Composition containing renal-tropic AAV and method of use thereof

Abstract

Recombinant adeno-associated virus (AAV) vectors are a major gene delivery platform, and the clinical use of several AAV-mediated therapies has recently been approved. Disclosed herein are compositions comprising AAV capsid proteins exhibiting improved tropism and improved transduction efficiency to renal cells and kidney-associated cells, as well as methods for using AAV particles and AAV vectors containing these AAV capsid proteins to efficiently deliver a gene or transgene of interest to target cells or tissues, and to treat subjects requiring treatment.

Description

[Technical Field]

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

[0002] II. Description of research or development provided for testing by the federal government. This invention was made with government support under National Institute of Health grants R01HL089221, UH3AR075336, and U01AI170064. The government has certain rights with respect to this invention.

[0003] III. Sequence List Reference The sequence listing, named "23-2075-WO-Sequence_Listing," submitted on March 25, 2024, as an XML file with a creation date of March 25, 2024, and a size of 7,706 kilobytes, is incorporated herein by reference in accordance with 37 CFR §1.52(e)(5).

[0004] IV. Fields This disclosure generally relates to gene delivery vehicles and related molecules that target the kidney. [Background technology]

[0005] V. Background of the Invention Chronic kidney disease (CKD) is estimated to affect 8–16% of the world's population and has increased by 31.7% in the last decade (Hill NR, et al. (2016) PLoS One. 11(7):e0158765; Evans M, et al. (2022) Adv. Ther. 39(1):33-43). Ultimately, CKD progresses to end-stage renal disease, in which case dialysis or kidney transplantation become 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 long-term sessions per week and is characterized by a low patient survival rate. Kidney transplantation has developed into a highly successful long-term therapy, but this field remains limited due to the shortage of donor organs 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, particularly cystinuria, polycystic kidney disease, and cystinic storage disorders, have underlying genetic etiologies that may be suitable for gene therapy or genome editing, thus highlighting the critical and unaddressed need for effective and safe kidney-targeted gene delivery vehicles (Peek JL, et al. (2023) Nat. Rev. Nephrol. 19(7):451-462; Rubin JD, 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).It is safe and effective, and it is safe and effective. [Preliminary Technology Documents] [Non-licensed literature]

[0006] [Non-licensed Document 1] Hill NR, et al. (2016) PLoS One. 11(7):e0158765 [Non-licensed Document 2] Evans M, et al. (2022) Adv. Ther. 39(1):33-43 [Non-licensed Document 3] Schrezenmeier E, et al. (2021) Genet Med. 23(7):1219-1224 [Non-licensed Document 4] Tonelli M, et al. (2011) Am. J. Transplant. 11(10):2093-2109 [Non-licensed Document 5] Malek SK, et al. (2011) Transpl. Int. 24(5):419-424 [Non-licensed Document 6] Keith DS, et al. (2016) Clin. J. Am. Soc. Nephrol. 11(4):684-693 [Non-licensed Document 7] Peek JL, et al. (2023) Nat. Rev. Nephrol. 19(7):451-462 [Non-licensed Document 8] Rubin JD, et al. (2020) Mol. Diagnosis Ther. 24(4):375-396 [Non-licensed Document 9] Peek JL, et al. (2022) Curr Opin Nephrol Hypertens. 31(2):175-179 [Non-licensed Document 10] Hildebrandt F. (2010) Lancet. 375 (9722) :1287-1295 [Overview of the project] [Means for solving the problem]

[0007] VI. Summary of the Invention AAV capsid proteins are disclosed herein. AAV capsid proteins having one or more substitutions in variable region IV (VR-IV) are disclosed herein. An adeno-associated virus (AAV) capsid protein is disclosed herein, wherein the AAV capsid protein comprises an amino acid sequence in which positions 452-458 are at least 85% identical to one of sequence numbers 04-23, and positions 452-458 of the AAV capsid protein are numbered with reference to sequence number 01.

[0008] An adeno-associated virus (AAV) capsid protein containing the sequence shown in Sequence ID No. 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein containing a sequence having at least 90% identity with the sequence shown in Sequence ID No. 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein containing one or more amino acid substitutions at positions 452-458 compared to Sequence ID No. 01 is disclosed herein.

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

[0010] An adeno-associated virus (AAV) capsid protein comprising the sequence shown in any one of SEQ ID NOs: 24 to 43 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising the sequence shown in SEQ ID NOs: 31, 35, or 43 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising the sequence shown in any one of SEQ ID NOs: 53 to 61 is disclosed herein.

[0011] AAV capsid proteins having at least 90% identity with the sequence shown in Sequence ID No. 03 are disclosed herein. AAV capsid proteins having one or more amino acid substitutions at positions 452-458 compared to Sequence ID No. 01 are disclosed herein. AAV capsids comprising an adeno-associated virus (AAV) capsid protein having one or more amino acid substitutions at positions 452-458 of Sequence ID No. 01 are disclosed herein, wherein the substitution at position 452 is any amino acid other than N; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or the substitution at position 458 is any amino acid other than Q. An AAV capsid containing an adeno-associated virus (AAV) capsid protein is disclosed herein, wherein the AAV capsid protein comprises the sequence shown in any one of SEQ ID NOs.24 to SEQ ID NOs.43. An AAV capsid containing an adeno-associated virus (AAV) capsid protein is disclosed herein, wherein the AAV capsid protein comprises the sequence shown in SEQ ID NOs.31 or SEQ ID NOs.35.

[0012] Disclosed herein is an AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises a sequence shown in any one of SEQ ID NOs. 53 to 61.

[0013] Nucleic acid molecules comprising nucleic acid sequences encoding the disclosed AAV capsid protein are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding the AAV capsid protein are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding the AAV capsid protein having one or more substitutions in variable region IV (VR-IV) are disclosed herein.

[0014] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein the amino acid sequence at positions 452-458 of the AAV capsid protein is at least 85% identical to one of sequence numbers 04-23, and the sequences at positions 452-458 of the AAV capsid protein are numbered with reference to sequence number 01.

[0015] This specification discloses nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein, which include a sequence having one or more substitutions compared to the sequence shown in Sequence ID No. 01.

[0016] A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in Sequence ID No. 03 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing a sequence having at least 90% identity to the sequence shown in Sequence ID No. 03 is disclosed herein.

[0017] This specification discloses nucleic acid molecules comprising a nucleic acid sequence encoding an AAV capsid protein, which includes one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01.

[0018] A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in any one of SEQ ID NOs: 24 to 43 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in SEQ ID NOs: 31, 35, or 43 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in any one of SEQ ID NOs: 53 to 61 is disclosed herein.

[0019] Disclosed herein is an AAV vector comprising a nucleic acid molecule containing a target gene and a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein the amino acid sequence at positions 452-458 of the AAV capsid protein is at least 85% identical to one of sequence numbers 04-23, and positions 452-458 of the AAV capsid protein are numbered with reference to sequence number 01.

[0020] AAV vectors comprising a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein are disclosed herein. AAV vectors comprising a transgene or heterologous nucleic acid for therapeutic proteins and / or therapeutic RNA are disclosed herein. AAV vectors comprising a transgene or heterologous nucleic acid for treating subjects with renal disease and / or renal impairment are disclosed herein. AAV particles comprising an AAV capsid containing a disclosed AAV capsid protein are disclosed herein. AAV particles comprising (i) an AAV capsid containing at least one disclosed AAV capsid protein and (ii) a vector genome are disclosed herein. AAV particles for use in a disclosed method are disclosed herein. AAV particles for use in a disclosed method for delivering a payload or a disclosed method for treating a subject are disclosed herein.

[0021] Pharmaceutical formulations comprising disclosed AAV particles or disclosed AAV vectors in a pharmaceutically acceptable carrier are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising nucleic acid sequences encoding an AAV capsid protein having at least 90% identity to the sequence shown in SEQ ID NO: 03 are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising nucleic acid sequences encoding an AAV capsid protein having one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01 are disclosed herein.

[0022] A method for generating AAV particles is disclosed herein, comprising the steps of: delivering a nucleic acid molecule containing a nucleic acid sequence encoding an AAV capsid protein to one or more cells; culturing one or more cells; and recovering AAV particles from one or more producing cells.

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

[0024] A method for generating AAV capsid proteins is disclosed herein, comprising the step of carrying out multiple rounds of evolution in one or more kidney or kidney-related models derived from one or more species.

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

[0026] A method for delivering a payload is disclosed herein, comprising the steps of contacting one or more target cells in an object requiring such delivery with disclosed AAV particles and expressing the encoded payload. A method for delivering a payload is disclosed herein, comprising the steps of contacting one or more target cells in an object requiring such delivery with a therapeutically effective amount of disclosed AAV particles comprising (i) 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 are methods for treating a subject, comprising administering a disclosed AAV particle or a disclosed AAV vector to a subject in need of such treatment in one or more therapeutic doses. Disclosed herein are methods for treating a subject, comprising administering a disclosed pharmaceutical formulation containing the disclosed AAV particle or a disclosed AAV vector to a subject in need of such treatment in one or more therapeutic doses. Disclosed herein are methods for treating a subject, comprising contacting one or more cells in the subject with a therapeutic dose of the disclosed AAV particle containing a payload. Disclosed herein are methods for treating a subject, comprising contacting one or more cells in the subject with a therapeutic dose of the disclosed AAV particle containing (i) 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. A method for treating a subject is disclosed herein, comprising the steps of: contacting one or more cells in the subject in a therapeutically effective amount of disclosed AAV particles comprising (i) 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 contact step enables the expression of the encoded payload, the encoded gene of interest, or the encoded transgene in one or more cells. VII. Brief Description of the Drawings [Brief explanation of the drawing]

[0028] [Figure 1A]Figures 1A–1E demonstrate that AAV.k variants can be obtained through multidimensional evolutionary strategies across different kidney model systems in vivo and ex vivo. Figure 1A is a schematic diagram of AAV capsid library evolution in non-human primate kidneys after intravenous administration in mice and pigs, in human kidney organoids, and after ex vivo perfusion via arterial and ureteral pathways. Figure 1B shows next-generation (NGS) sequencing of the AAV capsid library evolved from mouse kidneys. Black dots represent individual 7mer amino acid sequences, and brown dots represent AAV9. Figure 1C shows NGS of AAV capsid libraries evolved from pig renal cortex (blue) and medulla (orange). Zooming in on the upper right quadrant of the NGS plot shows enrichment of a specific 7mer amino acid sequence in pig renal cortex using AAV9, highlighted in brown. Figure 1D shows NGS of the AAV capsid library evolved from human kidney organoids. Black dots represent individual 7mer amino acid sequences, and brown dots represent AAV9. Figure 1E shows NGS of AAV capsid libraries evolved from ex vivo perfusion via arterial (blue) and ureteral (orange) pathways of non-human primate kidneys. The upper right quadrant zoom of the NGS plot shows the enrichment of specific 7mer amino acid sequences in ex vivo perfused non-human primate kidneys. AAV9 is highlighted in brown. In all plots, the X-axis represents the read depth of each sequence found in the library amplified from the specified tissue, and the Y-axis represents the enrichment factor change relative to the starting parent library. [Figure 1B-E]Figures 1A–1E demonstrate that AAV.k variants can be obtained through multidimensional evolutionary strategies across different kidney model systems in vivo and ex vivo. Figure 1A is a schematic diagram of AAV capsid library evolution in non-human primate kidneys after intravenous administration in mice and pigs, in human kidney organoids, and after ex vivo perfusion via arterial and ureteral pathways. Figure 1B shows next-generation (NGS) sequencing of the AAV capsid library evolved from mouse kidneys. Black dots represent individual 7mer amino acid sequences, and brown dots represent AAV9. Figure 1C shows NGS of AAV capsid libraries evolved from pig renal cortex (blue) and medulla (orange). Zooming in on the upper right quadrant of the NGS plot shows enrichment of a specific 7mer amino acid sequence in pig renal cortex using AAV9, highlighted in brown. Figure 1D shows NGS of the AAV capsid library evolved from human kidney organoids. Black dots represent individual 7mer amino acid sequences, and brown dots represent AAV9. Figure 1E shows NGS of AAV capsid libraries evolved from ex vivo perfusion via arterial (blue) and ureteral (orange) pathways of non-human primate kidneys. The upper right quadrant zoom of the NGS plot shows the enrichment of specific 7mer amino acid sequences in ex vivo perfused non-human primate kidneys. AAV9 is highlighted in brown. In all plots, the X-axis represents the read depth of each sequence found in the library amplified from the specified tissue, and the Y-axis represents the enrichment factor change relative to the starting parent library.

