiple freeze/thaw cycles, sonication, microfluidization, and treatment with chemicals (e.g., detergents and/or proteases), or any combination thereof.

[0219] In an aspect, a disclosed method can further comprise purifying the AAV particles. In an aspect, “purified” can comprise preparing AAV particles devoid of at least some of the other components that can also be present where the AAV particles naturally occur or are initially prepared from. In an aspect, isolated AAV particles can be prepared using a purification technique to enrich it from a source mixture (e.g., culture lysate or production culture supernatant). In an aspect of a disclosed method, AAV particles can be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anionic exchange filtration; tangential flow filtration (TFF) for concentrating the AAV particles; AAV capture by apatite chromatography; heat inactivation of helper virus; AAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and AAV capture by anionic exchange chromatography, cationic exchange chromatography, affinity chromatography, or any combination thereof. In an aspect, enrichment can be measured in a variety of ways known to the art, including by the proportion of DNase-resistant particles (DRPs) or genome copies (gc) present in a solution, or by infectivity. In an aspect, enrichment can be measured in relation to a second, potentially interfering substance present in the source mixture (e.g., contaminants, including production culture contaminants or in-process contaminants, including helper virus, media components, etc.). In an aspect, a disclosed method of generating and/or making AAV particles can comprise validating the purity of the AAV particles and/or the functionality of the AAV particles.

[0220] Disclosed herein is a method of generating AAV capsid proteins, the method comprising performing multiple rounds of evolution in one or more kidney or kidney-related models from one or more species. In an aspect of a disclosed method, generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 425-458) of SEQ ID NO:01. In an aspect, a disclosed method can further comprise packaging the initial library of capsid proteins into an AAV vector using triple plasmid transfection. Triple plasmid transfection is known to the art and discussed supra. In an aspect, a disclosed first round of evolution can comprise intravenously administering to mice the AAV vector comprising the initial variant capsid library. In an aspect, a disclosed second round of evolution can comprise intravenously administering to pigs an AAV vector comprising the variant capsid library generated in the first round of evolution. In an aspect, a disclosed third round of evolution can comprise transducing differentiated human kidney organoids with the variant capsid library generated in the second round of evolution. In an aspect, a disclosed fourth round of evolution can comprise perfusing ex vivo a non-human primate kidney with the variant capsid library generated in the third round of evolution.

[0221] In an aspect, a disclosed method can further comprise generating an initial library of capsid proteins identified via serial evolution. In an aspect, a disclosed parental or a disclosed wild-type capsid protein can comprise the sequence set forth in SEQ ID NO:01, SEQ ID NO:62, SEQ ID NO:67, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:82, SEQ ID NO:87, SEQ ID NO:92, SEQ ID NO:97, SEQ ID NO: 102, SEQ ID NO: 107, SEQ ID NO: 112, SEQ ID NO: 117, SEQ ID NO: 122, SEQ ID NO: 127, SEQ ID NO: 132, or SEQ ID NO: 137.

[0222] In an aspect, a disclosed method can further comprise assessing sequence diversity of the variant capsid library generated in the one or more rounds of evolution. In an aspect, a disclosed method can further comprise calculating the percent representation and fold enrichment of each evolved variant capsid library when compared to the parental capsid library. In an aspect, a disclosed method can further comprise ranking the amino acid sequences based on percent representation and fold enrichment to identify one or more candidate capsid proteins. In an aspect, a disclosed method can further comprise characterizing the one or more candidate capsid proteins.

[0223] In an aspect, one or more disclosed species can comprise Mus Musculus (mouse), Sus scrofa (pig), non-human primates (Macaca, macaque), or Homo sapiens (human), or any combination.

[0224] In an aspect, a disclosed method can comprise generating a disclosed AAV capsid protein including, for example, the AAV capsid protein comprising the sequence set forth in SEQ ID NO:24 - SEQ ID NO:43. In an aspect, a disclosed method can comprise generating a disclosed AAV capsid protein including, for example, the AAV capsid protein comprising the sequence set forth in SEQ ID NO: 53 - SEQ ID NO:61.

[0225] In an aspect, a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01, wherein positions 452 - 458 can comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect, a disclosed method can comprise generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01, wherein positions 452 - 458 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO: 191 - SEQ ID NO:8873.

2. METHODS OF DELIVERING A PAYLOAD, TRANSGENE, OR HETEROLOGOUS NUCLEIC ACID [0226] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells with a disclosed AAV particle; and expressing the encoded payload.

[0227] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.

[0228] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells in a subject in need thereof with a disclosed AAV particle; and expressing the encoded payload.

[0229] Disclosed herein is a method for delivering a payload, the method comprising contacting one or more target cells in a subject in need thereof with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a transgene or heterologous nucleic acid; and expressing the transgene or heterologous nucleic acid.

[0230] In an aspect, a disclosed payload can comprise a nucleic acid that is encapsidated in the AAV particle. For example, in an aspect of a disclosed method, a disclosed payload can encode a therapeutic RNA (e.g., an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA) or a therapeutic protein. In an aspect, the therapeutic RNA is a circular RNA (cirRNA). In an aspect, a disclosed payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA. In an aspect, a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload. In an aspect, a disclosed payload can encode a gene therapy product. A gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In an aspect, a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated. In an aspect, a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product. In an aspect, a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein. In an aspect, a disclosed therapeutic RNA can be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.

[0231] In an aspect, a disclosed payload can encode one or more a base-editing components and/or one or more gRNA targeting the region to be edited. In an aspect, a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule. In an aspect, a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).

In an aspect, a disclosed transgene or a heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed transgene or heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.

[0232] In an aspect, a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I.MX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. In an aspect, a disclosed transgene or heterologous nucleic acid can encode apolipoprotein LI, fibrocystin, myosin heavy chain 9, nephrocystin 1, poly cystin 1, polycystin 2, or any combination thereof. [0233] In an aspect, a disclosed method can improve and/or enhance gene transfer to one or more kidney cells or kidney-derived cell types when compared to an AAV particle having the wild-type capsid protein. In an aspect, a disclosed method can be used to effect widespread transduction of one or more kidney cells or kidney-derived cell types. In an aspect, a disclosed method can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than that of an AAV particle or AAV vector having the wild-type capsid protein. In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0234] In an aspect, a disclosed method can be used to improve and/or enhance gene transfer to any targeted region or targeted part of the kidney. In an aspect of disclosed method, improved and/or enhanced gene transfer to target kidney cells or target kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0235] In an aspect, a disclosed method can be used to treat a subject in need thereof. In an aspect, a disclosed method can be used in a method of delivering gene therapy to a subject in need thereof.

[0236] In an aspect, a disclosed method can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life. In an aspect, a disclosed method can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s kidney disease and/or kidney disorder. In an aspect, a disclosed method can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed method can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0237] In an aspect, a disclosed method can be used to improve kidney function in the subject. In an aspect, a disclosed method can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed method can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed method can be used to reduce the risk of developing inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed method can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney -related cell types.

[0238] In an aspect, a disclosed method can be used to treat a subject in need thereof. In an aspect, a subject can be any age and can be male or female. In an aspect of a disclosed method, a subject can be treatment-naive. In an aspect, a subject can have received treatment prior to the contacting step and/or administering step. In an aspect, a subj ect can need a kidney transplant or a subject can have already received a kidney transplant. In an aspect, a subject in need thereof can have one or more kidneys diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0239] In an aspect, a disclosed method can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, the contacting step allows for expression of the payload in the one or more target cells. In an aspect, the contacting step allows for expression of the transgene or the heterologous nucleic acid in the one or more target cells.

[0240] In an aspect of a disclosed method of delivering a payload, a disclosed AAV particle can comprise a disclosed AAV capsid protein. In an aspect, a disclosed AAV capsid protein can comprise any AAV capsid protein disclosed herein.

[0241] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x IO¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed AAV particle or disclosed vector can be administered at a dose of about 1 x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg or about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of at least about 1 x IO¹⁰, at least about 5 x IO¹⁰, at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of no more than about 1 x IO¹⁰, no more than about 5 x IO¹⁰, no more than about 1 x 10¹¹, 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 10¹⁴ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of about l x 10¹² vg/kg. In an aspect, a disclosed AAV particle or a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a disclosed AAV particle or a disclosed vector 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. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range determined by a skilled person.

[0242] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can be by delivered retrograde ureteral infusion and/or renal arterial administration and can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. [0243] In an aspect of a disclosed method, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme (e.g., NPHS1, NPHS2, PIXUII. CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1E) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60- 70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pretreatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0244] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0245] In an aspect of a disclosed method, contacting a cell can comprising methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof .

[0246] In an aspect, administering can comprise retrograde ureteral infusion, renal arterial administration, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra- CSF, intrathecal, intraventricular, intrahepatic, hepatic intra-arterial, hepatic portal vein (HPV), or in utero administration. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered in combination with RNAi, antisense oligonucleotides, siRNA, shRNA, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more replacement enzymes, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed AAV vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can comprise IV administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject including retrograde ureteral infusion and/or arterial route. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0247] In an aspect, a disclosed method can employ an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be employed with a kidney (or part thereof) obtained for a subject. In an aspect, a kidney can be obtained from a donor subject and can be subjected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into a subj ect in need thereof. In an aspect, a kidney can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, and can be returned to the subject in need thereof. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be applied to other relevant tissues in the subject in need thereof.

[0248] In an aspect, a disclosed method of delivering a payload can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome.

[0249] In an aspect, a disclosed therapeutic agent can be an enzyme or a recombinant enzyme. In an aspect, a therapeutically effective amount of a disclosed replacement enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight. In an aspect, a disclosed enzyme or disclosed recombinant enzyme can be therapeutically effective when the dose comprises about 0.01 mg/kg, about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 15 mg/kg, about 20 mg/kg, about 25 mg/kg, about 30 mg/kg, about 35 mg/kg, about 40 mg/kg, about 45 mg/kg, about 50 mg/kg, about 55 mg/kg, about 60 mg/kg, about 65 mg/kg, about 70 mg/kg, about 75 mg/kg, about 80 mg/kg, about 85 mg/kg, about 90 mg/kg, about 95 mg/kg, or about 100 mg/kg body weight.

[0250] In an aspect of a disclosed method delivering a payload, the administering step can treat a subject in need thereof. In an aspect, treating a subject can comprising administering one or more times to the subject one or more additional therapies. In an aspect, a disclosed method of delivering a payload can further comprise monitoring the subj ect for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0251] In an aspect, a disclosed method of delivering a payload can further comprise administering to the subject a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway. [0252] In an aspect, a disclosed method of delivering a payload can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0253] In an aspect, a disclosed of improving and/or enhancing transgene efficacy and/or expression method can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0254] In an aspect, a method of delivering a payload can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, aBTK inhibitor, an anti -IGF 1R antibody, a CD 19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0255] In an aspect, a disclosed method can further comprise administering lipid nanoparticles (LNPs). In an aspect, LNPs can be organ-targeted (such as, for example, kidney celles or kidney-derived cells). In an aspect, LNPs can be targeted to one or more parts or regions of the kidney. For example, in an aspect, mRNA therapy with LNP encapsulation for systemic delivery to a subject has the potential to restore the functionality and/or structural integrity of a missing, deficient, and/or mutant protein or enzyme.

[0256] In an aspect, a disclosed method of delivering a payload can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general.

[0257] In an aspect, a disclosed method can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV particle or a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells and/or a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response.

[0258] In an aspect, a disclosed method of delivering a payload can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject. [0259] In an aspect, a disclosed method can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0260] In as aspect, a disclosed method can comprise concurrent administration of one or more of the following: one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, one or more disclosed therapeutic agents, one or more disclosed immune modulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressive agents, one or more disclosed compounds that exert therapeutic effect against B cells, one or more disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0261] In an aspect, a disclosed immune modulator can be administered prior to or after the administration of a disclosed therapeutic agent. In an aspect, a disclosed method of delivering a payload can further comprise generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0262] For example, in an aspect, a disclosed method of delivering a payload can further comprise generating a disclosed AAV particle or a disclosed AAV vector. In an aspect, generating a disclosed AAV particle or a disclosed viral vector can comprise generating an AAV particle or an AAV particle or an AAV vector or a recombinant AAV (such as those disclosed herein).

[0263] In an aspect, a disclosed method of delivering a payload can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof. [0264] In an aspect of a disclosed method, a payload can comprise one or more base-editing components and one or more sgRNA targeting the region to be edited.

[0265] In an aspect, a disclosed method of delivering a payload can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0266] In an aspect, a disclosed method of delivering a payload can further comprise generating and/or validating one or more disclosed enzymes or disclosed recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0267] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0268] In an aspect, a disclosed method of delivering a payload can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subj ect with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof. [0269] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0270] In an aspect, a disclosed method can further comprise measuring and/or determining a subject’s pre-treatment level of one or more clinical and/or metabolic indicators (such as, for example, the expression of NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I MIX IB, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, CO/.4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2,PKHD1 , or DZIP1L) or the measure of a subject’s kidney function. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of one or more clinical and/or metabolic indicators.

[0271] In an aspect, a disclosed method of delivering a payload, a transgene, or a heterologous nucleic acid can further comprise reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed method of treating a subject can further comprise improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of delivering a payload, a transgene, or a heterologous nucleic acid can further comprise reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of delivering a payload, a transgene, or a heterologous nucleic acid can further comprise reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of delivering a payload, a transgene, or a heterologous nucleic acid can further comprise enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed method of treating a subject can extend and/or improve the life expectancy of a subject.

3. METHODS OF TREATING A SUBJECT

[0272] Disclosed herein is a method of treating a subject, the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector. Disclosed herein is a method of treating a subject, the method comprising administering one or more times to a subject in need thereof a therapeutically effective amount of a disclosed pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector. Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle comprising a payload. Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subj ect with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid.

[0273] Disclosed herein is a method for treating a subject, the method comprising contacting one or more cells in a subject with a therapeutically effective amount of a disclosed AAV particle (i) comprising a disclosed variant capsid protein and (ii) encoding a payload, a transgene, or a heterologous nucleic acid; and expressing the encoded payload, the encoded transgene, or the encoded heterologous nucleic acid, wherein the contacting step allows for expression of the encoded payload, the encoded gene of interest, or the encoded transgene in the one or more cells.

[0274] In an aspect, a subject can be any age and can be male or female. In an aspect of a disclosed method, a subject can be treatment-naive. In an aspect, a subject can have received treatment prior to the contacting step and/or administering step. In an aspect, a subject can need a kidney transplant or a subject can have already received a kidney transplant. In an aspect, a subject in need thereof can have one or more kidney diseases and/or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0275] In an aspect, a disclosed AAV particle can comprise a disclosed AAV capsid protein such as those, for example, describe supra. For example, in an aspect, a disclosed AAV particle used in a disclosed method of treating a subject can comprise an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43.

[0276] In an aspect, a disclosed AAV particle used in a disclosed method of treating a subject can comprise an AAV capsid protein comprising the sequence set forth in any one of SEQ ID NO:53 - SEQ ID NO:61. For example, a disclosed AAV particle used in a disclosed method of treating a subject can comprise an AAV capsid protein comprising the sequence set forth in SEQ ID NO:03. For example, a disclosed AAV particle used in a disclosed method of treating a subject can comprise an AAV capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. For example, a disclosed AAV particle used in a disclosed method of treating a subject can comprise an AAV capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:OL In an aspect, the substitutions at positions 452 - 458 relative to SEQ ID NO:01 can comprise the sequence set forth in any of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO:191 - SEQ ID NO:8873. In an aspect, the substitutions at positions 452 - 458 relative to SEQ ID NO:01 can comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect, a disclosed AAV capsid protein can comprise any AAV capsid protein disclosed herein.

[0277] In an aspect, a disclosed payload can comprise a nucleic acid that is encapsidated in the AAV particle. For example, in an aspect of a disclosed method, a disclosed payload can encode a therapeutic RNA or a therapeutic protein. In an aspect, a disclosed payload nucleic acid can encode a polypeptide, an inhibitory RNA, an antibody or antibody reagent, an oligonucleotide, or a miRNA. In an aspect, a disclosed payload can encode a messenger RNA (mRNA) can be encoded by a disclosed payload. In an aspect, a disclosed payload can encode a gene therapy product. A gene therapy product can comprise a polypeptide, RNA molecule, or other gene product that, when expressed in a target cell, provides a desired therapeutic effect. In an aspect, a gene therapy product can comprise a substitute for a non-functional gene that is absent or mutated. In an aspect, a disclosed payload nucleic acid can encode a transgene having a beneficial or desirable gene product. In an aspect, a disclosed transgene or a heterologous nucleic acid can encode a therapeutic RNA or a therapeutic protein. In an aspect, a disclosed therapeutic RNA can be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA. [0278] In an aspect, a disclosed payload can encode one or more a base-editing components and/or one or more gRNA targeting the region to be edited. In an aspect, a disclosed transgene or disclosed heterologous nucleic acid can encode a gene-editing molecule. In an aspect, a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA).

In an aspect, a disclosed transgene or a heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed transgene or heterologous nucleic acid can encode a missing, deficient, and/or mutant protein or enzyme.

[0279] In an aspect, a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I MIX IB, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, CO/.4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. In an aspect, a disclosed transgene or heterologous nucleic acid can encode apolipoprotein LI, fibrocystin, myosin heavy chain 9, nephrocystin 1, poly cystin 1, poly cystin 2, or any combination thereof. [0280] In an aspect, a disclosed method of treating a subject can improve and/or enhance gene transfer to one or more kidney cells or kidney -derived cell types when compared to an AAV particle having the wild-type capsid protein. In an aspect, a disclosed method of treating a subject can be used to effect widespread transduction of one or more kidney cells or kidney- derived cell types. In an aspect, a disclosed method of treating a subject can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than that of an AAV particle or AAV vector having the wild-type capsid protein. In an aspect, the increase and/or the improvement in transduction efficiency can comprise a 10% increase and/or improvement, a 20% increase and/or improvement, a 30% increase and/or improvement, a 40% increase and/or improvement, a 50% increase and/or improvement, a 60% increase and/or improvement, a 70% increase and/or improvement, a 80% increase and/or improvement, a 90% increase and/or improvement, a 100% increase and/or improvement, or more than a 100% increase and/or improvement.

[0281] In an aspect, a disclosed method of treating a subject can be used to improve and/or enhance gene transfer to any targeted region or targeted part of the kidney. In an aspect of disclosed method, improved and/or enhanced gene transfer to target kidney cells or target kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0282] In an aspect, a disclosed method can be used to treat a subject in need thereof. In an aspect, a disclosed method can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect, a disclosed method of treating a subject can be used to improve and/or can be used to enhance the quality of the subject’s life when compared to a pre-treatment level. In an aspect, a disclosed method of treating a subject can be used to improve the subject’s quality of life by at least 50% when compared to the subject’s pre-treatment quality of life.

[0283] In an aspect, a disclosed method of treating a subject can be used to diminish and/or decrease one or more symptoms associated with and/or related to the subject’s kidney disease and/or kidney disorder. In an aspect, a disclosed method of treating a subject can be used to prevent an undesired physiological change, disease, pathological condition, or disorder from occurring in the subject. In an aspect, a disclosed method of treating a subject can be used to inhibit a physiological change, disease, pathological condition, or disorder, z.e., arresting its development, in the subject. In an aspect, a disclosed method of treating a subject can be used to relieve a physiological change, disease, pathological condition, or disorder, z.e., causing regression of the disease, in the subject.

[0284] In an aspect, a disclosed method of treating a subject can be used to improve kidney function in the subject. In an aspect, a disclosed method of treating a subject can be used to decrease the risk for acute kidney failure and/or chronic kidney failure in the subject. In an aspect, a disclosed method of treating a subject can be used to reduce the risk of kidney infection in the subject. In an aspect, a disclosed method of treating a subject can be used to reduce the risk of developing inflammation of one or more parts or regions of the kidney in the subject. For example, in an aspect, inflammation can affect the kidney’s filtering units (e.g., glomerulonephritis) and/or the kidney’s tubules and surrounding structures (e.g., interstitial nephritis). In an aspect, a disclosed method of treating a subject can be used to repair diseased and/or dysfunctional kidney cells, kidney-derived cell types, and/or kidney -related cell types. In an aspect, a disclosed method can be used to reduce the subject’s need for a kidney transplant and/or reduce the subject’s risk of rejection of a transplanted kidney.

[0285] In an aspect, a disclosed method of treating a subject can be used in a method of delivering gene therapy to a subject in need thereof. In an aspect of a disclosed method, restoring the activity and/or functionality of a missing, deficient, and/or mutant protein or enzyme (e.g., NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I MIX IB, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, CO/.4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) can comprise a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount of restoration when compared to a pre-existing level such as, for example, a pre-treatment level. In an aspect, the amount of restoration can be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% more than a pre-existing level such as, for example, a pre-treatment level. In an aspect, restoration can be measured against a control level or a reference level (e.g., determined, for example, using one or more subjects not having a missing, deficient, and/or mutant protein or enzyme). In an aspect, restoration can be a partial or incomplete restoration. In an aspect, restoration can be complete or near complete restoration such that the level of expression, activity, and/or functionality is similar to that of a wild-type or control level.

[0286] In an aspect, following the administering step, the payload is expressed in the one or more target cells in the subject. In an aspect, following the administering step, the transgene or heterologous nucleic acid is expressed in the one or more target cells in the subject.

[0287] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹⁰ vg/kg to about 2 x 10¹⁴ vg/kg. In an aspect, for example, a disclosed AAV particle or disclosed vector can be administered at a dose of about l x 10¹¹ to about 8 x 10¹³ vg/kg or about 1 x 10¹² to about 8 x 10¹³ vg/kg or about 1 x 10¹³ to about 6 x 10¹³ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of at least about 1 x 10¹⁰, at least about 5 x 10¹⁰, at least about 1 x 10¹¹, at least about 5 x 10¹¹, at least about 1 x 10¹², at least about 5 x 10¹², at least about 1 x 10¹³, at least about 5 x 10¹³, or at least about 1 x 10¹⁴ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of no more than about 1 x 10¹⁰, no more than about 5 x 10¹⁰, no more than about 1 x 10¹¹, 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 10¹⁴ vg/kg. In an aspect, a disclosed AAV particle or a disclosed AAV vector can be administered at a dose of about 1 x 10¹² vg/kg. In an aspect, a disclosed AAV particle or a disclosed vector can be administered at a dose of about 1 x 10¹¹ vg/kg. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total.

[0288] In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷vg per subject total. In an aspect, a disclosed AAV particle or disclosed vector 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. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range determined by a skilled person. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector comprise a range of about 1 x 10¹² vg per subject total, about 1 x 10¹³ vg per subject total, about 1 x 10¹⁴ vg per subject total, about 1 x 10¹⁵ vg per subject total, about 1 x 10¹⁶ vg per subject total, or about 1 x 10¹⁷ vg per subject total. In an aspect, a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector can comprise a range of about 1 x 10¹² vg per subject total to about 1 x 10¹⁷ vg per subject total.

[0289] In an aspect of a disclosed method, techniques to monitor, measure, and/or assess the restoring one or more aspects of cellular homeostasis and/or cellular functionality can comprise qualitative (or subjective) means as well as quantitative (or objective) means. These means are known to the skilled person. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed supra.

[0290] In an aspect of a disclosed method, contacting a target cell can comprise methods known to the art. For example, contacting can comprise administering to a subject one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed AAV particles, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0291] In an aspect, administering can comprise retrograde ureteral infusion, renal arterial administration, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intra- CSF, intrathecal, intraventricular, intrahepatic, hepatic intra-arterial, hepatic portal vein (HPV), or in utero administration. In an aspect, administering can comprise retrograde ureteral infusion and/or arterial route. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered in combination with RNAi, antisense oligonucleotides, miRNA, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more replacement enzymes, one or more immune modulators, and/or a gene editing system. In an aspect, a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can be administered via LNP administration. In an aspect, a disclosed composition, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, and/or a disclosed AAV vector can be concurrently and/or serially administered to a subject via multiple routes of administration. For example, in an aspect, administering a disclosed composition, a disclosed enzyme or disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed pharmaceutical formulation, a disclosed AAV particle, and/or a disclosed AAV vector can comprise IV administration. In an aspect, a disclosed method can employ multiple routes of administration to the subject. In an aspect, a disclosed method can employ multiple routes of administration to the subject including retrograde ureteral infusion and/or arterial route. In an aspect, a disclosed method can employ a first route of administration that can be the same or different as a second and/or subsequent routes of administration.

[0292] In an aspect, a disclosed method of treating a subject can employ an ex vivo perfusion protocol. In an aspect, an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be employed with a kidney (or part thereof) obtained for a subject. In an aspect, a kidney can be obtained from a donor subject and can be subjected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into a subject in need thereof. In an aspect, a kidney can be obtained from the subject in need thereof, can be subjected to an ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, and can be returned to the subject in need thereof. In an aspect, a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be applied to other relevant tissues in the subject in need thereof. [0293] In an aspect, a disclosed method of treating a subj ect can further comprise administering to the subject a therapeutically effective amount of a therapeutic agent. A therapeutic agent can be any disclosed agent that effects a desired clinical outcome. In an aspect, a disclosed therapeutic agent can be an enzyme or a recombinant enzyme. In an aspect, a therapeutically effective amount of a disclosed replacement enzyme or disclosed recombinant enzyme can comprise about 0.01 mg/kg body weight to about 100 mg/kg body weight.

[0294] In an aspect, a disclosed method can comprise administering one or more times to the subject one or more additional therapies. In an aspect, a disclosed method of delivering a payload can further comprise monitoring the subject for adverse effects. In an aspect, in the absence of adverse effects, the method can further comprise continuing to treat the subject. In an aspect, in the presence of adverse effects, the method can further comprise modifying the treating step. Methods of monitoring a subject’s well-being can include both subjective and objective criteria (and are discussed supra). Such methods are known to the skilled person.

[0295] In an aspect, a disclosed method of treating a subj ect can further comprise administering to the subj ect a therapeutically effective amount of an agent that can correct one or more aspects of a dysregulated metabolic or enzymatic pathway. In an aspect, such an agent can comprise an enzyme for enzyme replacement therapy. In an aspect, a disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In an aspect, a disclosed method can comprise replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway. In an aspect, dysregulated metabolic or enzymatic pathways emanate from or exist in one or more of the subject’s kidneys.

[0296] In an aspect, a disclosed method of treating a subj ect can further comprise administering one or more immune modulators. In an aspect, a disclosed immune modulator can be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In an aspect, a disclosed immune modulator can be bortezomib or SVP-Rapamycin. In an aspect, a disclosed immune modulator can be Tacrolimus. In an aspect, a disclosed immune modulator such as methotrexate can be administered at a transient low to high dose. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.1 mg/kg body weight to about 0.6 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at a dose of about 0.4 mg/kg body weight. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for 3 to 5 or greater cycles, with up to three days per cycle. In an aspect, a disclosed immune modulator can be administered at about a daily dose of 0.4 mg/kg body weight for a minimum of 3 cycles, with three days per cycle. In an aspect, a person skilled in the art can determine the appropriate number of cycles. In an aspect, a disclosed immune modulator can be administered as many times as necessary to achieve a desired clinical effect.

[0297] In an aspect, a disclosed method of treating a subj ect can further comprise administering one or more immunosuppressive agents. In an aspect, an immunosuppressive agent can be, but is not limited to, azathioprine, methotrexate, sirolimus, anti -thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or a combination thereof. In an aspect, a disclosed method can comprise administering one or more immunosuppressive agents more than 1 time. In an aspect, a disclosed method can comprise administering one or more one or more immunosuppressive agents repeatedly over time. In an aspect, a disclosed method can comprise administering to the subject a compound that targets or alters antigen presentation or humoral or cell mediated or innate immune responses.