[0029] [Figure 2A-C]Figures 2A–2I show that AAV.k13 and AAV.k20 transduce mouse kidneys more efficiently than AAV9 after systemic administration. Figure 2A shows intravenous injection of AAV9, AAV.k13, or AAV.k20, packaged in a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter, at a dose of 5 × 10¹³ vg / kg into 8-week-old C57 / B6 mice. Organs were collected 30 days after injection. Figure 2B shows representative images of native mCherry fluorescence in mouse kidneys for mock, AAV9, AAV.k13, or AAV.k20. Scale bar = 1 mm. Figure 2C shows representative images of native mCherry fluorescence and lotus tetragonolobus lectin (LTL) staining of proximal tubules for AAV9, AAV.k13, or AAV.k20. Figure 2D shows a representative image of immunofluorescence labeling of the S1 and S2 segments of the proximal tubule in mouse kidneys. Scale bar = 70 μm. Figure 2E shows a representative image of immunofluorescence of nephrin in mouse kidneys. Scale bar = 130 μm. Figure 2F shows a representative image of immunofluorescence of collecting ducts labeled with dolichos biflorus agglutinin (DBA) in mouse kidneys. Scale bar = 130 μm. Figure 2G shows the vector genome copy number per μg of DNA for kidneys, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as the logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2H shows the vector genome copy number per μg of DNA for the liver, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as a logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2I shows the quantification of native mCherry fluorescence intensity normalized to the LTL signal in the kidney, for AAV.k13 compared to AAV9 (p<0.0063) and for AAV.k20 compared to AAV9 (p<0.0001).The multiplier change is indicated above the significance level. Statistical significance for the mCherry quantitative analysis was determined by one-way ANOVA and Tukey's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001;ns, not significant. [Figure 2D-F]Figures 2A–2I show that AAV.k13 and AAV.k20 transduce mouse kidneys more efficiently than AAV9 after systemic administration. Figure 2A shows intravenous injection of AAV9, AAV.k13, or AAV.k20, packaged in a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter, at a dose of 5 × 10¹³ vg / kg into 8-week-old C57 / B6 mice. Organs were collected 30 days after injection. Figure 2B shows representative images of native mCherry fluorescence in mouse kidneys for mock, AAV9, AAV.k13, or AAV.k20. Scale bar = 1 mm. Figure 2C shows representative images of native mCherry fluorescence and lotus tetragonolobus lectin (LTL) staining of proximal tubules for AAV9, AAV.k13, or AAV.k20. Figure 2D shows a representative image of immunofluorescence labeling of the S1 and S2 segments of the proximal tubule in mouse kidneys. Scale bar = 70 μm. Figure 2E shows a representative image of immunofluorescence of nephrin in mouse kidneys. Scale bar = 130 μm. Figure 2F shows a representative image of immunofluorescence of collecting ducts labeled with dolichos biflorus agglutinin (DBA) in mouse kidneys. Scale bar = 130 μm. Figure 2G shows the vector genome copy number per μg of DNA for kidneys, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as the logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2H shows the vector genome copy number per μg of DNA for the liver, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as a logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2I shows the quantification of native mCherry fluorescence intensity normalized to the LTL signal in the kidney, for AAV.k13 compared to AAV9 (p<0.0063) and for AAV.k20 compared to AAV9 (p<0.0001).The multiplier change is indicated above the significance level. Statistical significance for the mCherry quantitative analysis was determined by one-way ANOVA and Tukey's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001;ns, not significant. [Figure 2G-I]Figures 2A–2I show that AAV.k13 and AAV.k20 transduce mouse kidneys more efficiently than AAV9 after systemic administration. Figure 2A shows intravenous injection of AAV9, AAV.k13, or AAV.k20, packaged in a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter, at a dose of 5 × 10¹³ vg / kg into 8-week-old C57 / B6 mice. Organs were collected 30 days after injection. Figure 2B shows representative images of native mCherry fluorescence in mouse kidneys for mock, AAV9, AAV.k13, or AAV.k20. Scale bar = 1 mm. Figure 2C shows representative images of native mCherry fluorescence and lotus tetragonolobus lectin (LTL) staining of proximal tubules for AAV9, AAV.k13, or AAV.k20. Figure 2D shows a representative image of immunofluorescence labeling of the S1 and S2 segments of the proximal tubule in mouse kidneys. Scale bar = 70 μm. Figure 2E shows a representative image of immunofluorescence of nephrin in mouse kidneys. Scale bar = 130 μm. Figure 2F shows a representative image of immunofluorescence of collecting ducts labeled with dolichos biflorus agglutinin (DBA) in mouse kidneys. Scale bar = 130 μm. Figure 2G shows the vector genome copy number per μg of DNA for kidneys, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as the logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2H shows the vector genome copy number per μg of DNA for the liver, calculated by normalizing the mCherry copy number to the total number of μg of DNA input for qPCR quantification and plotting it as a logarithm of vg per μg of DNA. Each dot represents an individual mouse. Figure 2I shows the quantification of native mCherry fluorescence intensity normalized to the LTL signal in the kidney, for AAV.k13 compared to AAV9 (p<0.0063) and for AAV.k20 compared to AAV9 (p<0.0001).The multiplier change is indicated above the significance level. Statistical significance for the mCherry quantitative analysis was determined by one-way ANOVA and Tukey's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001;ns, not significant.

[0030] [Figure 3A-D]Figures 3A–3H show that AAV.k13 and AAV.k20 transduce 2D and 3D human kidney organoids more efficiently than AAV9. Figure 3A shows representative images of immunofluorescence labeling of mCherry and proximal tubules using Lrp2 in 2D human kidney organoids transduced with 1 × 10¹⁰ vg / well of AAV9, AAV.k13, or AAV.k20, which packaged a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 100 μm. Figure 3B shows a schematic diagram of 3D human kidney organoids that were differentiated and transduced on day 14 with AAV at 1 × 10¹¹ vg / well or 1 × 10¹² vg / well. Five days after transduction, organoids were harvested for frozen sectioning and DNA / RNA analysis. Figure 3C shows assays analyzing the uptake of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. The vector genome copy number per μg of DNA for organoids in both transduction assays was calculated by plotting the mCherry copy number as a logarithm of vg per μg of DNA, normalized to the total μg of DNA input for qPCR quantification. Each dot represents a single organoid. Figure 3D shows assays analyzing gene expression of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. mCherry mRNA levels were normalized to the housekeeping gene RPL13A from RT-qPCR quantification. Figure 3E shows representative images of mCherry immunofluorescence labeling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well. Scale bar = 100 μm. Figure 3F shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well.The comparison of AAV.k13 with AAV9 was significant (p<0.0445), and similarly, the comparison of AAV.k20 with AAV9 was also significant (p<0.0175). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3G shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced using AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well. The comparison of AAV.k13 with AAV9 was not significant (ns), but the comparison of AAV.k20 with AAV9 was significant (p<0.0053). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3H presents representative images of immunofluorescence labeling of mCherry and immunofluorescence labeling of the proximal tubule using LTL in 3D human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well, which package a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 130 μm. The row below the dashed box shows a zoomed-in region focusing on the localization of mCherry in the proximal tubule. The scale bar is 30 μm. Statistical significance of the quantitative analysis of mCherry compared to AAV9 was determined by one-way ANOVA and Dunnett's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001; ns, not significant. [Figure 3E-G]Figures 3A–3H show that AAV.k13 and AAV.k20 transduce 2D and 3D human kidney organoids more efficiently than AAV9. Figure 3A shows representative images of immunofluorescence labeling of mCherry and proximal tubules using Lrp2 in 2D human kidney organoids transduced with 1 × 10¹⁰ vg / well of AAV9, AAV.k13, or AAV.k20, which packaged a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 100 μm. Figure 3B shows a schematic diagram of 3D human kidney organoids that were differentiated and transduced on day 14 with AAV at 1 × 10¹¹ vg / well or 1 × 10¹² vg / well. Five days after transduction, organoids were harvested for frozen sectioning and DNA / RNA analysis. Figure 3C shows assays analyzing the uptake of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. The vector genome copy number per μg of DNA for organoids in both transduction assays was calculated by plotting the mCherry copy number as a logarithm of vg per μg of DNA, normalized to the total μg of DNA input for qPCR quantification. Each dot represents a single organoid. Figure 3D shows assays analyzing gene expression of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. mCherry mRNA levels were normalized to the housekeeping gene RPL13A from RT-qPCR quantification. Figure 3E shows representative images of mCherry immunofluorescence labeling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well. Scale bar = 100 μm. Figure 3F shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well.The comparison of AAV.k13 with AAV9 was significant (p<0.0445), and similarly, the comparison of AAV.k20 with AAV9 was also significant (p<0.0175). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3G shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced using AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well. The comparison of AAV.k13 with AAV9 was not significant (ns), but the comparison of AAV.k20 with AAV9 was significant (p<0.0053). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3H presents representative images of immunofluorescence labeling of mCherry and immunofluorescence labeling of the proximal tubule using LTL in 3D human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well, which package a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 130 μm. The row below the dashed box shows a zoomed-in region focusing on the localization of mCherry in the proximal tubule. The scale bar is 30 μm. Statistical significance of the quantitative analysis of mCherry compared to AAV9 was determined by one-way ANOVA and Dunnett's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001; ns, not significant. [Figure 3H]Figures 3A–3H show that AAV.k13 and AAV.k20 transduce 2D and 3D human kidney organoids more efficiently than AAV9. Figure 3A shows representative images of immunofluorescence labeling of mCherry and proximal tubules using Lrp2 in 2D human kidney organoids transduced with 1 × 10¹⁰ vg / well of AAV9, AAV.k13, or AAV.k20, which packaged a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 100 μm. Figure 3B shows a schematic diagram of 3D human kidney organoids that were differentiated and transduced on day 14 with AAV at 1 × 10¹¹ vg / well or 1 × 10¹² vg / well. Five days after transduction, organoids were harvested for frozen sectioning and DNA / RNA analysis. Figure 3C shows assays analyzing the uptake of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. The vector genome copy number per μg of DNA for organoids in both transduction assays was calculated by plotting the mCherry copy number as a logarithm of vg per μg of DNA, normalized to the total μg of DNA input for qPCR quantification. Each dot represents a single organoid. Figure 3D shows assays analyzing gene expression of selected AAVs at 1 × 10¹¹ vg / well (left) or 1 × 10¹² vg / well (right) in human kidney organoids. mCherry mRNA levels were normalized to the housekeeping gene RPL13A from RT-qPCR quantification. Figure 3E shows representative images of mCherry immunofluorescence labeling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well. Scale bar = 100 μm. Figure 3F shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹¹ vg / well.The comparison of AAV.k13 with AAV9 was significant (p<0.0445), and similarly, the comparison of AAV.k20 with AAV9 was also significant (p<0.0175). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3G shows the quantification of mCherry immunofluorescence intensity normalized to DAPI signaling in human kidney organoids transduced using AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well. The comparison of AAV.k13 with AAV9 was not significant (ns), but the comparison of AAV.k20 with AAV9 was significant (p<0.0053). The calculated magnification change in transduction between capsids is indicated above the significance. Figure 3H presents representative images of immunofluorescence labeling of mCherry and immunofluorescence labeling of the proximal tubule using LTL in 3D human kidney organoids transduced with AAV9, AAV.k13, or AAV.k20 at 1 × 10¹² vg / well, which package a self-complementary cassette encoding mCherry driven by a chicken-beta-actin hybrid (Cbh) promoter. The scale bar is 130 μm. The row below the dashed box shows a zoomed-in region focusing on the localization of mCherry in the proximal tubule. The scale bar is 30 μm. Statistical significance of the quantitative analysis of mCherry compared to AAV9 was determined by one-way ANOVA and Dunnett's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001; ns, not significant.

[0031] [Figure 4A]Figures 4A to 4D show that AAV.k13 and AAV.k20 transduce more efficiently than AAV9 into transplanted porcine kidneys. Figure 4A (left) shows a schematic diagram of a nephrectomy performed on a 50 kg pig. Briefly, the kidney graft was flushed with a cryogenic solution and stored on ice. During the cryogenic storage period, AAV.k13-scCbh-mCherry was administered via the renal artery at 5.9 × 10¹² vg over 2 hours, and also via the ureteral route at 5.9 × 10¹² vg. After static cryogenic storage, the kidney was transplanted back into the same pig and retrieved 14 days after transplantation. Immunohistochemical testing (IHC) of mCherry, which stains positive signals brown, was performed on biopsy materials of porcine kidney tissue. Figure 4A (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4B (left) shows that a nephrectomy was performed on a 48.9 kg pig and that ex vivo mechanical perfusion was initiated for 2 hours. Subsequently, AAV.k20-scCbh-mCherry was perfused at 6.1 × 10¹² vg via the arterial route for 2 hours, and 6.1 × 10¹² vg was administered retrogradely via the ureteral route. After mechanical perfusion, the kidney was transplanted back into the same pig and retrieved 9 days after transplantation. Figure 4B (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4C (left) shows that a nephrectomy was performed on a 42 kg pig and that ex vivo mechanical perfusion was initiated. In this experiment, AAV.k20-scCbh-mCherry was administered at 7.5 × 10¹² vg via the ureteral route for 2 hours. After mechanical perfusion, the kidneys were transplanted into the same pigs and retrieved 9 days after transplantation. Figure 4C (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4D (left) shows that nephrectomy was performed on a 40-50 kg pig and that mechanical perfusion was initiated for 2 hours. A total of 7 × 10¹² vg of AAV9, AAV.k13, or AAV.k20 packaged with scCbh-mCherry was administered via the ureteral route over 2 hours. After mechanical perfusion, the kidneys were transplanted into the same pigs and retrieved 9 days after transplantation.Figure 4D (right) shows representative IHC images from AAV9, AAV.k13, and AAV.k20 after kidney transplantation and retrieval. [Figure 4B-D]Figures 4A to 4D show that AAV.k13 and AAV.k20 transduce more efficiently than AAV9 into transplanted porcine kidneys. Figure 4A (left) shows a schematic diagram of a nephrectomy performed on a 50 kg pig. Briefly, the kidney graft was flushed with a cryogenic solution and stored on ice. During the cryogenic storage period, AAV.k13-scCbh-mCherry was administered via the renal artery at 5.9 × 10¹² vg over 2 hours, and also via the ureteral route at 5.9 × 10¹² vg. After static cryogenic storage, the kidney was transplanted back into the same pig and retrieved 14 days after transplantation. Immunohistochemical testing (IHC) of mCherry, which stains positive signals brown, was performed on biopsy materials of porcine kidney tissue. Figure 4A (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4B (left) shows that a nephrectomy was performed on a 48.9 kg pig and that ex vivo mechanical perfusion was initiated for 2 hours. Subsequently, AAV.k20-scCbh-mCherry was perfused at 6.1 × 10¹² vg via the arterial route for 2 hours, and 6.1 × 10¹² vg was administered retrogradely via the ureteral route. After mechanical perfusion, the kidney was transplanted back into the same pig and retrieved 9 days after transplantation. Figure 4B (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4C (left) shows that a nephrectomy was performed on a 42 kg pig and that ex vivo mechanical perfusion was initiated. In this experiment, AAV.k20-scCbh-mCherry was administered at 7.5 × 10¹² vg via the ureteral route for 2 hours. After mechanical perfusion, the kidneys were transplanted into the same pigs and retrieved 9 days after transplantation. Figure 4C (right) shows a representative IHC image with labeling of the proximal tubule, distal tubule, and glomerulus. Figure 4D (left) shows that nephrectomy was performed on a 40-50 kg pig and that mechanical perfusion was initiated for 2 hours. A total of 7 × 10¹² vg of AAV9, AAV.k13, or AAV.k20 packaged with scCbh-mCherry was administered via the ureteral route over 2 hours. After mechanical perfusion, the kidneys were transplanted into the same pigs and retrieved 9 days after transplantation.Figure 4D (right) shows representative IHC images from AAV9, AAV.k13, and AAV.k20 after kidney transplantation and retrieval.

[0032] [Figure 5A-D] Figure 5A shows the results of analyzing, comparing, and plotting unique sequences in NGS of AAV capsid libraries evolved from porcine renal cortex (blue) and medulla (orange). The porcine renal cortex had 12,304 unique sequences, while the porcine renal medulla had 10,068 unique sequences. There were 1,138 overlapping sequences. Figure 5B shows that analysis of the top 100 consensus motifs indicates that AAV was enriched with residues 452-458 (VP1 numbered) in terms of evolution in the porcine cortex and medulla. Figure 5C shows the results of analyzing, comparing, and plotting unique sequences in amplicon-EZ sequencing of AAV capsid libraries evolved from non-human primate renal perfusion via arterial (blue) and ureteral (orange) pathways. The arterial pathway had 4,552 unique sequences, while the ureteral pathway had 4,711 unique sequences. There were 5,913 overlapping sequences. Figure 5D shows that, based on the analysis of the top 100 consensus motifs, AAV enriches residues 452-458 (VP1 numbered) with respect to ex vivo NHP arterial and ureteral evolution.

[0033] [Figure 6A] Figure 6A shows an analysis of amino acid preferences for the parental and evolved libraries at each individual position in VR-IV (452-458). The libraries are plotted against amino acids. Blue and red represent the minimum and maximum enriched residues, respectively, and white represents the 50th percentile. [Figure 6B]Figure 6B shows the analysis of amino acid preferences at all positions in VR-IV(452–458) for the parent library and each evolved library. The positions within VR-IV(452–458) are plotted against amino acids for the parent library and each evolved library. Blue and red represent the minimum and maximum enriched residues, respectively, and white represents the 50th percentile.

[0034] [Figure 7] Figure 7 shows a comparison of the production yields of multiple recombinant AAV9, AAV.k13, and AAV.k20 in adherent and suspension systems. Purification yields are plotted as the number of vector genomes per liter of medium. Each combination of symbol and color represents a different transgene cassette used for production. Blue triangles represent the self-complementary Cbh-mCherry cassette, orange circles represent the self-complementary Cbh-PDL1 cassette, and purple squares represent the single-stranded CBA-luciferase cassette. Statistical significance of the analysis was determined by one-way ANOVA and Tukey's post-hoc test. *p<0.05;**p<0.01;***p<0.001;****p<0.0001;ns, not significant.

[0035] [Figure 8] Figure 8 shows additional representative images of immunofluorescence labeling of mCherry in mouse kidneys and immunofluorescence labeling of collecting tubules using DBA. Scale bar = 130 μm.

[0036] [Figure 9A-B]Figure 9A shows the extraction of RNA from undifferentiated iPSCs and human kidney organoids under different experimental conditions. RT-qPCR was performed on mRNA levels of OCT4, a marker expressed in stem cells, and normalized to human beta-actin. Figure 9B shows additional representative images of immunofluorescence labeling of mCherry and proximal tubules using LTL in transduced human kidney organoids using AAV9, AAV.k13, or AAV.k20 packaged with self-complementary Cbh-mCherry at 1 × 10¹² vg / well. Scale bar = 130 μm.

[0037] [Figure 10A-B] Figures 10A and 10B show TH1 cells transduced using a 500K MOI (Minimum Observed Area) for each AAV capsid packaged with a self-complementary Cbh-mCherry cassette. Cells were seeded in 24-well plates at a rate of 2 × 10⁵ cells per well, and imaging was performed 18 hours and 4 days after transduction.

[0038] [Figure 11A-B] Figures 11A and 11B show cells seeded at 2 × 10⁵ cells per well in a 24-well plate, and subjected to a luciferase assay 24 hours after transduction. Cells were transduced using a 500K MOI, which is the reference MOI for each AAV capsid containing a single-stranded CBA-luciferase cassette.

[0039] [Figure 12] Figure 12 shows the transduction of each AAV capsid, which packaged a single-stranded CBA-luciferase cassette, into kidney organoids using 1 × 10¹¹ vg / well. AAV.k20 showed superior performance compared to AAV in transduction into human kidney organoids.

[0040] [Figure 13A]Figures 13A–13C demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Figure 13A shows the locations of 10 biopsy samples obtained from the left and right kidneys. Figure 13B shows the transduction efficiency of AAV.k20 after in situ delivery. Figure 13C shows the distribution of the AAV.k20 viral genome. [Figure 13B] Figures 13A–13C demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Figure 13A shows the locations of 10 biopsy samples obtained from the left and right kidneys. Figure 13B shows the transduction efficiency of AAV.k20 after in situ delivery. Figure 13C shows the distribution of the AAV.k20 viral genome. [Figure 13C] Figures 13A–13C demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Figure 13A shows the locations of 10 biopsy samples obtained from the left and right kidneys. Figure 13B shows the transduction efficiency of AAV.k20 after in situ delivery. Figure 13C shows the distribution of the AAV.k20 viral genome.

[0041] [Figure 14A-C] Figures 14A to 14F show the in vivo distribution of various AAV9, AAV.k13, and AAV.k20 in mice after IV injection (Figures 14A to 14C). Luciferase assays were also performed for each of these AAVs (Figures 14D to 14F). [Figure 14D-F] Figures 14A to 14F show the in vivo distribution of various AAV9, AAV.k13, and AAV.k20 in mice after IV injection (Figures 14A to 14C). Luciferase assays were also performed for each of these AAVs (Figures 14D to 14F).