[0298] In an aspect, a disclosed method of treating a subj ect can further comprise administering a compound that exerts a therapeutic effect against B cells and/or a compound that targets or alters antigen presentation or humoral or cell mediated immune response. In an aspect, a disclosed compound can be rituximab, methotrexate, intravenous gamma globulin, anti CD4 antibody, anti CD2, an anti-FcRN antibody, a BTK inhibitor, an anti-IGFIR antibody, a CD19 antibody (e.g., inebilizumab), an anti-IL6 antibody (e.g., tocilizumab), an antibody to CD40, an IL2 mutein, or a combination thereof. Also disclosed herein are Treg infusions that can be administered as a way to help with immune tolerance (e.g., antigen specific Treg cells to AAV). [0299] In an aspect, a disclosed method of treating a subject can comprise repeating a disclosed administering step such as, for example, repeating the administering of a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV particle or a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immune modulator, a disclosed proteasome inhibitor, a disclosed immunosuppressive agent, a disclosed compound that exerts a therapeutic effect against B cells, a disclosed compound that targets or alters antigen presentation or humoral or cell mediated immune response, or any combination thereof.

[0300] In an aspect, a disclosed method of treating a subject can comprise modifying one or more of the disclosed steps. For example, modifying one or more of steps of a disclosed method can comprise modifying or changing one or more features or aspects of one or more steps of a disclosed method. For example, in an aspect, a method can be altered by changing the amount of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration of one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject, or by changing the duration of time one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof are administered to a subject.

[0301] In an aspect, a disclosed method of treating a subject can be altered by changing the amount of one or more disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immune modulators, disclosed proteasome inhibitors, disclosed immunosuppressive agents, disclosed compounds that exert therapeutic effect against B cells and/or disclosed compounds that targets or alters antigen presentation or humoral or cell mediated immune response administered to a subject.

[0302] For example, in an aspect, a disclosed method of treating a subject can further comprise generating a disclosed AAV particle or a disclosed AAV vector. In an aspect, generating a disclosed AAV particle or a disclosed viral vector can comprise generating an AAV particle or AAV particle or AAV vector or a recombinant AAV (such as those disclosed herein) using a method disclosed herein.

[0303] In an aspect, a disclosed method of treating a subject can further comprise generating and/or validating one or more of the disclosed nucleic acid molecules, one or more AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0304] In an aspect, a disclosed method of treating a subject can further comprise gene editing one or more relevant genes (such as, for example, a missing, deficient, and/or mutant protein or enzyme), wherein editing includes but is not limited to single gene knockout, loss of function screening of multiple genes at one, gene knockin, or a combination thereof.

[0305] In an aspect of a disclosed method, a payload can comprise one or more base-editing components and one or more sgRNA targeting the region to be edited. [0306] In an aspect, a disclosed method of treating a subj ect can further comprise administering an oligonucleotide therapeutic agent. A disclosed oligonucleotide therapeutic agent can comprise a single- stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, noncoding RNA (ncRNA), an antisense molecule, miRNA, a morpholino, a peptide-nucleic acid (PNA), or an analog or conjugate thereof. In an aspect, a disclosed oligonucleotide therapeutic agent can be an ASO or an RNAi. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise one or more modifications at any position applicable. In an aspect, a disclosed oligonucleotide therapeutic agent can comprise a CRISPR-based endonuclease. In an aspect, a disclosed endonuclease can be Cas9. In an aspect, a disclosed Cas9 can be from Staphylococcus aureus or Streptococcus pyogenes.

[0307] In an aspect of a disclosed method, a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be delivered and/or administered prior to, concurrent, or after the delivery and/or administration of enzyme replacement therapy, protein replacement, gene therapy, a recombinant product, or any combination thereof.

[0308] In an aspect, a disclosed method of treating a subject can further comprise plasmapheresis and immunosuppression. In an aspect, a disclosed method can comprise using immunosuppression to decrease the T cell, B cell, and /or plasma cell population, decrease the innate immune response, inflammatory response, and antibody levels in general. In an aspect, antibodies can be to one or more components of a disclosed AAV particle or a disclosed AAV vector or to the product encoded by a disclosed transgene, heterologous nucleic acid, or payload.

[0309] In an aspect, a disclosed method of treating a subject can further comprise reducing and/or minimizing vector-mediated immunotoxicity and/or transgene immunogenicity (e.g., the ability to induce specific immunity). In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the efficacy of the recombinant product encoded by the transgene. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subj ect with one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof. In an aspect, vector-mediated immunotoxicity and/or transgene immunogenicity can decrease and/or reduce the ability and/or likelihood of re-dosing a subject with gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof.

[0310] In an aspect, a disclosed method can further comprise administering one or more times one or more disclosed enzymes or recombinant enzymes, one or more of the disclosed nucleic acid molecules, one or more disclosed AAV particles, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

In an aspect, a disclosed method of treating a subject can further comprise measuring and/or determining a subject’s pre-treatment level of one or more clinical and/or metabolic indicators (such as, for example, the expression of NPHSL NPHS2, PLCE1, CI)2AP._i LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) or the measure of a subject’s kidney function. In an aspect, a disclosed method can further comprise measuring and/or determining one or more times a subject’s level of one or more clinical and/or metabolic indicators.

[0311] In an aspect, a disclosed method of treating a subject can further comprise reducing the risk of rejection of one or more solid organ transplants. In an aspect, a disclosed method of treating a subject can further comprise improving the viability of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of treating a subject can further comprise reducing the risk of developing graft vs. host disease (GVHD) following transplantation of one or more solid organs (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of treating a subject can further comprise reducing the risk of rejection of one or more solid organ transplants (such as, for example, (i) a donor kidney or (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof). In an aspect, a disclosed method of treating a subject can further comprise enhancing transplant efficiency of one or more solid organs (such as, for example, (i) a donor kidney, (ii) a donor kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation into the subject in need thereof, or (iii) the subject’s own kidney that has been treated via a disclosed ex vivo perfusion protocol employing a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof prior to implantation back into the subject in need thereof). In an aspect, a disclosed method of treating a subject can extend and/or improve the life expectancy of a subject.

D. MISCELLANEOUS

[0312] Disclosed herein an adeno- associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03. Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01. In an aspect, positions 452 - 458 comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO: 191 - SEQ ID NO:8873. In an aspect, positions

452 - 458 comprise a sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23 or SEQ ID NO: 191 - SEQ ID NO:8873.

[0313] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position

453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q.

[0314] Disclosed herein is an adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43.

[0315] In an aspect, a disclosed variant can be used to improve and/or enhance gene transfer to one or more kidney cells or kidney-derived cell types when compared to the wild-type capsid protein. In an aspect, disclosed kidney cells or kidney-derived cell types can comprise kidney epithelial cells and/or kidney endothelial cell types.

[0316] In an aspect, disclosed kidney cells or kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0317] In an aspect, a disclosed variant can be used to improve and/or enhance gene transfer to any region or part of the kidney. In an aspect, a disclosed region or part of the kidney can comprise the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof. In an aspect, a disclosed AAV capsid protein can be used to improve the correlation in doseresponse (e.g., thereby improving efficiency).

[0318] Disclosed herein is an AAV capsid comprising a disclosed AAV capsid protein. Disclosed herein is an AAV vector comprising a vector genome encapsidated by an AAV capsid comprising a disclosed AAV capsid protein or encapsidated by a disclosed AAV capsid. In an aspect, a disclosed vector genome can comprise a first inverted terminal repeat (ITR) and a second ITR. In an aspect, a disclosed vector genome can comprise a nucleic acid sequence encoding a transgene or a payload between the first ITR and the second ITR. In an aspect, a disclosed nucleic acid sequence encoding a transgene, a heterologous nucleic acid, or a payload can be operably linked to a promoter. In an aspect, a disclosed transgene, a heterologous nucleic acid, or payload can encode a therapeutic RNA or a therapeutic protein. In an aspect, the therapeutic RNA is a circular RNA (cirRNA). In an aspect, a disclosed therapeutic RNA can be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA. In an aspect, a disclosed transgene, heterologous nucleic acid, or payload can encode a missing, deficient, and/or mutant protein or enzyme. In an aspect, a disclosed missing, deficient, and/or mutant protein or enzyme can be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, I. X HR SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COI.4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof.

[0319] In an aspect, a disclosed transgene, heterologous nucleic acid, or payload can encode apolipoprotein LI, fibrocystin, myosin heavy chain 9, nephrocystin 1, polycystin 1, polycystin 2, or any combination thereof. In an aspect, a disclosed transgene, heterologous nucleic acid, or payload can encode a gene-editing molecule. In an aspect, a disclosed gene-editing molecule can comprise a nuclease or a single guide RNA (sgRNA). In an aspect, a disclosed AAV particle or a disclosed AAV vector can be used to improve and/or enhance gene transfer to one or more kidney cells or kidney -derived cell types when compared to an AAV particle or an AAV vector having a wild-type capsid protein. In an aspect, disclosed kidney cells or kidney- derived cell types can comprise kidney epithelial cells and/or kidney endothelial cell types. In an aspect, disclosed kidney cells or kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0320] Disclosed herein is a nucleic acid molecule, comprising a nucleic acid sequence encoding a disclosed adeno-associated virus (AAV) capsid protein. Disclosed herein is a pharmaceutical formulation comprising a disclosed AAV particle or a disclosed AAV vector and at least one pharmaceutically acceptable carrier. Disclosed herein is a method of delivering a transgene or a payload to a target cell in a subject, the method comprising administering to the subject a therapeutically effective amount of a disclosed AAV particle or a disclosed AAV vector, or a disclosed pharmaceutical formulation.

[0321] In an aspect, a disclosed target cell can be a kidney cell or a kidney-derived cell type.

[0322] In an aspect, disclosed kidney cells or kidney-derived cell types can comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof. In an aspect, a disclosed target cell can be any region or part of the kidney. In an aspect, a disclosed region or part of the kidney can comprise the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

[0323] Disclosed herein is a method of treating a subject in need thereof, the method comprising administering to a subject having a kidney disease or kidney disorder a therapeutically effective amount of a disclosed AAV vector or a disclosed AAV particle, or a disclosed pharmaceutical formulation. In an aspect, a disclosed kidney disease or disorder can comprises Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof. In an aspect, a disclosed method can further comprise administering one or more additional therapeutic agents. In an aspect, a disclosed method can further comprise monitoring the subject for adverse effects. In an aspect, following the administering step, one or more symptoms of the subject’s kidney disease or kidney disorder can be improved and/or alleviated. In an aspect, following the administering step, one or more aspects of the subject’s cellular homeostasis and/or cellular functionality can be restored and/or improved.

[0324] Disclosed herein is a method of generating AAV capsid proteins, the method comprising performing multiple rounds of evolution in one or more kidney or kidney-related models using one or more species. In an aspect, a disclosed method can further comprise generating an initial library of capsid proteins. In an aspect, generating the initial library of capsid proteins can comprise using saturation mutagenesis of variable region IV (corresponding to amino acids 425-458) of SEQ ID NO:OL In an aspect, a disclosed method can further comprise packaging the initial library of capsid proteins into an AAV vector using triple plasmid transfection. In an aspect, a first round of evolution can comprise intravenously administering to mice the AAV vector comprising the initial variant capsid library. In an aspect, a disclosed second round of evolution can comprise intravenously administering to pigs an AAV vector comprising the variant capsid library generated in the first round of evolution. In an aspect, a disclosed third round of evolution can comprise transducing differentiated human kidney organoids with the variant capsid library generated in the second round of evolution. In an aspect, a disclosed fourth round of evolution can comprise perfusing ex vivo a non-human primate kidney with the variant capsid library generated in the third round of evolution. In an aspect, a disclosed method can further comprise assessing sequence diversity of the variant capsid library generated in the one or more rounds of evolution. In an aspect, a disclosed method can further comprise calculating the percent representation and fold enrichment of each evolved variant capsid library when compared to the parental capsid library. In an aspect, a disclosed method can further comprise ranking the amino acid sequences based on percent representation and fold enrichment to identify one or more candidate capsid proteins. In an aspect, a disclosed method can further comprise characterizing the one or more candidate capsid proteins. In an aspect, the one or more species can comprise Mus Musculus (mouse), Sus scrofa (pig), non-human primates (Macaca, macaque), o Homo sapiens (human), or any combination.

[0325] Disclosed herein is a plasmid encoding a disclosed AAV capsid protein. Disclosed herein is a cell line comprising a disclosed AAV capsid protein. In an aspect, a disclosed cell line can further comprise a vector genome and an AAV rep gene.

[0326] Disclosed herein is a method of making an AAV vector the method comprising culturing a disclosed cell line under conditions such that it produces the AAV vector; and harvesting the AAV vector from the cell.

[0327] Disclosed herein is an AAV capsid library comprising one or more disclosed capsid proteins. Disclosed herein is an AAV capsid library comprising one or more of the capsid proteins made by a disclosed method. Disclosed herein is an AAV capsid library comprising (i) a first AAV capsid protein comprising the sequence set forth in SEQ ID NO:01, and (ii) one or more adeno-associated virus (AAV) capsid proteins comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q.

TABLE 5 - SEQUENCES IN SEQUENCE LISTING

IX. EMBODIMENTS

[0328] This disclosure provides the following non-limiting embodiments.

[0329] Embodiment 1. An adeno-associated virus (AAV) capsid protein comprising the sequence set forth in SEQ ID NO: 03.

[0330] Embodiment 2. An adeno-associated virus (AAV) capsid protein comprising a sequence having at least 90% identity to the sequence set forth in SEQ ID NO:03.

[0331] Embodiment 3. An adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 relative to SEQ ID NO:01.

[0332] Embodiment 4. The AAV capsid protein of Embodiment 3, wherein positions 452 - 458 comprise the sequence set forth in any one of SEQ ID NO:04 - SEQ ID NO:23.

[0333] Embodiment 5. An adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q.

[0334] Embodiment 6. An adeno-associated virus (AAV) capsid protein comprising the sequence set forth in any one of SEQ ID NO:24 - SEQ ID NO:43.

[0335] Embodiment 7. The AAV capsid protein of any one of Embodiments 1 - 6, wherein the variant demonstrates improved and/or enhanced gene transfer to one or more kidney cells or kidney-derived cell types when compared to the wild-type capsid protein.

[0336] Embodiment 8. The AAV capsid protein of Embodiment 7, wherein kidney cells or kidney-derived cell types comprise kidney epithelial cells and/or kidney endothelial cell types. [0337] Embodiment 9. The AAV capsid protein of Embodiment 7, wherein kidney cells or kidney-derived cell types comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof.

[0338] Embodiment 10. The AAV capsid protein of any one of Embodiments 1 - 6, wherein the variant demonstrates improved and/or enhanced gene transfer to any region or part of the kidney.

[0339] Embodiment 11. The AAV capsid protein of Embodiment 10, wherein the region or part of the kidney comprises the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

[0340] Embodiment 12. The AAV capsid protein of any one of Embodiments 1 - 11, wherein the AAV capsid protein demonstrates an improved correlation in dose-response.

[0341] Embodiment 13. An AAV capsid, comprising: the AAV capsid protein of any one of Embodiments 1 - 12.

[0342] Embodiment 14. An AAV vector, comprising: a vector genome encapsidated by an AAV capsid comprising the AAV capsid protein of any one of Embodiments 1 - 12 or encapsidated by the AAV capsid of Embodiment 13.

[0343] Embodiment 15. The AAV vector of Embodiment 14, wherein the vector genome comprises a first inverted terminal repeat (ITR) and a second ITR.

[0344] Embodiment 16. The AAV vector of Embodiment 15, wherein the vector genome comprises a nucleic acid sequence encoding a transgene or a payload between the first ITR and the second ITR.

[0345] Embodiment 17. The AAV vector of Embodiment 16, wherein the nucleic acid sequence encoding a transgene or a payload is operably linked to a promoter.

[0346] Embodiment 18. The AAV vector of Embodiment 16, wherein the transgene or payload encodes a therapeutic RNA or a therapeutic protein.

[0347] Embodiment 19. The AAV vector of Embodiment 18, wherein the therapeutic RNA is an antisense oligonucleotide, siRNA, shRNA, or mRNA.

[0348] Embodiment 20. The AAV vector of Embodiment 16, wherein the transgene or payload encodes a missing, deficient, and/or mutant protein or enzyme.

[0349] Embodiment 21. The AAV vector of Embodiment 20, wherein the missing, deficient, and/or mutant protein or enzyme is encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, C0Q2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, or any combination thereof.

[0350] Embodiment 22. The AAV vector of Embodiment 16, wherein the transgene or payload encodes apolipoprotein LI, fibrocystin, myosin heavy chain 9, nephrocystin 1, poly cystin 1, polycystin 2, or any combination thereof.

[0351] Embodiment 23. The AAV vector of Embodiment 16, wherein the transgene or payload encodes a gene-editing molecule.

[0352] Embodiment 24. The AAV vector of Embodiment 23, wherein the gene-editing molecule comprises a nuclease or a single guide RNA (sgRNA).

[0353] Embodiment 25. The AAV vector of any one of Embodiments 16 - 24, wherein the vector demonstrates improved and/or enhanced gene transfer to one or more kidney cells or kidney-derived cell types when compared to an AAV vector having a wild-type capsid protein. [0354] Embodiment 26. The AAV vector of Embodiment 25, wherein kidney cells or kidney- derived cell types comprise kidney epithelial cells and/or kidney endothelial cell types.

[0355] Embodiment 27. The AAV vector of Embodiment 25, wherein kidney cells or kidney- derived cell types comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof

[0356] Embodiment 28. A nucleic acid molecule, comprising: a nucleic acid sequence encoding the adeno-associated virus (AAV) capsid protein of any one of Embodiments 1 - 12. [0357] Embodiment 29. A pharmaceutical formulation comprising the AAV vector of any one of Embodiments 13 - 27 and at least one pharmaceutically acceptable carrier.

[0358] Embodiment 30. A method of delivering a transgene or a payload to a target cell in a subject, the method comprising: administering to the subject a therapeutically effective amount of the AAV vector of any one of Embodiments 17 - 27 or the pharmaceutical formulation of Embodiment 29.

[0359] Embodiment 31. The method of Embodiment 30, wherein the target cell is a kidney cell or a kidney-derived cell type.

[0360] Embodiment 32. The method of Embodiment 31, wherein kidney cells or kidney- derived cell types comprise glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct principal cells, collecting duct transitional cells, collecting duct intercalated cells, or cells in the vas afferens, vas efferens, ascending vasa recta, descending vasa recta, convoluted and straight proximal tubules, descending thin limb, ascending thin limb and thick ascending limb of loop of Henle, macular densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubules, or in any combination thereof

[0361] Embodiment 33. The method of Embodiment 30, wherein the target cell is any region or part of the kidney.

[0362] Embodiment 34. The method of Embodiment 33, wherein the region or part of the kidney comprises the adrenal glands, the cortex, the medulla, the renal column, the pyramid, the renal pelvis, the major calyx, the minor calyx, the papillae, the ureter, or any combination thereof.

[0363] Embodiment 35. Amethod of treating a subject inneed thereof, the method comprising: administering to a subj ect having a kidney disease or kidney disorder a therapeutically effective amount of the AAV vector of any one of Embodiments 17 - 27 or the pharmaceutical formulation of Embodiment 29.

[0364] Embodiment 36. The method of Embodiment 35, wherein the kidney disease or disorder comprises Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis complex (TSC), or any combination thereof.

[0365] Embodiment 37. The method of any one of Embodiments 35 - 36, further comprising administering one or more additional therapeutic agents.

[0366] Embodiment 38. The method of any one of Embodiments 35 - 37, further comprising monitoring the subject for adverse effects.

[0367] Embodiment 39. The method of any one of Embodiments 35 - 38, wherein following the administering step, one or more symptoms of the subject’s kidney disease or kidney disorder are improved and/or alleviated.

[0368] Embodiment 40. The method of any one of Embodiments 35 - 39, wherein following the administering step, one or more aspects of the subject’s cellular homeostasis and/or cellular functionality are restored and/or improved. [0369] Embodiment 41. A method of generating AAV capsid proteins, the method comprising: performing multiple rounds of evolution in one or more kidney or kidney -related models using one or more species.

[0370] Embodiment 42. The method of Embodiment 41, further comprising generating an initial library of variant capsid proteins.

[0371] Embodiment 43. The method of Embodiment 42, wherein generating the initial library of variant capsid proteins comprises using saturation mutagenesis of variable region IV (corresponding to amino acids 425-458) of SEQ ID NO:01.

[0372] Embodiment 44. The method of Embodiments 42 or 43, further comprising packaging the initial library of variant capsid proteins into an AAV vector using triple plasmid transfection.

[0373] Embodiment 45. The method of Embodiment 44, wherein a first round of evolution comprises intravenously administering to mice the AAV vector comprising the initial variant capsid library.

[0374] Embodiment 46. The method of Embodiment 45, wherein a second round of evolution comprises intravenously administering to pigs an AAV vector comprising the variant capsid library generated in the first round of evolution.

[0375] Embodiment 47. The method of Embodiment 46, wherein a third round of evolution comprises transducing differentiated human kidney organoids with the variant capsid library generated in the second round of evolution.

[0376] Embodiment 48. The method of Embodiment 47, wherein a fourth round of evolution comprises perfusing ex vivo a non-human primate kidney with the variant capsid library generated in the third round of evolution.

[0377] Embodiment 49. The method of any one of Embodiment 45 - 48, further comprising assessing sequence diversity of the variant capsid library generated in the one or more rounds of evolution.

[0378] Embodiment 50. The method of Embodiment 49, further comprising calculating the percent representation and fold enrichment of each evolved variant capsid library when compared to the parental capsid library.

[0379] Embodiment 51. The method of Embodiment 50, further comprising ranking the amino acid sequences based on percent representation and fold enrichment to identify one or more candidate variant capsid proteins.

[0380] Embodiment 52. The method of Embodiment 51, further comprising characterizing the one or more candidate variant capsid proteins. [0381] Embodiment 53. The method of Embodiment 41, wherein the one or more species comprise Mus Musculus (mouse), Sus scrofa (pig), non-human primates (Macaca, macaque), or Homo sapiens (human), or any combination.

[0382] Embodiment 54. A plasmid encoding the AAV capsid variant protein of any one of Embodiments 1 - 12.

[0383] Embodiment 55. A cell line comprising the AAV capsid protein of any one of Embodiments 1 - 12.

[0384] Embodiment 56. The cell line of Embodiment 55, wherein the cell line further comprises a vector genome and an AAV rep gene.

[0385] Embodiment 57. A method of making an AAV vector, the method comprising:

[0386] culturing the cell line of Embodiments 55 or 56 under conditions such that it produces the AAV vector; and harvesting the AAV vector from the cell.

[0387] Embodiment 58. An AAV capsid library, comprising: one or more of the capsid proteins of Embodiment 3.

[0388] Embodiment 59. An AAV capsid library, comprising: one or more of the capsid proteins made by the method of any one of Embodiments 41 - 53.

[0389] Embodiment 60. An AAV capsid library, comprising: (i) a first AAV capsid protein comprising the sequence set forth in SEQ ID NO:01, and (ii) one or more adeno-associated virus (AAV) capsid proteins comprising one or more amino acid substitutions at positions 452 - 458 of SEQ ID NO:01, wherein the substitution at position 452 is any amino acid other than N; wherein the substitution at position 453 is any amino acid other than G; wherein the substitution at position 454 is any amino acid other than S; wherein the substitution at position 455 is any amino acid other than G; wherein the substitution at position 456 is any amino acid other than Q; wherein the substitution at position 457 is any amino acid other than N; and/or wherein the substitution at position 458 is any amino acid other than Q.

X. EXAMPLES

[0390] The following is a description of various methods and materials used in the studies. These Examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention. These Examples are not intended to limit the scope of the disclosed invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, temperatures, etc.).

[0391] Chronic kidney disease (CKD) is estimated to affect 8-16% of the population worldwide and has increased by 31.7% over the last ten years. (Hill NR, et al. (2016) PLoS One. 1 l(7):e0158765; Evans M, et al. (2022) Adv. Ther. 39(l):33-43). Ultimately, CKD progresses to end-stage renal disease, where dialysis or kidney transplantation are the only viable options for renal replacement therapy. (Schrezenmeier E, et al. (2021) Genet Med. 23(7): 1219-1224; Tonelli M, et al. (2011) Am. J. Transplant. 11(10):2093-2109). Maintenance dialysis therapy requires multiple prolonged sessions per week and is characterized by poor patient survival. Kidney transplant has developed into a successful long-term therapy, but the field remains limited by donor organ scarcity and the need for lifelong immunosuppression. (Malek SK, et al. (2011) Transpl. Int. 24(5):419-424; Keith DS, et al. (2016) Clin. J. Am. Soc. Nephrol. 11(4):684-693). Many kidney diseases such as cystinuria, polycystic kidney disease, and cystinosis amongst others have underlying genetic etiologies that may be amenable by gene therapy or genome editing, underscoring the crucial unmet need for an effective and safe kidney-targeting gene delivery vehicle. (Peek JL, et al. (2023) Nat. Rev. Nephrol. 19(7):451- 462; Rubin J D, et al. (2020) Mol. Diagnosis Ther. 24(4):375-396; Peek JL, et al. (2022) Curr Opin Nephrol Hypertens. 31(2): 175-179; Hildebrandt F. (2010) Lancet. 375(9722): 1287- 1295). Gene delivery to the kidney, however, has proven difficult, limiting the translation of genetic medicines to over 10% of the global population suffering from chronic kidney disease. [0392] Within this framework, recombinant adeno -associated virus (AAV) vectors constitute a promising technology platform for human gene therapy. (Pupo A, et al. (2022) Mol. Ther. 30(12):3515-3541; Wang D, et al. (2019) Nat. Rev. Drug Discov. 18(5):358-378).

[0393] With the approval of five AAV-based products by the FDA for treating ocular, hematological and neuromuscular disorders, there are -200 completed or active gene therapy clinical trials registered at ClinicalTrials.gov as of September 2023. However, little progress has been made to date in achieving effective therapeutic gene transfer to kidneys. Due to the intrinsic filtering function and complex physiology of kidneys, successful gene delivery has proven challenging. (Rubin J D, et al. (2020) Mol. Diagnosis Ther. 24(4):375-396; Rubin JD. et al. (2019) Hum. Gene Ther. 30(12): 1559-1571).

[0394] Here, new cross-species compatible, AAV-kidney (AAV.k) variants, were evolved by cycling AAV capsid libraries across different kidney systems. To cycle AAV capsid libraries, this robust approach utilized (i) intravenous dosing in mice and pigs, (ii) ex vivo machine perfusion in isolated rhesus macaque kidneys using both ureteral and arterial delivery, and (iii) human organoid cultures. By employing a multi-model and multi-species evolution strategy in vivo and ex vivo, novel AAV.k variants were created and evolved, thereby serving as delivery vehicles for therapeutic kidney gene transfer. Multiple new variants, notably, AAV.kl3 and AAV.k20 were enriched following sequential intravenous (IV) cycling through mouse and pig kidneys, ex vivo cycling in human organoid cultures, and ex vivo machine perfusion in isolated non-human primate (NHP) kidneys. Following IV administration, these vectors display robust and widespread transduction in murine kidneys, with selective tropism for proximal tubules. Markedly higher transgene expression compared to parental AAV9 vectors was also observed in proximal tubule epithelial cells within human organoid cultures and in transplanted pig kidneys, underscoring potential for clinical translation.