[0042] [Figure 15A-C]Figures 15A–15F demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Here, Figure 15A shows the sites of 10 biopsy specimens of transduced kidneys. Figures 15B–15C show the in vivo distribution of the AAV.k20 viral genome. Transduction efficiency of AAV.k20 after in situ delivery. Figures 15D–15E show mCherry expression assessed by qPCR. Figure 15F is a Western blot for vinculin (housekeeping gene) and mCherry (target transgene), showing that the variability in mCherry levels in these biopsy specimens correlates with mCherry RNA levels. [Figure 15D-E] Figures 15A–15F demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Here, Figure 15A shows the sites of 10 biopsy specimens of transduced kidneys. Figures 15B–15C show the in vivo distribution of the AAV.k20 viral genome. Transduction efficiency of AAV.k20 after in situ delivery. Figures 15D–15E show mCherry expression assessed by qPCR. Figure 15F is a Western blot for vinculin (housekeeping gene) and mCherry (target transgene), showing that the variability in mCherry levels in these biopsy specimens correlates with mCherry RNA levels. [Figure 15F] Figures 15A–15F demonstrate the ability of AAV.k20 to effectively transduce non-human primate kidneys. Here, Figure 15A shows the sites of 10 biopsy specimens of transduced kidneys. Figures 15B–15C show the in vivo distribution of the AAV.k20 viral genome. Transduction efficiency of AAV.k20 after in situ delivery. Figures 15D–15E show mCherry expression assessed by qPCR. Figure 15F is a Western blot for vinculin (housekeeping gene) and mCherry (target transgene), showing that the variability in mCherry levels in these biopsy specimens correlates with mCherry RNA levels. [Modes for carrying out the invention]

[0043] VIII. Detailed Description of the Invention This disclosure describes nucleic acid molecules, viral vectors, viral capsid proteins, viral particles, plasmids, cells, kits, pharmaceutical formulations, and compositions thereof, as well as methods for using the disclosed compositions. Since synthesis methods and reagents can naturally vary, it should be understood that embodiments of the invention are not limited to specific synthesis methods or specific reagents unless otherwise specified. It should also be understood that the terminology used herein is merely for describing specific embodiments and is not limiting. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but examples of methods and materials are given herein.

[0044] All publications referenced herein are incorporated herein by reference to disclose and describe methods and / or materials in relation to the cited publications. Publications considered herein are provided solely for their disclosures prior to the filing date of this application. Nothing herein should be construed as accepting that the present invention is not prior to such publications based on prior art.

[0045] A.Definition Before disclosing and describing the compounds, compositions, articles, systems, devices, vectors, and / or methods described herein, it should be understood that, as synthesis methods and reagents are naturally subject to change, the compounds, compositions, articles, systems, devices, vectors, and / or methods described herein are not limited to any particular synthesis method unless otherwise specified, nor are they limited to any particular reagent unless otherwise specified. It should also be understood that the terminology used herein is merely for describing specific embodiments and is not limiting. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but examples of methods and materials are given herein.

[0046] This disclosure describes the concept of the invention with reference to specific examples. However, it is intended to encompass all modifications, equivalents, and substitutions that are consistent with the concept of the invention as described in this disclosure.

[0047] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” refer to multiple subjects unless the context clearly indicates otherwise.

[0048] The phrase "consisting essentially of" limits the claims to the components described in the composition or the steps described in the method, and does not substantially affect the basic and novel features (one or more) of the claimed composition or method. The phrase "consisting of" excludes any components, steps, or elements not described in the claims. The phrase "comprising" is synonymous with "including," "containing," or "characterized by," and is comprehensive or open-ended. "Comprising" does not exclude additional undescribed components or steps.

[0049] In some contexts, when referring to any numerical value, the term "approximately" means a value that falls within ±10% of the specified value.

[0050] In this specification, a range may be expressed as "about" one particular value and / or "about" another particular value. Where such a range is expressed, further aspects may encompass "about" one particular value and / or "about" another particular value. Similarly, where a value is expressed as an approximation by using the antecedent "about", it will be understood that a particular value forms further aspects. Furthermore, it will be understood that each endpoint of a range is important both in relation to the other endpoint and independently of the other endpoint. It will also be understood that several values ​​are disclosed in this specification, and for each value, in addition to the value itself, "about" that particular value is also disclosed in this specification. For example, where the value "10" is disclosed, "about 10" is also disclosed. It will also be understood that each unit between two particular units is also disclosed. For example, where 10 and 15 are disclosed, 11, 12, 13, and 14 are also disclosed.

[0051] In some embodiments, the terms “approximately” or “about” refer to a value similar to the explicitly stated reference value when applied to one or more values ​​of interest. In some embodiments, the terms “approximately” or “about” refer to a range of values ​​that, unless otherwise specified or clearly indicated by the context, 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 than 1% of the explicitly stated reference value in any direction.

[0052] In some embodiments, the term "in vitro" refers to events or experiments that occur or are conducted in an artificial environment, such as in a petri dish, test tube, or cell culture, rather than within a multicellular organism. In other embodiments, the term "in vivo" refers to events or experiments that occur or are conducted within a multicellular organism.

[0053] In some aspects, the term “equivalent” means, in the context of a particular value and a reference value, that particular value is identical to the reference value, or its deviation from the reference value (above or below the reference value) is no more than 10%.

[0054] In this specification and the last claim, any reference to parts by weight of a particular element or component in a composition indicates a weight relationship between that element or component in the composition and any other element or component or article expressed in parts by weight. Thus, in a compound containing 2 parts by weight of component X and 5 parts by weight of component Y, X and Y exist in a weight ratio of 2:5, and such a ratio exists regardless of whether the compound contains additional components.

[0055] In some embodiments, the terms “as needed” or “as required” mean that the events or circumstances described thereafter may or may not occur, and that the cases in which such events or circumstances occur and the cases in which such events or circumstances do not occur are encompassed in the description. In some embodiments, the disclosed method may include, as needed, one or more additional steps, such as a step of repeating the administration step or a step of modifying the administration step.

[0056] In some embodiments, the term “subject” refers to the target of administration. In some embodiments, the subject may be a human. The term “subject” encompasses domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mice, rabbits, rats, guinea pigs, fruit flies, etc.). Thus, the subjects of the methods disclosed herein may be vertebrates, e.g., mammals, fish, birds, reptiles, or amphibians. Alternatively, the subjects of the methods disclosed herein may be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cows, cats, guinea pigs, or rodents. This term does not indicate a specific age or sex and therefore encompasses adult subjects and juvenile subjects as well as fetuses, regardless of sex. In some embodiments, the subject may be a human patient. In some embodiments, the subject may be a diseased Individuals may have illnesses, disabilities, infections, symptoms, and / or complications, or may be suspected of having a disease, disease, disability, infection, symptoms, and / or complications, or may be at risk of developing a disease, disability, infection, symptoms, and / or complications. For example, individuals may have risk factors for developing a disease, disability, infection, symptoms, and / or complications. Individuals may have risks resulting from genetic predisposition, occupation (e.g., healthcare workers, miners), attendance at certain places (e.g., schools), attendance at social events (e.g., sporting events, concerns, religious events, political rallies and events, social justice rallies, marches and events, etc.), use of public transport or public services, or exposure to natural and man-made disasters (e.g., Chernobyl, the 9 / 11 attacks, etc.).

[0057] In some embodiments, subjects may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0058] In some embodiments, the subject may have a genetic disorder. In some embodiments, the subject may have a missing, defective, and / or mutated protein or enzyme. The protein or enzyme may be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof.

[0059] In some embodiments, “effective amount” and “amount effective” may refer to an amount sufficient to achieve a desired outcome, such as the treatment and / or prevention of a disease, disorder, infection, symptom, and / or complication, or suspected disease, disorder, infection, symptom, and / or complication (e.g., those associated with renal disease and / or renal impairment). In some embodiments, an effective amount may be sufficient to alleviate and / or improve one or more symptoms and / or complications associated with renal disease and / or renal impairment. In some embodiments, “therapeutic effective amount” may refer to an amount (i.e., vector genome / body weight or vg / kg) sufficient to achieve a desired therapeutic outcome or to have an effect against an undesirable symptom, but generally insufficient to cause adverse side effects. In some embodiments, “therapeutic dose” means an amount of the disclosed composition that (i) treats a particular disease, disorder, and / or infection; (ii) reduces, improves, or eliminates one or more symptoms of a particular disease, condition, and / or disorder; or (iii) delays the onset of one or more symptoms of a particular disease, condition, and / or disorder as described herein. A specific therapeutic dose level for any particular patient depends on a variety of factors, including the disorder and severity of the disorder being treated; a particular disclosed composition and / or pharmaceutical preparation containing one or more disclosed compositions, or the method of use; the patient’s age, weight, overall health, sex, and diet; time of administration; route of administration; elimination rate of the disclosed composition and / or pharmaceutical preparation containing one or more disclosed compositions used; duration of treatment; drugs used in combination with or concurrently with the disclosed composition and / or pharmaceutical preparation containing one or more disclosed compositions used, as well as other factors similarly well known in the field of medicine. For example, starting a dose of a disclosed composition and / or a pharmaceutical preparation containing one or more disclosed compositions at a level lower than necessary to achieve a desired therapeutic effect, and gradually increasing the dose until the desired effect is achieved, falls well within the scope of the art of the art.If desired, the effective daily dose may then be divided into multiple doses for administration. Consequently, a single dose of the disclosed composition and / or a pharmaceutical preparation or method containing one or more of the disclosed compositions may contain such amounts or submultiples to constitute a daily dose. Dosage may be adjusted by the individual physician in case of any contraindications. Dosage may vary and may be administered once or multiple times a day for one day or several days. Guidance on appropriate dosages for a given class of pharmaceutical products can be found in the literature. In various further embodiments, preparations may be administered in a “preventive effective dose.” A preventive effective dose is an amount effective in preventing a disease, disorder, infection, symptom, and / or complication.

[0060] In some embodiments, “control” refers to a standard or reference condition for which the results are compared. In some embodiments, a control is used simultaneously with the test variable or subject to provide a comparison. In some embodiments, a control is a control based on past history of previous experiments, a previously known result or quantity, or a record that already exists in other forms. A control may be a positive control or a negative control.

[0061] In some embodiments, the term “diagnosed” means that a person is subjected to a physical examination by a skilled person, e.g., a physician, and is found to have a disease, disorder, infection, condition, and / or comorbidity (e.g., related to renal disease and / or renal impairment) that can be diagnosed or treated by one or more of the disclosed capsid proteins, disclosed AAV particles, disclosed vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, and / or disclosed methods. For example, “suspected to have” could mean that a person is subjected to a physical examination by a skilled person, e.g., a physician, and is found to have a condition (e.g., renal disease and / or renal impairment) that could potentially be treated by one or more of the disclosed capsid proteins, disclosed vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, and / or disclosed methods.