[0395] AAV.kl3 and AAV.k20 variants are promising vectors for gene therapy and genome editing to treat kidney diseases and enhance kidney transplant outcomes.

METHODS AND MATERIALS USED IN EXAMPLES

1. Study Design.

[0396] The approach employed here involved cycling an AAV9 VR-IV library intravenously in mice and pig, on human kidney organoids, and in ex vivo machine perfusion in NHP kidney via arterial and ureteral routes. All mouse, pig, and NHP protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at Duke University and at North Carolina State College of Veterinary Medicine. During the study, the care and use of animals was conducted in accordance with the guidelines of the USA National Research Council, the US Department of Agriculture (Animal Welfare Act; Public Law 99-198) and those of the Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011).

2. AAV Capsid Libraries and Recombinant Vector Production.

[0397] The AAV9 VR-IV plasmid library was designed, constructed, and produced in-house. Recombinant AAV vectors packaging different genome cassettes were produced by triple plasmid transfection in HEK293 cells, purified, and analyzed. (Gonzalez TJ, et al. (2022) Nat. Commun. 13(1):5947; Gonzalez T J, et al. (2023) Nat. Protoc. 18(1 l):34I3-3459).

3. Cycling of AAV Capsid Libraries in Kidney Model Systems.

[0398] AAV VR-IV libraries were produced as described in the section above. The first round of evolution was performed in mice following intravenous administration. The kidney was harvested three days post-injection and dissected. Half of the sagittal dissection was used for genomic DNA isolation. AAV genomes were amplified from mouse genomic DNA using primers targeting AAV9 Cap.

TABLE 6 - PRIMER SEQUENCES

[0399] PCR amplicons were digested and ligated into the AAV library plasmid backbone and used to generate the round 2 AAV9 VR-IV library. Schematics were created in part using BioRender. The second round of evolution occurred in pigs following intravenous administration. Briefly, Landrace, Yorkshire and Duroc cross-strain pigs (n = 2) were used for AAV library cycling and evolution. Studies were performed in 3-week-old newly weaned piglets weighing approximately 7 kgs. Male and female piglets were injected systemically via the heart vena cava with lel3 vg/kg of the AAV VR-IV library. Kidneys were harvested 3 days post-injection and prepared for library amplification. Pig kidneys were harvested 3 days post injection and dissected into multiple cortex and medulla regions before genomic DNA isolation. The AAV VR-IV library region was amplified and ligated into the AAV library plasmid backbone and used to produce the next round of AAV VR-IV library.

[0400] The third round of evolution occurred on differentiated human kidney organoids, where the pig cortex and medulla AAV capsid libraries were pooled at a 1 : 1 ratio and used to transduce organoids. Genomic DNA was isolated 24 hours post-transduction, and the AAV VR-IV library region was amplified and used to generate the next round of AAV capsid library. [0401] The final round of evolution was performed using an ex vivo NHP kidney machine perfusion system. Briefly, kidneys from rhesus macaques, weighing 4 kg - 8 kg, were perfused using the method described above. AAV capsid library generated from evolving on human kidney organoids was perfused for four hours via the arterial route and ureteral route of two different NHP kidneys. NHP kidneys were flushed and dissected before extracting genomic DNA. All kidneys were perfused with PBS to remove AAV variants that may be surface bound, but not internalized by kidney cell types. Additional details for ex vivo kidney perfusion of AAV vectors in pigs and NHPs are outlined below. The final evolved library was prepared as previously described. Next-generation sequencing was performed on libraries to track the progress of the evolution.

4. High-Throughput Sequencing Analysis and Identification of Newly Evolved AAV.k Variants.

[0402] AAV capsid library vectors were produced from AAV library plasmids that were generated after every step in the evolution. Evolved viral libraries were DNAse-I treated to extract viral genomes from capsids and Illumina adapters were added via PCR. For all but the NHP. the first PCR adds Illumina adapters using primers specific to the amplicon, while the second PCR adds the Illumina indexing adapters. For NHP sequencing the first PCR with Illumina adapters was done followed by amplicon EZ sequencing (2 x 250 bp configuration) (Genewiz). Libraries were then prepared for sequencing with the Illumina NovaSeq 6000 S- Prime Reagent Kit (2 x 150 bp configuration) and sequenced on the Illumina NovaSeq System. PCR products were purified using the PureLink PCR Micro Kit (Invitrogen) and concentrations were quantified using Qubit spectrometer (Thermo Fisher Scientific). NGS reads were analyzed with an in-house Python script that counts and ranks nucleotides sequences of the library regions. These nucleotide sequences are then translated to amino acid sequences, which are also counted and ranked.

5. Mouse Studies.

[0403] All experiments were approved by the Institutional Animal Care and Use Committees at Duke University. In this study, C57BL/6J mice were housed in a temperature controlled and enriched environment, with a 12-hour light/dark cycle, and provided water and standard chow.

6. Intravenous Administration in Wild-Type C57/B6 Mice.

[0404] For all mouse studies, 8-10-week-old male and female adult mice were systemically administered with AAV via the tail vein. The wild-type C57/B6 mouse colony was bred and maintained at Duke University. For the AAV capsid library evolution in C57/B6 mice (n = 2), AAV was injected systemically at 2el3 vg/kg. Kidneys were harvested 3 days post-injection and prepared for library amplification. For the mCherry reporter studies (n = 3), C57/B6 mice were injected systemically at a dose of 5el3vg/kg of AAV. Kidneys were harvested 4 weeks post injection for all gene transfer studies in C57/B6 mice.

7. Ex Vivo Kidney Perfusion of AAV Vectors in Pigs.

[0405] Machine perfusion of porcine kidneys was conducted using an automated and portable perfusion platform that was specifically developed by BioMed Innovations Inc., Organ Bank. The organ was perfused with a solution made of human albumin, bicarbonate-based dialysate (B. Braun Medical Inc., Melsungen, Germany), calcium gluconate, heparin, multivitamins, dexamethasone, and piperacillin/tazobactam at a temperature between 22 °C and 25 °C. An additional nutritional supplement called Clinimix (Baxter International Inc., Deerfield, IL, USA) and regular insulin were continuously infused into the organ along with verapamil. The perfusate was oxygenated using a mixture of carbogen, which consists of 95% oxygen (O2) and 5% carbon dioxide (CO2), at a flow rate of 2 L/min - 3 L/min. The renal artery was cannulated and linked to the device; however, the renal vein was intentionally left open to facilitate drainage. The ureter was cannulated similarly. A pressure-controlled pump was used to progressively elevate the mean arterial pressure to 70 mmHg after a 30-minute warming-up time. Different AAV vectors were delivered either through the ureter route or the ureter and renal artery route; subsequently, the ureter was clamped to obstruct the outflow of urine.

[0406] For transplant studies, nephrectomy was performed in pigs and ex vivo machine perfusion was initiated using the conditions described above. During machine perfusion, AAV vectors packaging a self-complementary Cbh-mCherry cassette were administered via the arterial route or ureteral route. Following the machine perfusion period, kidney transplantation was performed by auto-transplantation back into the same pig. Kidneys were harvested 9-14 days post-transplant.

8. Histology and Immunohistochemistry Staining of Pig Kidney Tissues for mCherry.

[0407] Biopsies from harvested kidneys were fixed in 10% formalin and embedded in paraffin. Immunohistochemical stains were performed using chicken anti-mCherry (1 : 1000; Novus Biologies NBP2-25158). Immunohistochemistry was performed using a horseradish peroxidase conjugated anti-chicken secondary antibody and DAB as the chromogenic substrate. Whole slide digital images were taken using the Aperio AT Turbo digital slide scanner system (Leica Biosystems) and viewed with Imagescope (Leica Biosytems) digital pathology software.

9. iPSC-Derived 2D Kidney Organoid Differentiation and Staining.

[0408] iPSCs (WTC-11 cell line; Coriell, GM25256) were maintained in mTeSRl medium (STEMCELL Technologies 85850) with daily medium changes. When iPSCs reached 70-80% confluence, the iPSCs were dissociated using Accutase (Gibco Al 110501) and passaged onto 6-well plates pre-coated with 1% geltrex (Gibco A1413202). iPSCs were differentiated into kidney organoids (Lian E, et al. (2023) STAR Protoc. 4: 102314; Xu Y, et al. (2022) Nat Gen. 54(11): 1690-1701; Freedman BS, et al. (2015) Nat Commun. 6:8715). Briefly, 24-well plates were pre-coated with 300 pL of DMEM-F12 containing 0.2 mg/mL Matrigel (Coming 356230). iPSCs were then dissociated and plated as single cells at 2,000 cells per well in 500 pL of mTeSRl medium containing 10 pM Y-27632 (STEMCELL Technologies 72302). The next day, the media was replaced with 500 pL of mTeSRl containing 0.2 mg/mL Matrigel to produce scattered, isolated spheroid colonies. An mTeSRl media change was performed 24 hours later. Three days after plating, hPSC-derived spheroids were treated with 12 pM CHIR99021 (Sigma 1046) in 1,000 pL of advanced RPMI (Gibco 12633012) + l x Glutamax (Gibco 35050061) + Pen-strep (Gibco 15140122). 36 to 42 hours later, media was then changed to 1,000 pL RB (Advanced RPMI + l x Glutamax + Pen-strep + l x B27 Supplement (Gibco 17504001)). RB changes were then performed every 2-3 days post-differentiation. 1E10 viral genomes of AAV9, AAV.kl3 or AAV.k20 packaging self-complementary CBh- mCherry were added to the organoids at day 10 post-differentiation in 1,000 pL mTeSRl for 48 hours then exchanged with fresh mTeSRl without virus. 5 days post virus addition, organoids were fixed with 4% paraformaldehyde for 15 min at room temperature. After fixing, organoids were washed in PBS then blocked in 10% donkey serum + 0.3% Triton-X-100 in PBS for 1 hour. Organoids were then incubated overnight at 4 °C with primary antibodies in PBS containing 1% BSA + 0.3% Triton-X-100 + 0.1 mM Calcium Chloride. Organoids were then washed and incubated with secondary antibodies and 2 pg/mL DAPI overnight at 4 °C in the same antibody buffer and imaged the next day after washing using an inverted fluorescence microscope. Primary antibodies used were LRP2 (1 : 100; rabbit; Abeam 76969), Nephrin (1 : 100, sheep; R&D Systems AF4269-SP), and mCherry (1 : 100; rat; Invitrogen Ml 1217). Secondary antibodies used at 1 :400 were donkey anti-Rabbit 488 (Invitrogen A21206), donkey anti-Sheep 594 (Invitrogen Al 1016), and donkey anti -Rat 647 (Invitrogen A48272).

10. iPSC-Derived 3D Human Kidney Organoid Differentiation and Processing.

[0409] A human EE0000002 DMl-iPSC cell line available through the National Institute of Neurological Disorders and Stroke (NINDS) Human Cell and Data Repository (NHCDR) was used in combination with an established protocol to generate differentiated human kidney organoids. (Przepiorski A, et al. (2021) J. Vis. Exp. (170): 10.3791/62452). At Day 14, kidney organoids were evenly distributed in a 6-well plate, about 10 organoids/well, and prepared for transduction assays. Kidney organoids were transduced at Day 14 with either lei 1 vg or lel2 vg of AAV vector packaging a self-complementary Cbh-mCherry cassette by adding directly into the well. Media was replenished with fresh Stage II media 24 hours post-transduction. Kidney organoids were harvested every five days post-transduction and placed in either 4% PFA for 24 hours or RNAlater (Invitrogen). Organoids placed in 4% PFA were rinsed three times with PBS and subsequently placed in 30% sucrose on a tube rotator at 4°C for 24 hours. Organoids were then embedded in Tissue-Tek O.C.T Compound (Sakura) molds and carefully dipped in liquid nitrogen-chilled 2-methyl butane until frozen blocks were formed. Frozen organoid blocks were sectioned at 8 pm on the Leica CM1520 cryostat and mounted on Superfrost Plus Microscope Slides (Fisherbrand) and stored at -80 °C. DNA and RNA were extracted from organoids kept in RNAlater using the DNA/RNA/Protein Extraction Kit (IB I) and kept at -20 °C and -80 °C, respectively.

11. iPSC-Derived 3D Human Kidney Organoid Immunostaining.

[0410] Cryosectioned organoids were allowed to come to room temperature for 10 minutes and rinsed once in PBS. Organoids were then incubated in 2% BSA + 0.5% Triton X-100 in PBS for 1 hour at room temperature. Organoids were then quickly rinsed in PBS and incubated overnight at 4 °C with primary antibodies in PBS containing 2% BSA + 0.5% Triton-X-100. Organoids were then washed three times in PBS and incubated with secondary antibodies in PBS containing 2% BSA + 0.5% Triton-X-100 at room temperature for 1 hour. Organoids were then washed three times in PBS and mounted using ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen). Slides were then imaged using an Echo Revolve microscope or Zeiss 880 Airyscan Fast Inverted Confocal at Duke University Light Microscopy Core Facility. Primary antibody used was RFP (1 :200; rabbit; Rockland 600-401- 379). Secondary antibody used was anti-rabbit Alexa Fluor 594 (1 :500; Invitrogen A-l 1012) and LTL-FL (1 :200; Vector Laboratories FL-1321-2).

12. Determination of Vector Genome Uptake in Organoids by Quantitative qPCR.

[0411] After extracting DNA, vector genomes were quantified via quantitative PCR, using a self-complementary Cbh-mCherry plasmid standard and primers targeting an mCherry amplicon (see Table 6). The uptake of viral genomes is represented as the ratio of vector genomes per microgram of DNA extracted for both lei 1 vg/well and lel2 vg/well conditions. Quantitative PCR reactions were carried out using a Roche Light-Cycler 480 and SYBR Green I Master (Roche Applied Sciences).

13. Determination of AAV Vector Transcripts in Organoids by Quantitative RT-PCR.

[0412] Extracted RNA from organoids was subjected to DNAse treatment using TURBO DNA-free kit (Invitrogen). Equal amounts of DNAse-treated RNA were used for cDNA synthesis using the High-Capacity RNA-to-cDNA kit (Applied Biosystems). Newly synthesized cDNA was used for quantitative PCR using primers specific to mCherry and human RPL13 A (see Table 6). Quantitative RT-PCR reactions were carried out using a Roche Light-Cycler 480 and SYBR Green I Master (Roche Applied Sciences).

14. Determination of Vector Genome Biodistribution by Quantitative qPCR.

[0413] After extracting DNA from mouse kidneys, vector genomes were quantified via quantitative PCR, using a self-complementary Cbh-mCherry plasmid standard and primers targeting an mCherry amplicon (see Table 6). The biodistribution of viral genomes is represented as the ratio of vector genomes per microgram of DNA extracted. Quantitative PCR reactions were carried out using a Roche Light-Cycler 480 and SYBR Green I Master (Roche Applied Sciences).

15. Immunostaining of Mouse Kidney Tissues.

[0414] Frozen sections were allowed to come to room temperature for a few seconds then fixed in 4% PFA for 15 minutes. After washing with PBS, sections were blocked for 1 hourin Power Block (Biogenex Laboratories HK0855K) with 10% donkey serum then incubated overnight at 4 °C with primary antibodies in PBS containing 5% donkey serum + 2.5% BSA + 0.05% Tween. After washing, sections were then incubated with secondary antibodies and 0.25 pg/mL DAPI for one hour at room temperature in the same antibody buffer. After washing the sections with PBS, coverslips were mounted in ProLong Gold without DAPI (Invitrogen P36934). After curing, slides were imaged using an inverted fluorescence microscope. Primary antibodies used were SGLT1 (1 : 1000; rabbit; Izumi Kaji lab 576-610), SGLT2 (1 : 100, mouse; Santa Cruz sc-393350), and mCherry (1 :500; rat; Invitrogen Ml 1217). Secondary antibodies used at 1 :500 were donkey anti-Rabbit 488 (Invitrogen A21206), donkey anti-Rat 594 (Invitrogen A21209), and donkey anti-Mouse 647 (Invitrogen A31571).

[0415] All C57/B6 mouse kidneys were post fixed in 10% formalin overnight washed 3x in IX PBS before sucrose treatment. Harvested kidney organoids and post-fixed mouse kidneys were incubated in 30% sucrose on a tube rotator for 48 hours at 4 °C. Specimens were then embedded in Tissue-Tek O.C.T Compound (Sakura) compound and snap frozen in liquid nitrogen-chilled 2-methylbutane and cryosectioned on a Leica CM1520 cryostat and mounted on Superfrost Plus Microscope Slides (Fisherbrand) and stored at -80 °C. Cryosectioned mouse kidneys were allowed to come to room temperature for 15 minutes and rinsed once in PBS. Mouse kidneys were then incubated in blocking buffer (5% normal goat serum, 0.1% Triton X-100 in IX PBS) for 1 hour at room temperature. Mouse kidneys were then quickly rinsed three times in PBS and incubated overnight at 4 °C with primary antibodies diluted in blocking buffer. Mouse kidney tissues were then rinsed three times in PBS and incubated with secondary antibodies diluted in blocking buffer at room temperature for 1 hour. Sections were then washed three times in PBS and followed by treatment with Vector TrueView Autofluorescence Quenching Kit (Vector Laboratories). Sections were then quickly washed in PBS and mounted using ProLong Gold Antifade Mountant with DNA Stain DAPI (Invitrogen). Slides were then imaged using an Echo Revolve microscope or Zeiss 880 Airyscan Fast Inverted Confocal at Duke University Light Microscopy Core Facility. Primary antibody used was RFP (1 :200; rabbit; Rockland 600-401-379) and mouse nephrin (1 :200; goat; R&D Systems AF3159). Secondary antibody used was anti -rabbit Alexa Fluor 594 (1 :500; Invitrogen A-l 1012), anti-goat Alex Fluor 488 (1 :500; Invitrogen A-l 1055), DBA-FL (1 :200; Vector Laboratories FL-1031) and LTL-FL (1 :200; Vector Laboratories FL-1321-2).

[0416] To quantify total fluorescence intensity for the mCherry studies in mice, the following equation was used: total fluorescence intensity = Integrated Density - (Area of tissue region X mean fluorescence of background readings). Total fluorescence intensity of mCherry was then normalized to LTL intensity. All mouse staining quantification was performed using at least two sections and taking three images of each section for every mouse (n = 3). Similarly, total fluorescence intensity for the mCherry studies in human kidney organoids was performed using the same approach. For organoids, mCherry expression was normalized to DAPI.

16. Statistical Analysis.

[0417] GraphPad Prism Software (Version 10.0.2; GraphPad Software Inc) was used for statistical analysis. All values are presented as mean ± standard deviation. For data sets with four groups, significance was determined by one-way ANOVA, with Tukey’s post-test unless otherwise noted. Where indicated, */? < 0.05; **/? < 0.01; ***/? < 0.001; ****/? < 0.0001; ns is not significant.

EXAMPLE 1 CYCLING CAPSID LIBRARIES ACROSS DIFFERENT KIDNEY SYSTEMS YIELDED NEW AAV VARIANTS

[0418] To engineer AAV-kidney (AAV.k) variants, AAV9-based capsid libraries were generated. These capsid libraries were then subjected to sequential evolution first in mice, then in pigs via intravenous administration, which was then followed by infectious cycling ex vivo on human kidney organoids and finally ex vivo machine perfusion through isolated NHP kidneys via arterial and ureteral routes. (FIG. 1A). Capsid libraries were constructed via saturation mutagenesis of AAV9 variable region IV, which correspond to amino acids 452-458 (VP1 subunit numbering). This surface epitope has an important role as it is involved with the three-fold symmetry axis in cellular uptake, transduction, and neutralizing antibody recognition. (Gonzalez TJ, et al. (2022) Nat. Commun. 13(1):5947; Adachi K, et al. (2014) Nat. Commun. 5:3075; Emmanuel SN, et al. (2022) J. Virol. 96(3):e0125121; Tse LV, et al. (2017) Proc. Natl. Acad. Sci. USA. 114(24):E4812-E4821; Havlik LP, et al. (2020) J. Virol. 94(19):e00976-20; Chan KY, et al. (2017) Nat. Neurosci. 20(8): 1172-1179).

[0419] Wild-type AAV capsid libraries packaging genomes consisting of AAV2 Rep and AAV9 mutant Cap, flanked by AAV2 ITRs, were sequentially cycled. Enriched variants were amplified from mouse, pig, and (isolated) NHP kidneys. Enriched variants were also isolated from human organoid cultures. NGS analysis of each step in the library cycling process plotted as a function of fold enrichment (compared to parental library) and read depth enabled the identification of clones enriched across species (FIG. IB - FIG. IE). Notably, with respect to pig kidney isolated after intravenous library dosing, specific variants enriched in the kidney cortex vs. the medulla were identified (FIG. 1C, FIG. 5A - FIG. 5B). Of the -12,300 and 10,000 unique sequences that were enriched in the cortex and medulla, respectively, only -1100 overlapping variants were determined. Moreover, further analysis in ex vivo machine perfused NHP kidneys enabled comparison between clones enriched via ureteral vs. arterial delivery routes (FIG. IE, FIG. 5C - FIG. 5D). Variants having substitutions at positions 452 - 458 relative to SEQ ID NO:01 are represented in SEQ ID NO: 191 - SEQ ID NO:8873.

[0420] In this scenario, 4,552 unique sequences were enriched from the arterial route and 4,711 unique sequences were enriched from the ureteral route. There were also 5,913 overlapping variants. Deeper analysis of NGS data from theoretical, parental (packaged into AAV) and evolved libraries at each step of the evolution process was performed by calculating amino acid frequency at each position in the variable region IV loop (FIG. 6A - FIG. 6B). The parental library shows a broad distribution of amino acids for each position, with some structural bias for Gly, Ala, Vai, Ser and Arg residues as illustrated by red color intensity. Cys residues, which are prone to disulfide formation and aromatic side chain hydrophobic amino acids such as Trp, Tyr and Phe are generally not preferred from a structural compatibility standpoint. As the evolution progresses with increasing in complexity of the model, a strong deviation in amino acid preferences from mouse to higher organisms was observed. Striking preferences for basic Arg residues, but not Lys in positions 456 and 458 were observed for AAV variants enriched in human organoid and NHP kidney tissues. The different routes of administration (arterial vs ureteral), however, did not display notably different trends in amino acid preferences as discussed earlier. In contrast, subtle yet possibly meaningful differences were observed for long chain hydrophilic residues, i.e., Asp, Glu, Asn and Gin between the different regions (cortex vs. medulla) in pig kidneys. The structure-function correlates of such preferences are examined via additional mutational analysis.

EXAMPLE 2

AAV.K13 AND AAV.K20 WERE MORE POTENT THAN AAV9 VECTORS IN C57/B6 MICE AND PREFERENTIALLY TRANSDUCED PROXIMAL TUBULES

[0421] Two dominant variants, AAV.kl3 and AAV.k20, showed overlapping enrichment profiles across the various cycling parameters. These two variants were then compared to wildtype AAV9 by determining vector genome titers following research scale production. Regardless of transgene choice, no significant difference in total yields of AAV.kl3 and AAV.k20 when compared to AAV9 were observed (FIG. 7). First, four (4) weeks postadministration, the transduction profiles of AAV.kl3, AAV.k20, and AAV9 vectors packaging a self-complementary Cbh-mCherry cassette administered intravenously at a dose of 5el3 vg/kg at were examined (FIG. 2A). While no significant difference in vector genomes was noted in mouse kidneys and liver (FIG. 2G, FIG. 2H), kidney cryosections showed widespread and robust transduction with AAV.kl3 and AAV.k20 vectors when compared to AAV9 (FIG. 2B, demonstrated via native mCherry fluorescence). Using immunofluorescence microscopy, specific kidney markers were then used to determine localization of mCherry expression. Using anti-nephrin immunofluorescence, no localization of mCherry expression was observed in glomeruli, specifically, podocytes (FIG. 2E). Moreover, no localization of mCherry expression was observed in collecting ducts by Dolichos biflorus agglutinin (DBA)-staining (FIG. 2F, FIG. 8) However, robust transduction of proximal tubules was observed based on the co-localization of mCherry with lotus tetragonolobus lectin (LTL)-staining (FIG. 2C). Semi -quantitative assessment of relative fluorescence (mCherry to LTL) revealed AAV.kl3 had ~3-fold higher fluorescence intensity when compared to AAV9 while AAV.k20 had 6-fold higher fluorescence intensity when compared to AAV9 (FIG. 21). To further characterize proximal tubule regions transduced by AAV.kl3 and AAV.k20, anti-SGLTl and anti-SGLT2 antibodies were used to stain the SI and S2 segments of proximal tubules, respectively (FIG. 2D) Based on co-localization with SGLT2 staining, a relatively higher level of mCherry expression was detected in early proximal tubule segments.

[0422] In another experiment, AAV9, AAV.kl3 and AAV.k20 packaging a single-stranded CBA promoter driving the expression of luciferase (an enzyme that produces bioluminescence) was delivered IV at a total dose of 1E12 vg to 8-week-old C57/B6 mice via tail vein injection (200 pL volume). Four (4) weeks post-injection, the kidneys, hearts, and livers were harvested. [0423] DNA extractions were performed on all tissues to determine the distribution of AAV9, AAV.kl3, and AAV.k20 viral genomes. The mock for each tissue was set as baseline. For both heart and liver, there was no significant difference in number of viral genomes across all capsids. Meanwhile, AAV.k20 has about 3-fold less viral genomes than AAV9 in kidneys. (FIG. 14A - FIG. 14C)

[0424] To evaluate transduction efficiency, a luciferase assay was performed on these tissues. Mock tissues were considered baseline. There was no significant difference in luciferase activity in heart and liver samples for AAV.kl3 and AAV.k20 compared to AAV9. AAV.kl3 and AAV.k20 significantly outperformed AAV9 by 40-fold and 23-fold, respectively, in mouse kidneys (FIG. 14D - FIG. 14F) These experiments highlighted the ability of variant AAVs (i.e., AAV.k variants) to effectively transduce C57/B6 mice, further supporting its translational properties.

EXAMPLE 3

AAV.K13 AND AAV.K20 TRANSDUCED 2D AND 3D HUMAN KIDNEY ORGANOIDS WITH HIGH EFFICIENCY [0425] Briefly, at 10 days post-differentiation, human iPSCs that were embedded in Matrigel as single cells and differentiated into 2D kidney organoids were transduced with AAV9 and AAV.k variants. (Lian E, et al. (2023) STAR Protoc. 4(2): 102314; Xu Y, et al. (2022) Nat. Genet. 54(11): 1690-1701; Freedman BS, et al. (2015) Nat. Commun. 6:8715). Higher and selective mCherry expression (AAV.k20 > AAV.kl3 » AAV9) that co-localized with Lrp2, a transmembrane protein expressed on the surface of proximal tubular epithelial cells, was observed (FIG. 3 A). (Perez-Gomez MV, et al. (2020) Clin. Kidney J. 13(3):281 -286). Further, 3D human kidney organoids were generated in suspension and validated by quantitation of OCT4 transcript levels, a well-established sternness marker (FIG. 9A). (Shi G, et al. (2010) Stem Cell Res. Ther. 1(5):39).