[0062] The words “treat,” “treating,” or “treatment” refer to therapeutic or medical actions aimed at slowing, improving, and / or reducing undesirable physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, beneficial or desired clinical outcomes, whether detectable or undetectable, include, but are not limited to, relief of symptoms, reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or temporary relief of the condition, and remission (whether partial or complete remission). “Treatment” may also mean extending survival compared to the survival expected without treatment. Treatment does not necessarily have to result in the complete elimination of infection, and may reduce or minimize complications, side effects, and / or the progression of disease, disorder, infection, symptoms, and / or complications. The success or failure of a treatment can be monitored by physical examination of the subject as well as by cytopathological detection techniques, DNA detection techniques, and / or mRNA detection techniques. The words “treat,” “treating,” or “treatment” encompass palliative care, i.e., treatment designed to alleviate symptoms rather than cure a disease, pathological condition, or disorder; preventive care, i.e., treatment directed to minimize or partially or completely inhibit the occurrence of the associated disease, pathological condition, or disorder; and supportive care, i.e., treatment used to complement another specific treatment directed to improve the associated disease, pathological condition, or disorder.In various embodiments, these terms encompass all treatments of subjects, including mammals (e.g., humans), and include (i) preventing the development of undesirable physiological changes, diseases, pathological conditions, or disorders in subjects who may have a predisposition to disease but have not yet been diagnosed with it; (ii) inhibiting physiological changes, diseases, pathological conditions, or disorders, i.e., preventing their occurrence; or (iii) mitigating physiological changes, diseases, pathological conditions, or disorders, i.e., causing disease regression. For example, in some embodiments, treatment for an infection can reduce the severity of an established infection in a subject by 1% to 100% compared to a control (e.g., a subject without the disease, disorder, infection, symptoms, and / or complications). In some embodiments, treatment may mean reducing the severity of an established disease, disorder, infection, symptom, and / or complication by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, treatment may mean reducing one or more symptoms by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. It is understood that treatment does not necessarily mean the cure, complete elimination, or eradication of a disease, disorder, infection, symptom, and / or complication. However, in some embodiments, treatment may mean the cure, complete elimination, or eradication of a disease, disorder, infection, symptom, and / or complication.

[0063] Methods and techniques for monitoring a subject's response to a disclosed method may include qualitative (or subjective) and quantitative (or objective) means. In some embodiments, qualitative (or subjective) means may include the subject's own perspective. For example, the subject may report how they feel, whether they have experienced improvement and / or regression, whether they have experienced relief or worsening of one or more symptoms, or a combination thereof. In some embodiments, quantitative (or objective) means may include, but are not limited to, methods and techniques including: (i) fluid analysis (e.g., aqueous humor and vitreous fluid, bile, blood, serum, breast milk, cerebrospinal fluid, cerumen (earwax), digestive fluids, endolymph and perilymph, female ejaculate, gastric juice, mucus (including nasal secretions and sputum), peritoneal fluid, pleural fluid, saliva, sebum (skin) (ii) Testing of the subject's bodily fluids, including oil, semen, sweat, synovial fluid, tears, vaginal secretions, vomit, and urine; (ii) Imaging (e.g., conventional X-ray, ultrasound, 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, colonoscopy, cystoscopy, hysteroscopy, arthroscopy, laparoscopy, mediastinoscopy, and thoracoscopy); (iv) Analysis of organ activity (e.g., electrocardiogram (ECG), electroencephalogram (EEG), and pulse oximetry); (v) Biopsy (e.g., taking tissue samples for microscopic evaluation); and (vi) Genetic testing.

[0064] "Patient" refers to a person suffering from a disease, disorder, infection, condition, and / or complication (e.g., kidney disease and / or kidney impairment). In some embodiments, patient may refer to a person who has been diagnosed with or is suspected of having a disease, disorder, infection, condition, and / or complication (e.g., relating to kidney disease and / or kidney impairment). In some embodiments, patient may refer to a person who has been diagnosed with or is suspected of having an established disease, disorder, infection, condition, and / or complication and is seeking or receiving treatment.

[0065] In some embodiments, the terms “prevent,” “preventing,” or “prevention” refer to preventing, avoiding, removing, deterring, stopping, or interfering with something from happening, particularly through prior measures. Where “reduce,” “inhibit,” or “prevent” are used herein, it is understood that the use of the other two words is also expressly disclosed unless otherwise indicated. In some embodiments, the intention is to prevent disease, disorder, infection, symptom, and / or complications. The words “prevent,” “preventing,” and “prevention” also refer to prophylactic or preventive measures to protect or prevent the progression of complications in subjects who do not have complications associated with a given infection (e.g., those with renal disease and / or renal impairment). Individuals for whom prevention is needed include individuals with an infection.

[0066] In some embodiments, the terms “administering” and “administration” refer to any method of providing one or more of the disclosed capsid proteins, disclosed AAV particles, disclosed vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, and / or disclosed methods. Such methods are well known to those skilled in the art and include, but are not limited to, retrograde ureteral infusion, renal artery administration, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ophthalmic administration, intraocular administration, ear drops, interutero administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including injectable administrations such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration may be continuous or intermittent. Administration may be carried out by one or more ex vivo methods, such as ex vivo perfusion protocols. In one embodiment, an ex vivo perfusion protocol can be used in conjunction with a kidney (or a portion thereof) obtained for a subject. In one embodiment, a kidney can be obtained from a donor subject, subjected to an ex vivo perfusion protocol, and then implanted in a subject in need. In one embodiment, a kidney can be obtained from a subject in need, subjected to an ex vivo perfusion protocol, and then returned to a subject in need.

[0067] In some embodiments, the term “ex vivo” may generally refer to activities performed outside of a living organism or subject, such as experiments, modifications, differentiations, manipulations, and / or measurements performed in or on living tissue in an artificial environment outside of a living organism. In some embodiments, ex vivo experiments, ex vivo modifications, ex vivo differentiations, ex vivo manipulations, and / or ex vivo measurements may be performed by making minimal changes to natural conditions. In some embodiments, “ex vivo” may include living cells, tissues, or organs (e.g., kidneys or kidney-related) obtained from a subject or donor subject requiring it and cultured and / or maintained and / or perfused in laboratory equipment, usually under sterile conditions, generally for a limited duration (e.g., several 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 longer, depending on the circumstances and / or desired characteristics). In one embodiment, tissues, cells, or organs can be collected, frozen, and later thawed for ex vivo processing.

[0068] In some embodiments, the terms “perfume” or “perfuse” may refer to the action of causing a fluid to flow through the lumen of a hollow structure, for example, causing a fluid to flow from an artery or other vessel supplying the tissue’s vascular bed through the tissue’s vascular bed. In some embodiments, the disclosed perfume fluid may be a body fluid, preservation fluid, or any other fluid suitable for perfuming a tissue, graft, or organ. The disclosed body fluid may be blood, lymph, plasma, serum, cerebrospinal fluid, urine, or any other body fluid. The disclosed preservation fluid may be any organ preservation fluid, including, but not limited to, solutions, saline, or mechanical perfusation solutions. In some embodiments, the disclosed fluid may include the disclosed AAV vector.

[0069] In some embodiments, “modifying the method” may include modifying or altering one or more features or aspects of one or more steps of the disclosed method. For example, in some embodiments, the method may be modified by changing the amount of one or more of the disclosed capsid protein, disclosed AAV particles, disclosed vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, or any combination thereof administered to a subject, or by changing the frequency of administration, or by changing the duration of administration to a subject or the duration between administrations.

[0070] In some aspects, “parallel” means (1) simultaneously in time, or (2) at different points in time within a common treatment schedule.

[0071] The term "contacting" may mean bringing together one or more of the disclosed capsid proteins, disclosed AAV particles, disclosed AAV vectors, disclosed nucleic acid molecules, disclosed compositions thereof, 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 the kidney) in a manner that allows one or more of the disclosed capsid proteins, disclosed AAV particles, disclosed AAV vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, or any combination thereof to exert a direct or indirect effect on the intended target or targeted area.

[0072] In some aspects, “determining” may refer to measuring or confirming the presence and severity of a disease, disorder, infection, symptom, and / or complication (e.g., relating to kidney disease and / or kidney impairment). Methods and techniques used to determine the presence and / or severity of a disease, disorder, infection, symptom, and / or complication are generally known in the field of medicine. For example, methods for identifying and / or diagnosing the presence, severity, or both of a disease, disorder, infection, symptom, and / or complication are well known in the art.

[0073] In some embodiments, the term “pharmaceutically acceptable carrier” refers to sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, as well as sterile powders for reconstitution in sterile injections or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters, such as ethyl oleate. In some embodiments, the pharmaceutical carrier used may be solid, liquid, or gaseous. In some embodiments, examples of solid carriers include lactose, clay, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, and stearic acid. In some embodiments, examples of liquid carriers include liquid sugar, peanut oil, olive oil, and water. In some embodiments, examples of gaseous carriers include carbon dioxide and nitrogen. Any convenient pharmaceutical medium may be used when preparing the disclosed compositions as oral dosage forms. For example, water, glycols, oils, alcohols, flavorings, preservatives, colorants, etc., can be used to form oral liquid preparations such as suspensions, elixirs, and solutions, while carriers such as starch, sugars, microcrystalline cellulose, diluents, granulators, lubricants, binders, and disintegrants can be used to form oral solid preparations such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units for which solid pharmaceutical carriers are used because they are easy to administer. If necessary, tablets can be coated by standard aqueous or non-aqueous techniques. Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersants. By including various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid, the action of microorganisms can be reliably prevented.It may also be desirable to include isotonic agents, such as sugars and sodium chloride. The inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, can result in sustained absorption of the injectable pharmaceutical form. Injectable depot formulations are prepared by forming a microcapsule matrix of the drug in biodegradable polymers such as polylactic acid-polyglycolide, poly(orthoester), and poly(acid anhydride). The rate of drug release can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Injectable depot formulations can also be prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues. Injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injection medium immediately before use. Suitable inert carriers include sugars such as lactose. It is desirable that at least 95% by weight of the active ingredient particles have an effective particle size within the range of 0.01 to 10 micrometers.

[0074] In some embodiments, the term “derivative” or “variant” means a compound having a structure derived from the structure of a parent compound (for example, a polypeptide having the sequence shown in any of SEQ ID NOs. 24 to 43 or a nucleic acid having the sequence shown in any of SEQ ID NOs. 171 to 190), whose structure is sufficiently similar to the structures disclosed herein, and which, based on that similarity, is expected by those skilled in the art to exhibit the same or similar activity and utility as the claimed compound, or, as a precursor, to induce the same or similar activity and utility as the claimed compound.

[0075] In one embodiment, the disclosed AAV genome or AAV vector, if present in a suitable producing cell and in the presence of AAV Rep and Cap proteins, can replicate and be packaged into AAV viral particles, particularly infectious viral particles.

[0076] In some embodiments, “genome particle (gp),” “genome equivalent,” or “genome copy” may refer to the number of virions containing the AAV DNA genome, regardless of viral titer, infectivity, or functionality.

[0077] In some embodiments, “adeno-associated virus” or “AAV” refers to a viral particle comprising at least one AAV capsid protein VP1, VP2, and / or VP3, preferably all three capsid proteins, and a capsid-formed polynucleotide AAV genome or AAV vector. The disclosed AAV may generally be recombinant AAV. The AAV may be an AAV that does not exist in nature. The AAV may include one or more heterologous polynucleotides, i.e., polynucleotides other than wild-type AAV polynucleotides, such as transgenes. An example of a transgene is a therapeutic gene.

[0078] In some embodiments, a “therapeutic gene” refers to a gene that, when expressed, produces a therapeutic gene product that confers a beneficial effect to the cells or tissues in which it exists, or to the mammal in which it is expressed. Examples of beneficial effects include improvement of signs or symptoms 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 genetic defects in cells or mammals. In some embodiments, therapeutic genes may be NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L.

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

[0080] In one embodiment, an "AAV reverse terminal repeat (ITR) sequence" or "ITR" may include a sequence of approximately 145 nucleotides present at both ends of the native single-stranded AAV genome.

[0081] In one embodiment, the “transgene” is a polynucleotide that encodes a gene delivered to a cell by the disclosed AAV vector.

[0082] In some embodiments, “gene” refers to a polynucleotide containing at least one open reading frame capable of encoding a particular gene product after transcription and sometimes after translation. The terms “gene” or “coding sequence” refer to a nucleotide sequence that codes for a gene product in vitro or in vivo. In some cases, a gene consists of or is essentially composed of a coding sequence, i.e., a sequence that codes for a gene product. In other cases, a gene includes additional non-coding sequences that enable, facilitate, or direct the expression of the encoded product by the cellular expression mechanism. Such sequences may include, but are not limited to, promoters, enhancers, transcription termination and / or poly(A) addition signals, and elements that affect the processing and / or stability of the transcript. A gene may or may not include regions preceding and following the coding region, e.g., a 5' untranslated (5'UTR) or “leader” sequence and a 3'UTR or “trailer” sequence, as well as intervening sequences (introns) between individual coding segments (exons).

[0083] A "gene product" is a molecule produced by the expression of a specific gene or a fragment thereof. Examples of gene products include polypeptides, aptamers, interfering RNA, and mRNA. A "gene product" may be a polypeptide, peptide, protein, or interfering RNA, including small interfering RNA (siRNA), miRNA, or small hairpin RNA (shRNA). In some embodiments, the disclosed gene product may be a therapeutic gene product, such as a therapeutic protein or therapeutic RNA (e.g., antisense oligonucleotide, ribozyme, siRNA, shRNA, or miRNA).

[0084] AAVs can be replication-competent or replication-incompetent. “Replication-competent” means that the virus or viral particle is infectious and capable of replicating in suitable infected cells. In some embodiments, the disclosed AAVs may be replication-incompetent.

[0085] In some embodiments, the term “viral vector” refers to a nucleic acid vector construct comprising at least one element of viral origin that can be packaged into a viral particle. Viral vectors may contain nucleic acids encoding polypeptides described herein (e.g., a transgene, a gene of interest, and / or a payload) instead of non-essential viral genes. Vectors and / or particles can be used to transfer nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.

[0086] In some embodiments, "AAV virus" or "AAV virus particle" refers to a viral particle composed of at least one AAV capsid protein, e.g., VP1 (generally all capsid proteins of wild-type AAV) and a capsid-formed polynucleotide AAV vector. If the particle contains heterologous polynucleotides (i.e., polynucleotides other than the wild-type AAV genome, e.g., a transgene targeted for delivery to mammalian cells), it is generally referred to as a "recombinant AAV vector particle" or simply an "AAV vector." Therefore, since such vectors are contained within AAV particles, the preparation of AAV particles always involves the preparation of AAV vectors.

[0087] In some embodiments, “viral capsid polypeptide” refers to the proteinaceous shell or coating of a viral particle. Viral capsid polypeptides enable the capsid polypeptide to be packaged or assembled into a viral particle eligible for delivery of nucleic acids to host cells. The capsid functions to capsidize, protect, transport, and release the viral genome into host cells. Capsids are generally composed of oligomeric structural subunits of the polypeptide of the viral capsid polypeptide.

[0088] In some embodiments, "capsid formation" means encapsulation within a viral capsid. For example, the AAV genome contains three duplicate sequences, VP1, VP2, and VP3, which encode capsid proteins, and these sequences begin at a single promoter, P40. The AAV capsid consists of a mixture of VP1, VP2, and VP3, with a total of 60 monomers arranged in an icosahedral symmetry in a 1:1:10 ratio.

[0089] In some embodiments, "packaging" refers to a series of intracellular events that result in the assembly and capsid formation of AAV particles.

[0090] In some embodiments, “payload” refers to a capsid-formed nucleic acid within a viral vector, such as an AAV vector. The payload nucleic acid may encode a polypeptide, inhibitory RNA, antibody or antibody reagent, oligonucleotide, or miRNA. In some embodiments, “payload” refers to one or more polynucleotides or polynucleotide regions encoded by or within the viral genome, or the expression product of a polynucleotide or modulating nucleic acid or regulatory nucleic acid encoding such a polynucleotide or polynucleotide region, such as a transgene, polypeptide, or multipolypeptide. In some embodiments, the disclosed payload may include any nucleic acid useful for modulating expression in target cells transduced with or in contact with AAV particles carrying the payload. In some embodiments, modulation may be by supplementation of the payload in the target cell or tissue. In some embodiments, modulation may be gene substitution by the payload in the target cell or tissue. In some embodiments, modulation may be by inhibition of the payload in the target cell or tissue using a modulating nucleic acid. In some embodiments, the disclosed payload may include a combination of coding nucleic acid sequences and non-coding nucleic acid sequences, and may be codon-optimized. In some embodiments, the payload may include one or more moduloable elements. In some embodiments, the disclosed payload may encode messenger RNA (mRNA) that can be encoded by the disclosed payload. In some embodiments, the disclosed payload may encode a gene therapy product. The gene therapy product may include a polypeptide, RNA molecule, or other gene product that, when expressed in target cells, produces a desired therapeutic effect. In some embodiments, the gene therapy product may include a substitute for a non-functional gene that is absent or mutated. In some embodiments, the disclosed payload nucleic acid may encode a transgene having a beneficial or desirable gene product.

[0091] In some embodiments, the term “polypeptide” refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. Peptides are relatively short polypeptides, generally between approximately 2 and 60 amino acids in length. Polypeptides as used herein generally contain amino acids, such as the 20 most commonly found L-amino acids in proteins. However, other amino acids and / or amino acid analogs known in the art may be used. One or more amino acids within a polypeptide can be modified by adding chemical entities, such as carbohydrate groups, phosphate groups, fatty acid groups, or linkers, for conjugation, functionalization, etc. Polypeptides in which non-polypeptide portions are linked covalently or noncovalently are still considered “polypeptides.” Exemplary modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis. In some embodiments, the terms “polypeptide sequence” or “amino acid sequence” may refer to the polypeptide material itself and / or sequence information (i.e., a set of letters or three-letter codes used as abbreviations for amino acid names) that biochemically characterize the polypeptide. Unless otherwise specified, polypeptide sequences presented herein are in the direction from the N-terminus to the C-terminus.

[0092] A variant amino acid or DNA sequence may 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 higher percentage identical to the native or reference sequence. The degree of homology (percent identity) between the native sequence and the variant sequence can be determined, for example, by comparing the two sequences using a computer program commonly used for this purpose, such as one freely available on the World Wide Web (e.g., BLASTp or BLASTn with default settings). Modifications to the native amino acid sequence can be achieved by any of several techniques known to those skilled in the art. A mutation can be introduced, for example, by synthesizing an oligonucleotide containing the variant sequence flanked by restriction sites that allow ligation with a fragment of the native sequence at a specific locus. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, site-directed mutagenesis (SMS) procedures using oligonucleotides can be used to produce altered nucleotide sequences in which specific codons are modified according to the desired substitutions, deletions, or insertions. Techniques for inducing such alterations are well-established. To improve the oxidative stability of the molecule and prevent abnormal crosslinking, any cysteine ​​residues that do not participate in maintaining the precise conformation of the polypeptide can also be substituted, generally with serine. Conversely, cysteine ​​bonds can also be added to polypeptides to improve their stability or facilitate oligomer formation.

[0093] In some embodiments, “polynucleotide sequence” may refer to sequence information (i.e., a series of letters used as abbreviations for bases) that biochemically characterizes the polynucleotide material itself and / or a particular nucleic acid. Polynucleotide sequences presented herein are oriented from 5' to 3' unless otherwise specified.

[0094] In some embodiments, when the term “corresponds” is used in relation to amino acid or polynucleotide sequences, it means that a given amino acid or polynucleotide sequence within one polypeptide or polynucleotide molecule has similar structural, functional, or both structural properties to an amino acid or polynucleotide sequence located at a similar position within another polypeptide or polynucleotide molecule. Homologs of a given polypeptide in different species “correspond” to each other, and similarly, regions or domains of homologous polypeptides from different species “correspond” to each other. Similarly, capsid polypeptides of viral vectors with different serotypes, including but not limited to adeno-associated virus (AAV) vectors, “correspond” to each other, and similarly, regions of such polypeptides defined, for example, by aligning their amino acid sequences, “correspond” to each other. While other alignment parameters can be used to define such regions, to avoid doubt, alignment can be performed using BLAST® (Basic Local Alignment Search Tool) with default parameters.

[0095] In some embodiments, the “promoter” or “promoter(s)” is publicly known in the art. Various promoter elements can be used depending on the desired level and tissue-specific expression. The promoter can be a tissue-specific promoter or a ubiquitous promoter, and can be a constitutive promoter or an inducible promoter, depending on the desired pattern of gene expression. The promoter may be native or exotic, and may be a natural or synthetic sequence. Exotic means that the transcription initiation region is not found in the wild-type host into which it is introduced.

[0096] "Tissue-specific promoters" are publicly known in the relevant technical field and include, but are not limited to, nerve-specific promoters, kidney-specific promoters, muscle-specific promoters, liver-specific promoters, skeletal muscle-specific promoters, and heart-specific promoters.

[0097] In some embodiments, a “ubiquitous / constitutive promoter” refers to a promoter that enables the continuous transcription of a gene in question. Ubiquitous / constitutive promoters are always active and can be used to express genes in a wide range of cells and tissues, including, but are not limited to, the liver, kidneys, skeletal muscle, cardiac muscle, smooth muscle, diaphragmatic muscle, brain, spinal cord, endothelial cells, intestinal cells, lung cells (e.g., smooth muscle cells or epithelial cells), peritoneal epithelial cells, and fibroblasts. Examples of ubiquitous / constitutive promoters include, but are not limited to, the CMV major early enhancer / chicken beta-actin promoter, the cytomegalovirus (CMV) major early promoter, the elongation factor 1-α (EF1α) promoter, the monkey vacuolated virus 40 (SV40) promoter, the AmpR promoter, the PγK promoter, the human ubiquitin C gene (Ubc) promoter, the MFG promoter, the human beta-actin promoter, the CAG promoter, the EGR1 promoter, the FerH promoter, the FerL promoter, the GRP78 promoter, the GRP94 promoter, the HSP70 promoter, the β-kin promoter, the mouse phosphoglycerate kinase (mPGK) or human PGK (hPGK) promoter, the ROSA promoter, the human ubiquitin B promoter, the Roussarcoma virus promoter, or any other native or synthetic ubiquitous / constitutive promoter.

[0098] In some embodiments, an "inducible promoter" refers to a promoter that can be regulated by positive or negative control. Factors that can modulate an inducible promoter include, but are not limited to, chemical agents (e.g., metallothionein promoters or hormone-inducible promoters), temperature, and light.