[0426] At 5 days post-transduction in differentiated 3D human kidney organoids, the transduction profiles of AAV.k variants and AAV9 vectors packaging self-complementary CBh-mCherry incubated at lei 1 vg or lel2 vg total were compared (FIG. 3B). While no significant difference in uptake of viral genomes for AAV9 and AAV.k variants was observed at either incubated titer (FIG. 3C), a significant difference in mRNA transcript levels derived from AAV.k variants was observed when compared to AAV9 (FIG. 3D). Native mCherry fluorescence from cryosections show widespread and robust transduction at lei 1 vg/well for AAV.kl3 and AAV.k20 when compared to AAV9 (FIG. 3E). Quantification of relative mCherry fluorescence to DAPI nuclear staining revealed that AAV.kl3 had ~8-fold higher fluorescence intensity when compared to AAV9 and that AAV.k20 had 12-fold higher fluorescence intensity when compared to AAV9 (FIG. 3F). At the higher dose of lel2 vg/well, a saturating effect, wherein AAV.kl3 and AAV.k20 had ~1.5- and 3-fold higher fluorescence intensity, respectively, was observed when compared to AAV9 (FIG. 3G). Immunofluorescence for mCherry and LTL was then performed on organoids transduced with AAV9 or AAV.k variants (FIG. 3H, FIG. 9B). While some sporadic and non-specific transduction was observed with AAV9, AAV.kl3 and AAV.k20 demonstrated more robust and selective mCherry expression colocalized with LTL staining on proximal tubules (FIG. 3H, bottom panel). These results support the potential translatability of AAV.k vectors from murine models to human kidney models enabling robust and selective transduction of proximal tubule epithelial cell types using a ubiquitous promoter.

EXAMPLE 4 AAV TRANSDUCED TH1 CELLS (HUMAN RENAL PROXIMAL TUBULE EPITHELIAL) [0427] TH1 (human proximal tubule epithelial) cells were seeded at 2E5 cells/well in a 24- well plate. AAV9, AAV.kl3, AAV.k20, and AAV.k41 packaged a self-complementary Cbh promoter driving the expression of mCherry (a red monomeric fluorescent protein) at an MOI (multiplicity of infection) of 500K. (FIG. 10A). Cells were then imaged 18 hours and 4 days post-transduction. At 18 hours, robust expression of mCherry was present, noted by the red- colored cells, with AAV.k41. Visually, intensity of mCherry expression is noted AAV.k41 > AAV.k20 > AAV.kl3 > AAV9 18 hours post-transduction. At 4 days post-transduction, AAV.k41 shows robust and widespread expression of mCherry. Intensity of mCherry expression is noted AAV.k41 > AAV.k20 > AAV.kl3 > AAV9 at 4 days post-transduction. (FIG. 10B). These experiments highlighted the ability of AAV.kl3, AAV.k20, and AAV.k41 to effectively transduce human renal proximal tubule epithelial cells, supporting its translational properties.

[0428] Then, TH1 (human proximal tubule epithelial) cells were seeded at 2e5 cells/well in a 24-well plate. (FIG. 11A). AAV9, AAV.kl3, AAV.k20, and AAV.k41 packaged a singlestranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence, at an MOI (multiplicity of infection) of 500K. A luciferase assay was performed 24 hours post-transduction to determine transduction efficiency. AAV.k41 outperformed AAV9, AAV.kl3, and AAV.k20 by roughly a 14-fold change. (FIG. 11B). These experiments highlighted the ability of AAV.k41’s to effectively transduce human renal proximal tubule epithelial cells, supporting its translational properties.

[0429] Differentiated human kidney organoids were transduced at el l vg/well with AAV9, AAV.kl3, or AAV.k20 packaging a single-stranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence. Three days post-transduction, a luciferase assay was performed to determine transduction of efficiency of AAV capsids. AAV.k20 significantly outperformed transduction of human kidney organoids compared to AAV9, and AAV.kl3. There was roughly a 6-fold increase of luciferase activity of AAV.k20 compared to AAV9. (FIG. 12). These experiments highlighted the ability of AAV.kl3 and AAV.k20 to effectively transduce human kidney organoids, supporting its translational properties.

[0430] AAV.k20 packaging a single-stranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence, was delivered to the kidneys of nonhuman primate in situ via the ureter following midline laparotomy at a dose of 3E12 vg/kidney. Both kidneys received AAV.k20 employing a standard protocol. Initially, the distal part of the ureter was isolated and clamped, followed by cannulation with a 22G angiocatheter. Subsequently, the renal artery was isolated and administered systemic heparin to ensure adequate anti coagulation throughout the procedure. The renal artery was then temporarily clamped to arrest the blood flow. Following AAV.k20 administration through the ureter, a 15- minute waiting period was ensued for optimal distribution and uptake of the viral vector within the kidney tissue before unclamping both renal artery and ureter to restore the blood flow and urinary drainage.

[0431] Four weeks post in situ delivery, liver, ureters, and kidneys were harvested. Five biopsies were taken from the mock kidney, liver, left ureter, and right ureter. Meanwhile, ten (10) biopsies were taken from both the left and right kidney (biopsy numbers noted in images of NHP kidney). (FIG. 13A). To evaluate AAV.k20 transduction efficiency, a luciferase assay was performed on these biopsies. Mock kidney was considered baseline. The liver had an average 5 -fold increase compared to baseline. The left ureter has an average 44-fold increase compared to baseline. The right ureter had an average 7-fold increase to baseline. The left kidney had an average 62-fold increase compared to baseline, while the right kidney had an average 84-fold increase compared to baseline. Both left and right kidneys demonstrated significant luciferase activity compared to baseline. (FIG. 13B). DNA extractions were performed on all biopsies to determine the distribution of AAV.k20 viral genomes. The mock kidney was set as baseline. While the liver biopsies had 1 x 10⁶ vg/ug of DNA, biopsies for left ureter, right ureter, left kidney, and right kidney averaged to about 5 x 10⁴ vg/ug of DNA (all well above mock kidney). (FIG. 13C).

[0432] Biodistribution and luciferase activity from each of the ten biopsies were plotted separately, where biopsy location is noted by biopsy number. Experiment highlighted the ability of AAV.k20 to effectively transduce nonhuman primate kidneys, supporting its translational properties.

EXAMPLE 5 AAV.K13 AND AAV.K20 SHOWED ROBUST AND WIDESPREAD TRANSDUCTION IN PIG KIDNEYS EX VIVO

[0433] To further assess translatability of AAV.k vectors in transplant applications, AAV.k vectors were delivered to pig kidney grafts, either through direct administration during cold storage or during ex vivo machine perfusion. Treated kidney grafts were then transplanted and assessed at 1-2 weeks post-transplant to determine transduction efficiency. In current clinical practice, kidney grafts are preserved by either static cold storage on ice or by ex vivo machine perfusion. Machine perfusion, although more complex, can have some advantages due to its ability to improve kidney viability, deliver targeted treatment, and even reduce immunogenicity. (Zulpaite R, et al. (2021) Front Med (Lausanne). 8:808719; Yuzefovych Y, et al. (2020) Front. Immunol. 11 :265; Samoylova ML, et al. (2019) Clin. Transplant. 33:el3716; Moers C, et al. (2009) N. Engl. J. Med. 360(17):7-19).

[0434] Due to their similar structure and renal physiology to humans, pigs have been particularly well studied for preclinical evaluation of kidney-focused therapeutics. (Locke JE, et al. (2023) JAMA Surg. 158(10): 1106-1108; Gutierrez K, et al. (2015) Front. Genet. 6:293; Koslowski S, et al. (2020) Int. J. Mol. Sci. 21(12):4537; Lian X, et al. (2019) Br. J. Pharmacol. 176(5):711-724). For AAV.kl3, the efficacy of delivery during static cold storage was assessed. Following nephrectomy, the pig kidney was flushed with preservation solution and stored on ice. Half of the total AAV.kl3 vector dose was administered via the arterial route and the other half through retrograde ureteral delivery. Here, the pig kidney graft was stored on ice for 2 hours prior to auto-transplantation in the same animal (FIG. 4A). Following transplantation, immunohistochemistry (IHC) of mCherry protein performed on biopsies of pig kidney graft tissue revealed prominent and widespread expression in proximal tubules with some expression in distal tubules, but no signal in the glomerulus.

[0435] Next, the transduction profile of the AAV.k20 vector was assessed using the same transplant model, but with AAV delivery during ex vivo machine perfusion (FIG. 4B). A similar pattern, albeit with a more robust expression profile, was observed with AAV.k20 vectors using a dual perfusion via both the arterial and ureteral route. Building on these results, the AAV.k20 vector was delivered via ureteral administration alone to assess potential clinical translatability. (Chung DC, et al. (2011) Nephron Extra. 1 (1 ):217-223). Following successful transplantation, IHC analysis reaffirmed robust expression throughout the proximal tubules of the graft as described earlier (FIG. 4C).

[0436] Given the success of retrograde ureteral delivery of the AAV.k20 vector in during ex vivo machine perfusion, a comparison study between AAV9 and the AAV.k variants was performed using the same model and experimental conditions (FIG. 4D). All vectors were administered at the same total dose and route during ex vivo machine perfusion, and kidney grafts were assessed 9 days following transplantation. IHC analysis for mCherry expression in kidneys perfused with AAV9 vector revealed little to no expression of mCherry in glomeruli and distal tubules. Most importantly, no expression was observed in proximal tubules with the AAV9 vector. In stark contrast with AAV9, AAV.kl3, and AAV.k20 vectors demonstrated robust and widespread expression throughout proximal tubules, with some expression in distal tubules. These results not only support the translatability of AAV.k vectors across different preclinical models, namely, murine, human organoid, and porcine kidneys, but also highlight the potential translatability of retrograde ureteral delivery of AAV vectors in the clinic for the treatment of genetic kidney diseases as well as transplant applications.

[0437] In another experiment, machine perfusion of nonhuman primate kidneys was conducted as described above. The nephrectomy was performed in nonhuman primate and ex vivo machine perfusion was initiated using the conditions described above. Here, during machine perfusion, AAV.k20 packaging a self-complementary Cbh promoter driving the expression of mCherry cassette was administered via the ureteral route. Following the machine perfusion period, kidney transplantation was performed by auto-transplantation back into the same nonhuman primate. Kidneys were harvested 4-weeks post-transplant. Ten biopsies were taken from the transduced kidney (FIG. 15A), while three biopsies were taken from the mock kidney, and one biopsy from the liver and spleen. DNA extractions were performed on all biopsies to determine the distribution of AAV.k20 viral genomes. The mock kidney was set as baseline. While no viral genomes were detected in the liver and spleen biopsies, the kidney biopsies had significant and varying amounts of viral genomes according to biopsy location, with the average being 374-fold difference compared to mock. (FIG. 15B - FIG. 15C).

[0438] RNA extractions were also performed on all biopsies and mCherry expression was determined by first making cDNA from the RNA samples and then running a qPCR. mCherry cDNA levels were normalized to GAPDH. Mock kidney, liver, and spleen show little to no mCherry expression. Meanwhile, the ten kidney biopsies show significant and varying amounts of mCherry RNA with the average being 2300-fold difference compared to mock. (FIG. 15D - FIG. 15E) For both biodistribution and mCherry expression, data is plotted with respect to biopsy. Protein extractions were also performed on all biopsies and a western blot probing for vinculin (housekeeping gene) and mCherry (gene of interest) was performed. (FIG. 15F). The last column on the western blot is a positive control (mCherry input). The varying degrees of mCherry correlate with mCherry RNA levels for those biopsies. These experiments highlighted the ability of AAV.k20 to effectively transduce nonhuman primate kidneys, further supporting its translational properties.

EXAMPLE 6 COMPARISON OF AAV.K VARIANTS TO WILD-TYPE AAV9

[0439] Capsid proteins are compared to wild-type AAV9 by determining vector genome titers following research scale production. First, four (4) weeks post-administration to mice, the transduction profiles of one or more AAV capsid proteins (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873) and AAV9 vectors packaging a self- complementary Cbh-mCherry cassette are administered intravenously at a dose of 5el3 vg/kg. AAV capsid proteins are tested in batches of 20 or more. The distribution of vector genomes is examined and quantified. Kidney and liver cryosections are examined for transduction with one or more AAV capsid proteins and then compared to AAV9. Using immunofluorescence microscopy, specific kidney markers are then used to determine localization of mCherry expression. Using anti-nephrin immunofluorescence, localization of mCherry expression is determined. An examination of the collecting ducts is done using Dolichos biflorus agglutinin (DBA)-staining. Transduction of proximal tubules is performed using co-localization of mCherry with lotus tetragonolobus lectin (LTL)-staining. Semi-quantitative assessment of relative fluorescence (mCherry to LTL) is performed for one or more AAV capsid proteins and AAV9.

[0440] To further characterize proximal tubule regions transduced by one or more AAV capsid proteins, anti-SGLTl and anti-SGLT2 antibodies are used to stain the SI and S2 segments of proximal tubules, respectively. Based on co-localization with SGLT2 staining, a mCherry expression in early proximal tubule segments is examined.

[0441] In another experiment, one or more AAV capsid proteins (those having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873) packaging a single-stranded CBA promoter driving the expression of luciferase (an enzyme that produces bioluminescence) are delivered IV at a total dose of lel2 vg to 8-week-old C57/B6 mice via tail vein injection (200 pL volume). Four (4) weeks post-injection, the kidneys, hearts, and livers are harvested. DNA extractions are performed on all tissues to determine the distribution of viral genomes. The mock for each tissue is set as baseline. To evaluate transduction efficiency, a luciferase assay is performed on these tissues. Mock tissues are considered baseline.

EXAMPLE 7 AAV.K TRANSDUCTION OF 2D AND 3D HUMAN KIDNEY ORGANOIDS

[0442] At 10 days post-differentiation, human iPSCs embedded in Matrigel as single cells and differentiated into 2D kidney organoids are transduced with AAV9 and AAV.k variants. The co-localization of mCherry expression with Lrp2, a transmembrane protein expressed on the surface of proximal tubular epithelial cells, is observed. Further, 3D human kidney organoids are generated in suspension and validated by quantitation of OCT4 transcript levels, a well- established sternness marker. At 5 days post-transduction in differentiated 3D human kidney organoids, the transduction profiles of AAV.k variants and AAV9 vectors packaging self- complementary CBh-mCherry incubated at el l vg or lel2 vg total are compared. Native mCherry fluorescence from cryosections is also quantified and compared to AAV9. Quantification of relative mCherry fluorescence to DAPI nuclear staining is quantified and compared to AAV9. Immunofluorescence for mCherry and LTL is then performed on organoids transduced with AAV9 or AAV.k variants.