[0099] As used herein, “isolated” biological components (e.g., nucleic acid molecules, proteins, or viruses) are substantially separated or purified from other biological components (e.g., other intrachromosomal and extrachromosomal DNA and RNA, proteins, and / or organelles). “Isolated” nucleic acids, proteins, and / or viruses include nucleic acids, proteins, and viruses purified by standard purification methods. The term also encompasses nucleic acids, proteins, and viruses prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids or proteins. The term “isolated” (or “purified”) is intended as a relative term, not requiring absolute purity. Thus, for example, isolated or purified nucleic acids, proteins, viruses, or other active compounds are isolated, either whole or partially, from accompanying nucleic acids, proteins, and other contaminants. In some embodiments, the term “substantially purified” refers to nucleic acids, proteins, viruses, or other active compounds isolated from cells, cell culture media, or other crude preparations and subjected to fractionation to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0100] "Sequence identity" and "sequence similarity" can be determined by aligning two peptide or nucleotide sequences using a global or local alignment algorithm. If those sequences are optimally aligned, they can then be referred to as "substantially identical" or "essentially similar." For example, sequence similarity or identity can be determined by searching databases such as FASTA and BLAST, but hits should be retrieved and aligned in pairs to compare sequence identity. Two proteins or two protein domains, or two nucleic acid sequences, may have "substantially identical" sequence identity if their percentage sequence identity is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or higher, preferably 90%, 95%, 98%, 99%, or higher. Such sequences are also referred to herein as "variants," e.g., other variants of the AAV capsid protein. It should be understood that sequences 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 one has additional nucleotides at the 3' and / or 5' ends are 100% identical.

[0101] A "codon-optimized" nucleic acid refers to a nucleic acid sequence in which the codons have been modified to optimize expression in a particular system (e.g., a particular species or group of species). For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a specific mammalian species (e.g., human cells). Codon optimization does not alter the amino acid sequence of the encoded protein.

[0102] This specification discloses components used to prepare one or more of the disclosed capsid proteins, disclosed vectors, disclosed nucleic acid molecules, disclosed compositions thereof, disclosed pharmaceutical formulations, and / or disclosed methods, which are used within the methods disclosed herein. These and other materials are disclosed herein, and combinations, subsets, interactions, groups, etc., of these materials are disclosed, but specific references to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed, and it is understood that each is specifically intended and described herein. For example, if a particular compound is disclosed and discussed, and several modifications that can be made to several molecules including the compound are discussed, then, unless otherwise indicated, all possible combinations and permutations of the compound and modifications are specifically intended. Thus, if classes A, B, and C and classes D, E, and F of molecules are disclosed, and an example of a combination of molecules, AD, is disclosed, then even if each is not described individually, it is understood that each is individually and collectively intended, i.e., combinations AE, AF, BD, BE, BF, CD, CE, and CF are disclosed. Similarly, any subset or combination of these is also disclosed. Therefore, for example, the subgroups AE, BF, and CE are also disclosed. This concept applies to all aspects of this application, including, but not limited to, steps of methods for producing and using the compositions of the present invention. Therefore, where various additional steps can be implemented, it is understood that each of these additional steps can be implemented in conjunction with any particular aspect or combination of aspects of the methods of the present invention.

[0103] B. Composition 1. AAV capsid protein AAV capsid proteins are disclosed herein. AAV capsid proteins having one or more substitutions in variable region IV (VR-IV) are disclosed herein.

[0104] An adeno-associated virus (AAV) capsid protein is disclosed herein, wherein the AAV capsid protein comprises an amino acid sequence in which positions 452-458 are at least 85% identical to one of sequence numbers 04-23, and positions 452-458 of the AAV capsid protein are numbered with reference to sequence number 01.

[0105] In some embodiments, the disclosed AAV capsid protein may contain 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 NOs: 24 to 43.

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

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

[0108] In some embodiments, the disclosed AAV capsid protein may be a variant of the parental wild-type capsid protein. In some embodiments, the disclosed parental wild-type capsid protein may be the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10. In some embodiments, the disclosed parental wild-type capsid protein may be VP1 of AAV9. In some embodiments, the disclosed parental wild-type capsid protein may be VP2 of AAV9. In some embodiments, the disclosed parental wild-type capsid protein may be VP3 of AAV9.

[0109] In some embodiments, the disclosed AAV capsid protein may improve gene transfer to and / or expression in one or more regions or parts of the kidney compared to the disclosed parental wild-type capsid protein. In some embodiments, gene transfer and / or expression may be improved by 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-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, or at least 14-fold. In some embodiments, the disclosed region or part of the kidney may be the adrenal gland, cortex, medulla, trabecula, pyramidal cavity, pelvis, major calyces, minor calyces, papilla, or ureter. In some embodiments, the disclosed region or part of the kidney may be the proximal tubule within the cortex.

[0110] An adeno-associated virus (AAV) capsid protein containing the sequence shown in Sequence ID No. 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein containing a sequence having at least 90% identity with the sequence shown in Sequence ID No. 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein containing one or more amino acid substitutions at positions 452-458 compared to Sequence ID No. 01 is disclosed herein.

[0111] In some embodiments of the disclosed AAV capsid protein, positions 452-458 may contain the sequence shown in any one of SEQ ID NOs. 04-23. In some embodiments of the disclosed AAV capsid protein, positions 452-458 may contain a sequence that is at least 85% identical to any one of SEQ ID NOs. 04-23. In some embodiments of the disclosed AAV capsid protein, positions 452-458 may contain the sequence shown in any one of SEQ ID NOs. 191-8873. In some embodiments of the disclosed AAV capsid protein, positions 452-458 may contain a sequence that is at least 85% identical to any one of SEQ ID NOs. 191-8873. In some embodiments of the disclosed AAV capsid protein, positions 452-458 may contain the sequence shown in SEQ ID NOs. 11, SEQ ID NOs. 15, or SEQ ID NOs. 23. In some embodiments of the disclosed AAV capsid protein, positions 452–458 may contain the sequence shown in any one of SEQ ID NOs. 44–52.

[0112] In some embodiments of the disclosed AAV capsid protein, positions 452–458 of the AAV capsid may include shortened substitutions, such as AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0113] An adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452-458 of Sequence ID No. 01 is disclosed herein, wherein the substitution at position 452 is any amino acid other than N; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or the substitution at position 458 is any amino acid other than Q. In some embodiments, one or more disclosed amino acid substitutions may include substitutions other than GVSLGGG.

[0114] In some embodiments of the disclosed AAV capsid protein, each of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, one or more of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, two or more of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, three or more of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, four or more of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, five or more of the positions 452 to 458 may have an amino acid substitution. In some embodiments of the disclosed AAV capsid protein, six or more amino acid substitutions may be present at positions 452–458. In some embodiments of the disclosed AAV capsid protein, seven or more amino acid substitutions may be present at positions 452–458.

[0115] An adeno-associated virus (AAV) capsid protein comprising the sequence shown in any one of SEQ ID NOs: 24 to 43 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising the sequence shown in SEQ ID NOs: 31, 35, or 43 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising the sequence shown in any one of SEQ ID NOs: 53 to 61 is disclosed herein. In some embodiments, the disclosed AAV capsid protein is not SEQ ID NO: 57.

[0116] Adeno-associated virus (AAV) capsid proteins are disclosed herein that include sequences having one or more substitutions compared to the sequences shown in SEQ ID NO: 62, SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 77, and SEQ ID NO: 82. Adeno-associated virus (AAV) capsid proteins are disclosed herein that include sequences having one or more substitutions compared to the sequences shown 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 one embodiment, the disclosed wild-type sequences (shown in Table 1 below) have one or more substitutions in one or more regions of the protein. [Table 1]

[0117] In some embodiments, one or more disclosed substitutions may include one substitution, two substitutions, three substitutions, four substitutions, five substitutions, six substitutions, seven substitutions, eight substitutions, nine substitutions, or ten substitutions. In some embodiments, one or more disclosed substitutions may include seven substitutions.

[0118] Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 65 or SEQ ID NO: 66 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 75 or SEQ ID NO: 76 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 80 or SEQ ID NO: 81 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 85 or SEQ ID NO: 86 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 90 or SEQ ID NO: 91 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 95 or SEQ ID NO: 96 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 100 or SEQ ID NO: 101 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 105 or SEQ ID NO: 106 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 110 or SEQ ID NO: 111 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 115 or SEQ ID NO: 116 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 120 or SEQ ID NO: 121 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 125 or SEQ ID NO: 126 are disclosed herein. Adeno-associated virus (AAV) capsid proteins containing the sequence shown in SEQ ID NO: 130 or SEQ ID NO: 131 are disclosed herein. Adeno-associated virus (AAV) capsid proteins comprising the sequence shown in SEQ ID NO: 135 or SEQ ID NO: 136 are disclosed herein. Adeno-associated virus (AAV) capsid proteins comprising the sequence shown in SEQ ID NO: 140 or SEQ ID NO: 141 are disclosed herein.For example, in one embodiment, the disclosed AAV capsid variant may include the sequences listed in Table 2 below. [Table 2-1] [Table 2-2]

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

[0120] An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 62 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 67 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 72 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 77 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 82 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 87 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 92 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 97 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 102 and comprises a sequence including one or more substitutions in variable region IV (VR-IV).An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 107 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 112 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 117 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity to the sequence shown in Sequence ID No. 122 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 127 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 132 and includes one or more substitutions in variable region IV (VR-IV). An adeno-associated virus (AAV) capsid protein is disclosed herein that has at least 85% identity with the sequence shown in Sequence ID No. 137 and includes one or more substitutions in variable region IV (VR-IV).

[0121] In some embodiments, the disclosed AAV capsid protein can be used to improve and / or enhance gene transfer into one or more renal cells, kidney-derived cell types, and / or kidney-associated cell types compared to wild-type AAV capsid protein. In some embodiments, the disclosed AAV capsid protein can be used to induce widespread transduction into one or more renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0122] In some embodiments, the disclosed AAV capsid protein can be used to transduce one or more renal cells, kidney-derived cell types, and / or kidney-associated cell types more efficiently than wild-type AAV capsid protein. In some embodiments, the increase and / or improvement in transduction efficiency may include 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, an 80% increase and / or improvement, a 90% increase and / or improvement, a 100% increase and / or improvement, or an increase and / or improvement greater than 100%.

[0123] In some embodiments, the disclosed AAV capsid protein can be used to improve and / or enhance gene transfer to any region or portion of the kidney. In some embodiments, the disclosed AAV capsid protein may exhibit improved dose-response correlation (e.g., thereby improving efficiency).

[0124] In one embodiment, the disclosed wild-type capsid protein may include the sequence shown in SEQ ID NO: 01. In another embodiment, the disclosed wild-type capsid protein may include the sequence shown 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 some embodiments, the disclosed renal cells, renal-derived cell types, and / or renal-associated cell types may include renal epithelial cells and / or renal endothelial cell types. In some embodiments, the renal cells, renal-derived cell types, and / or renal-associated cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, cells in the connecting tubule, or any combination thereof. In some embodiments, the disclosed region or portion of the kidney may include the adrenal gland, cortex, medulla, trabecula, pyramidal region, pelvis, major calyces, minor calyces, papillae, ureter, or any combination thereof.