EXAMPLE 8

AAV.K TRANSDUCTION OF TH1 CELLS (HUMAN RENAL PROXIMAL TUBULE EPITHELIAL)

[0443] TH1 (human proximal tubule epithelial) cells are seeded at 2e5 cells/well in a 24-well plate. AAV9 and AAV.k variants (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873) are packaged with a self-complementary Cbh promoter driving the expression of mCherry (a red monomeric fluorescent protein) at an MOI (multiplicity of infection) of 500K. Cells are then imaged 18 hours and 4 days post-transduction. At 18 hours, expression of mCherry is measured. At 4 days post-transduction, mCherry expression is also examined.

[0444] Then, TH1 (human proximal tubule epithelial) cells are seeded at 2e5 cells/well in a 24-well plate. AAV9 and AAV.k variants (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873) are packaged a single-stranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence, at an MOI (multiplicity of infection) of 500K. A luciferase assay is performed 24 hours post-transduction to determine transduction efficiency.

[0445] Differentiated human kidney organoids are transduced at 1 el 1 vg/well with AAV9 and AAV.k variants (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO: 8873) packaging a single-stranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence. Three days post-transduction, a luciferase assay is performed to determine transduction of efficiency of AAV capsids. The best performing AAV.k variants packaging a single-stranded CBA promoter driving the expression of luciferase, an enzyme that produces bioluminescence, is delivered to the kidneys of nonhuman primate in situ via the ureter following midline laparotomy at a dose of 3el2 vg/kidney. Both kidneys receive AAV.k employing a standard protocol. Initially, the distal part of the ureter is isolated and clamped, followed by cannulation with a 22G angiocatheter. Subsequently, the renal artery is isolated and administered systemic heparin to ensure adequate anti coagulation throughout the procedure. The renal artery is then temporarily clamped to arrest the blood flow. Following administration of the AAV.k variant through the ureter, a 15-minute waiting period is ensued for optimal distribution and uptake of the viral vector within the kidney tissue before unclamping both renal artery and ureter to restore the blood flow and urinary drainage. [0446] Four weeks post in situ delivery, liver, ureters, and kidneys are harvested. Five biopsies are taken from the mock kidney, liver, left ureter, and right ureter. Meanwhile, ten (10) biopsies are taken from both the left and right kidney. To evaluate transduction efficiency of one or more AAV.k variants, a luciferase assay is performed on these biopsies. Mock kidney is considered baseline. DNA extractions are performed on all biopsies to determine the distribution of the AAV.k variant viral genomes. The mock kidney is set as baseline. Biodistribution and luciferase activity from each of the ten biopsies are plotted separately.

EXAMPLE 9 AAV.K TRANSDUCTION IN PIG KIDNEYS EX VIVO

[0447] To further assess translatability of AAV.k vectors in transplant applications, AAV.k vectors (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873) are delivered to pig kidney grafts, either through direct administration during cold storage or during ex vivo machine perfusion. Treated kidney grafts are then transplanted and assessed at 1-2 weeks post-transplant to determine transduction efficiency. In current clinical practice, kidney grafts are preserved by either static cold storage on ice or by ex vivo machine perfusion (as described above). For AAV.k variants (having the substitution represented by any one of SEQ ID NO: 191 - SEQ ID NO:8873), the efficacy of delivery during static cold storage is assessed. Following nephrectomy, the pig kidney is flushed with preservation solution and stored on ice. Half of the total AAV.k vector dose is administered via the arterial route and the other half through retrograde ureteral delivery. Here, the pig kidney graft is stored on ice for 2 hours prior to auto-transplantation in the same animal. Following transplantation, immunohistochemistry (IHC) of mCherry protein is performed on biopsies of pig kidney graft tissue.

[0448] Next, the transduction profile of the AAV.k variant vector is assessed using the same transplant model, but with AAV delivery during ex vivo machine perfusion. The AAV.k variant vector is then delivered via ureteral administration alone to assess potential clinical translatability. Following successful transplantation, IHC analysis is performed.

[0449] Given the success of retrograde ureteral delivery of the AAV.k20 vector in during ex vivo machine perfusion (discussed above), a comparison study between AAV9 and the AAV.k variants is performed using the same model and experimental conditions. All AAV.k vectors are administered at the same total dose and route during ex vivo machine perfusion, and kidney grafts are then assessed 9 days following transplantation. IHC analysis for mCherry expression in kidneys perfused with AAV9 vector and the AAV.k variant vector is examined. [0450] In another experiment, machine perfusion of nonhuman primate kidneys is conducted as described above. The nephrectomy is performed in nonhuman primate and ex vivo machine perfusion is initiated using the conditions described above. Here, during machine perfusion, AAV.k variants packaging a self-complementary Cbh promoter driving the expression of mCherry cassette is administered via the ureteral route. Following the machine perfusion period, kidney transplantation is performed by auto-transplantation back into the same nonhuman primate. Kidneys are harvested 4-weeks post-transplant. Ten biopsies are taken from the transduced kidney, while three biopsies are taken from the mock kidney, and one biopsy from the liver and spleen. DNA extractions are performed on all biopsies to determine the distribution of AAV.k variant viral genomes.

RNA extractions are also performed on all biopsies and mCherry expression is determined by first making cDNA from the RNA samples and then running a qPCR. mCherry cDNA levels are normalized to GAPDH. For both biodistribution and mCherry expression, data is plotted with respect to biopsy. Protein extractions are also performed on all biopsies and a western blot probing for vinculin (housekeeping gene) and mCherry (gene of interest) is performed.

SUMMARY OF EXAMPLES

[0451] These experiments describe the significantly augmented AAV transduction efficiency in the kidney across multiple model systems using a cross-species evolution approach. In particular, two capsid variants, AAV.kl3 and AAV.k20, show promise for preclinical development based on improved gene transfer efficiency in mice following IV administration and in human kidney organoids. Furthermore, retrograde ureteral delivery of AAV.kl3 and AAVk.20 variants was demonstrated to a promising approach for clinical translation with the ability to achieve robust kidney gene transfer at nominal AAV doses. These findings have significant implications for therapeutic kidney gene transfer applications as well as the genetic manipulation of kidneys for organ transplant applications.

[0452] A key element of this successful approach beyond cross-species evolution is the multifaceted approach ranging from kidney organoids to different routes of administration (i.e., intravenous vs. arterial vs retrograde ureteral). In particular, consideration of the structural, anatomical and physiological differences in kidneys across species played a key role in selective pressure. While mouse kidneys are uni-papillary, pig kidneys are multi -papillary and share similar cortical and medullary structures with human kidneys. Therefore, high- throughput sequencing on variants enriched separately from the pig cortex and medulla were performed. Only ~2% of the sequences (including the lead variants AAV.kl3 and AAV.k20) were found to be enriched in both anatomical regions, with nearly half of the remaining unique sequences being recovered individually from the cortex and medulla. This observation can indicate differential preferences in the regional biodistribution of different AAV variants within the kidney. Strikingly, when ex vivo machine perfusion techniques were used in this evolution platform, NGS data revealed nearly a third of the enriched sequences (including AAV.kl3 and AAV.k20) overlapped between arterial and ureteral routes. The remaining unique sequences split approximately in half. These results can indicate that AAV capsid accessibility to proximal tubule epithelial cells was not particularly limited by the route of administration. Further analysis of region-specific and route-specific capsid variants in NHP and/or pig kidneys is likely to yield additional candidates enabling different preclinical development paths.

[0453] Comparison of AAV.kl3, AAV.k20, and AAV9 following intravenous administration in mice did not reveal any significant differences in biodistribution. Strikingly however, higher transgene expression was observed in kidneys with AAV.k variants, specifically within proximal tubules. This observation was translatable across human kidney organoids and transplanted pig kidneys as well. This preferential transduction profile can be explained by the fact that proximal tubules constitute a significant portion of the kidney, with proximal tubule epithelial cells being a highly abundant cell population. From an anatomical perspective, the renal artery branches into the afferent and efferent arterioles, which form the vascular network that encapsulates proximal tubules and the rest of the nephron. Due to glomerular filtration, large macromolecules that do not enter the Bowman’s capsule are typically returned to the bloodstream through the efferent arterioles, specifically the peritubular capillaries. This can offer a potential uptake pathway involving transcytotic uptake from capillaries into the basolateral membrane of proximal tubule epithelial cells. This is corroborated in part by immunocolocalization staining of the SI and S2 segments of the proximal tubules with mCherry in mouse kidney tissue. Studies focused on dissecting the mechanistic underpinnings of AAV.k variant transduction in kidneys involve isolation of different cell types within the kidney as well as tracking of viral capsids and genomes. Expanded cellular tropism using AAV.k vectors can be driven by transgene expression using specific promoters for other kidney cell types and/or mining for additional enriched capsids as outlined supra. Nevertheless, the evolved properties of AAV.k variants are distinct from those of parental AAV9, which does not appear to appreciably transduce proximal tubule epithelia. In addition, the propensity to transduce proximal tubule epithelia (or lack thereof) is contrasted by the transduction profile of AAV.k variants vs. AAV9 in human organoids. Efforts to understand the kidney cell entry and post-entry mechanisms of AAV.k capsids continues. [0454] A particularly exciting attribute of the examples provided herein is the ability of AAV.k variants to transduce the pig kidney when administered via the ureter. Widespread expression was observed in the proximal tubules for AAV.k variants, but little to no expression was observed with AAV9. This result, combined with the expression profile observed in murine kidney and human kidney organoids provides a clear path for preclinical development in disease models. Notable examples of renal diseases involving proximal tubules are polycystic kidney disease, cystinuria, cystinosis amongst others. Moreover, AAV.k variants can enable transgene expression (secreted or cell surface localized) in the kidney with significant implications for renal transplantation. When combined with machine perfusion of donor organs, which can maintain the kidney in a functioning state, AAV.k vectors can enable expression of immunomodulatory agents prior to transplantation. This approach has great potential value with regard to evaluation of strategies to mitigate the risks of transplant rejection.

Claims

XI. CLAIMS What is claimed is:

1. An adeno-associated virus (AAV) capsid protein, wherein positions 452-458 of the AAV capsid protein comprise an amino acid sequence that is at least 85% identical to any one of SEQ ID NO:04 - SEQ ID NO:23, wherein the positions 452-458 of the AAV capsid protein is numbered with reference to SEQ ID NO:01.

2. The AAV capsid protein of Claim 1, wherein the AAV capsid protein comprises an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to any one of SEQ ID NO:24 - SEQ ID NO:43.

3. The AAV capsid protein of Claim 1, wherein positions 452-458 of the AAV capsid protein comprise the amino acid sequence of SEQ ID NO: 11.

4. The AAV capsid protein of Claim 1, wherein positions 452-458 of the AAV capsid protein comprise the amino acid sequence of SEQ ID NO: 15.

5. The AAV capsid protein of Claim 2, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:31.

6. The AAV capsid protein of Claim 2, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:35.

7. The AAV capsid protein of any one of Claims 1-6, wherein the AAV capsid protein is a variant of a parental wild-type capsid protein.

8. The AAV capsid protein of Claim 7, wherein the parental wild-type capsid protein is a capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrhlO.

9. The AAV capsid protein of Claim 8, wherein the parental wild-type capsid protein is VP1 of AAV9.

10. The AAV capsid protein of any one of Claims 7-9, wherein the AAV capsid protein improves gene transfer and/or expression in one or more region(s) or part(s) of kidney when compared to the parental wild-type capsid protein.

11. The AAV capsid protein of Claim 10, wherein the gene transfer and/or expression is improved at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 2 times, at least 3 times, at least 4 time, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, or at least 14 times.

12. The AAV capsid protein of Claim 10 or Claim 11, wherein the region or part of kidney is adrenal glands, cortex, medulla, renal column, pyramid, renal pelvis, major calyx, minor calyx, papillae, or ureter.

13. The AAV capsid protein of Claim 12, wherein the region or part of kidney is the proximal tubule in the cortex.

14. A nucleic acid molecule, comprising: a nucleic acid sequence encoding the AAV capsid protein of any one of Claims 1-13.

15. An AAV vector, comprising:

(a) a gene of interest; and

(b) the nucleic acid molecule of Claim 14.

16. The AAV vector of Claim 15, wherein the gene of interest encodes a therapeutic RNA or a therapeutic protein.

17. The AAV vector of Claim 16, wherein the therapeutic RNA is a circular RNA (cirRNA).

18. The AAV vector of Claim 16 or 17, wherein the therapeutic RNA is an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.

19. The AAV vector of any one of Claims 15-18, wherein the gene of interest is operably linked to a promoter.

20. The AAV vector of any one of Claims 15-19, further comprising a first inverted terminal repeat (ITR) and a second ITR.

21. A pharmaceutical formulation comprising the AAV vector of any one of Claims 15-20 and at least one pharmaceutically acceptable carrier.

22. A method of treating a subject with a kidney disease or kidney disorder, the method comprising: administering to the subj ect a therapeutically effective amount of the AAV vector of any one of Claims 15-20 or the pharmaceutical formulation of Claim 21.

23. The method of Claim 22, wherein the kidney disease or kidney disorder is Alport syndrome,

Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystinosis, Dent disease types 1 and 2, distal renal tubular acidosis, Fabry disease, familial amyloidosis, Gitelman syndrome, Liddle syndrome, Lowe syndrome, nephronophthisis (NPHP) autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal renal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, or tuberous sclerosis complex (TSC).

24. The method of Claim 22 or Claim 23, wherein the AAV vector of any one of Claims 15-

20 or the pharmaceutical formulation of Claim 21 is administered via retrograde ureteral infusion.

25. The method of Claim 22 or 23, wherein the AAV vector of any one of Claims 15-20 or the pharmaceutical formulation of Claim 21 is administered via the arterial route.