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

[0127] In some embodiments, the disclosed AAV capsid protein can be used to reduce and / or decrease one or more symptoms associated with and / or related to renal disease and / or renal impairment in a subject. In some embodiments, the disclosed AAV capsid protein can be used to prevent the development of undesirable physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed AAV capsid protein can be used to inhibit, i.e., prevent the development of, physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed AAV capsid protein can be used to mitigate, i.e., induce disease regression in a subject.

[0128] In some embodiments, the disclosed renal diseases or disorders include Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittellmann syndrome, Liddle syndrome, Lowe syndrome, nephronophthys (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof.

[0129] In some embodiments, the disclosed AAV capsid protein can be used to improve renal function in a subject. In some embodiments, the disclosed AAV capsid protein can be used to reduce the risk of acute and / or chronic renal failure in a subject. In some embodiments, the disclosed AAV capsid protein can be used to reduce the risk of kidney infection in a subject. In some embodiments, the disclosed AAV capsid protein can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in a subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed AAV capsid protein can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0130] In some embodiments, the disclosed AAV capsid protein can be used to treat subjects in need of it. In some embodiments, the disclosed AAV capsid protein can be used in a method of delivering gene therapy to subjects in need of it. In some embodiments, subjects in need of it are one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronopharyngeal plasia (NPHP)). The disclosed AAV capsid protein may be incorporated into the disclosed AAV capsid. In some embodiments, the disclosed AAV capsid protein may exhibit improved tropism to one or more cell types and / or one or more tissue types (e.g., one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types). In some embodiments, the disclosed AAV capsid protein may exhibit improved transduction efficiency and / or properties when introduced into one or more cell types and / or one or more tissue types. In some embodiments, the disclosed AAV capsid variant may efficiently transduce one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types. In some embodiments, the disclosed AAV capsid protein may exhibit superior performance to parental wild-type AAV in one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0131] In some embodiments, the disclosed AAV capsid protein can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts. In some embodiments, the disclosed AAV capsid protein can be used in a method to improve the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed AAV capsid protein can be used in a method to reduce the risk of graft-versus-host disease (GVHD) occurring after transplantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed AAV capsid protein can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it).In some embodiments, the disclosed AAV capsid protein can be used in a method to enhance the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation and return to a subject in need). In some embodiments, the disclosed AAV capsid protein can be used in a method to extend and / or improve the life expectancy of a subject.

[0132] AAV capsids comprising the disclosed AAV capsid protein are disclosed herein. An AAV capsid protein comprising the sequence shown in SEQ ID NO: 03 is disclosed herein. An AAV capsid protein comprising a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 03 is disclosed herein. An AAV capsid protein comprising one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01 is disclosed herein. In some embodiments of the disclosed AAV capsid, positions 452-458 of the AAV capsid protein may comprise the sequence shown in any one of SEQ ID NOs: 04-23. In some embodiments of the disclosed AAV capsid, positions 452-458 may comprise a sequence that is at least 85% identical to any one of SEQ ID NOs: 04-23. In some embodiments of the disclosed AAV capsid, positions 452-458 may comprise the sequence shown in any one of SEQ ID NOs: 191-8873. In some embodiments of the disclosed AAV capsid, positions 452–458 may contain a sequence that is at least 85% identical to one of the sequence codes 191–8873. In some embodiments of the disclosed AAV capsid, positions 452–458 of the AAV capsid protein may contain the sequence shown in sequence code 11, sequence code 15, or sequence code 23.

[0133] In some embodiments, positions 452-458 of the AAV capsid may include shortened substitutions, 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 having one or more amino acid substitutions at positions 452-458 of Sequence ID No. 01, wherein the substitution at position 452 is any amino acid other than N; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or 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 has a sequence represented by any one of Sequence ID No. 24-43. An AAV capsid comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence shown in SEQ ID NO: 31 or SEQ ID NO: 35, is disclosed herein.

[0135] Disclosed herein are AAV capsids comprising an adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein comprises the sequence shown in any one of SEQ ID NOs. 53 to 61. In some embodiments, the disclosed AAV capsid protein is not SEQ ID NO. 57. In some embodiments of the disclosed AAV capsid, one or more amino acid substitutions in the AAV capsid protein may include substitutions other than GVSLGGG (SEQ ID NO. 50).

[0136] Libraries of AAV capsid proteins are disclosed herein. Libraries of AAV capsid proteins having one or more substitutions in variable region IV (VR-IV) are disclosed herein. Libraries of AAV capsid proteins containing the sequence shown in SEQ ID NO: 03 are disclosed herein. Libraries of AAV capsid proteins containing a sequence having at least 90% identity to the sequence shown in SEQ ID NO: 03 are disclosed herein. Libraries of AAV capsid proteins containing one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01 are disclosed herein. Libraries of AAV capsid proteins where positions 452-458 relative to SEQ ID NO: 01 may contain the sequence shown in any one of SEQ ID NOs from A library of AAV capsid proteins is disclosed herein, in which positions 452-458 relative to SEQ ID NO: 01 may contain at least 85% identical sequences to any one of SEQ ID NOs from 04 to 23. A library of AAV capsid proteins is disclosed herein, in which positions 452-458 relative to SEQ ID NO: 01 may contain sequences shown in any one of SEQ ID NOs from 191 to 8873. A library of AAV capsid proteins is disclosed herein, in which positions 452-458 relative to SEQ ID NO: 01 may contain at least 85% identical sequences to any one of SEQ ID NOs from 191 to 8873. A library of AAV capsid proteins is disclosed herein, in which the capsid proteins contain sequences shown in any one of SEQ ID NOs from 24 to 43 or SEQ ID NOs from 53 to 61.

[0137] 2. Nucleic acid molecules Nucleic acid molecules comprising nucleic acid sequences encoding the disclosed AAV capsid protein are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding the AAV capsid protein are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding the AAV capsid protein having one or more substitutions in variable region IV (VR-IV) are disclosed herein.

[0138] Disclosed herein is a nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein the amino acid sequence at positions 452–458 of the AAV capsid protein is at least 85% identical to one of SEQ ID NOs. 04–23, and the amino acid sequence at positions 452–458 of the AAV capsid protein is numbered with reference to SEQ ID NOs. 01. In some embodiments, the disclosed encoded AAV capsid protein may 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 one of SEQ ID NOs. 24–43.

[0139] This specification discloses nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein, which include a sequence having one or more substitutions compared to the sequence shown in Sequence ID No. 01.

[0140] A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in Sequence ID No. 03 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing a sequence having at least 90% identity to the sequence shown in Sequence ID No. 03 is disclosed herein.

[0141] Disclosed herein are nucleic acid molecules comprising nucleic acid sequences encoding an AAV capsid protein, which includes one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which positions 452-458 may contain the sequence shown in any one of SEQ ID NOs. 04-23. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which positions 452-458 may contain the sequence shown in any one of SEQ ID NOs. 191-8873. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which positions 452-458 may contain a sequence that is at least 85% identical to any one of SEQ ID NOs. 04-23. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which positions 452-458 may contain a sequence that is at least 85% identical to any one of SEQ ID NOs. 191-8873.

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

[0143] In one embodiment, the disclosed nucleic acid molecule may encode an AAV capsid protein in which the 452-458 positions of the AAV capsid may contain a shortened substitution, for example, AEG, GE, GGG, GGR, GGV, GIM, GQA, GRV, SD, SSM, SSR, SSS, VGG, VHL, VNL, VSK, VSR, VST, YSG, or YSR.

[0144] Disclosed herein are nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein, the sequence comprising a sequence having one or more substitutions compared to the sequence shown 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 one embodiment, the disclosed wild-type sequence (Table 1) has one or more substitutions within one or more regions of the protein. In one embodiment, the disclosed one or more substitutions may comprise one, two, three, four, five, six, seven, eight, nine, or ten substitutions. In one embodiment, the disclosed one or more substitutions may comprise seven substitutions.

[0145] Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 65 or SEQ ID NO: 66 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 70 or SEQ ID NO: 71 are disclosed herein. Adeno-associated virus (AAV) capsid proteins comprising the sequence shown in SEQ ID NO: 75 or SEQ ID NO: 76 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 80 or SEQ ID NO: 81 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 85 or SEQ ID NO: 86 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 90 or SEQ ID NO: 91 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 95 or SEQ ID NO: 96, are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 100 or SEQ ID NO: 101, are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 105 or SEQ ID NO: 106, are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 110 or SEQ ID NO: 111, are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 115 or SEQ ID NO: 116, are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 120 or SEQ ID NO: 121, are disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, including the sequence shown in SEQ ID NO: 125 or SEQ ID NO: 126, is disclosed herein.Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 130 or SEQ ID NO: 131 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 135 or SEQ ID NO: 136 are disclosed herein. Nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein containing the sequence shown in SEQ ID NO: 140 or SEQ ID NO: 141 are disclosed herein. For example, in one embodiment, the disclosed encoded AAV capsid variant may include the sequences shown in Table 2.

[0146] Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 62, excluding one or more substitutions in the region of SEQ ID NO: 63 or SEQ ID NO: 64. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 67, excluding one or more substitutions in the region of SEQ ID NO: 68 or SEQ ID NO: 69. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 72, excluding one or more substitutions in the region of SEQ ID NO: 73 or SEQ ID NO: 74. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 77, excluding one or more substitutions in the region of SEQ ID NO: 78 or SEQ ID NO: 79. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, with one or more substitutions in the region of SEQ ID NO: 83 or SEQ ID NO: 84. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, with one or more substitutions in the region of SEQ ID NO: 88 or SEQ ID NO: 89. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, with one or more substitutions in the region of SEQ ID NO: 93 or SEQ ID NO: 94. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, with one or more substitutions in the region of SEQ ID NO: 98 or SEQ ID NO: 97. Disclosed herein are nucleic acid molecules comprising a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, which includes the sequence shown in SEQ ID NO: 102, with the exception of one or more substitutions in the region of SEQ ID NO: 103 or SEQ ID NO: 104.Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 107, excluding one or more substitutions in the region of SEQ ID NO: 108 or SEQ ID NO: 109. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 112, excluding one or more substitutions in the region of SEQ ID NO: 113 or SEQ ID NO: 114. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 117, excluding one or more substitutions in the region of SEQ ID NO: 118 or SEQ ID NO: 119. An adeno-associated virus (AAV) capsid protein is disclosed herein that includes the sequence shown in SEQ ID NO: 122, excluding one or more substitutions in the region of SEQ ID NO: 123 or SEQ ID NO: 124. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 127, with one or more substitutions in the region of SEQ ID NO: 128 or SEQ ID NO: 129. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 132, with one or more substitutions in the region of SEQ ID NO: 133 or SEQ ID NO: 134. Nucleic acid molecules are disclosed herein that include a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, comprising the sequence shown in SEQ ID NO: 137, with one or more substitutions in the region of SEQ ID NO: 138 or SEQ ID NO: 139.

[0147] Disclosed herein are nucleic acid molecules comprising nucleic acid sequences encoding an adeno-associated virus (AAV) capsid protein, which have at least 85% identity to the sequences shown 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, and which have 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, the 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; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or the substitution at position 458 is any amino acid other than Q. In some embodiments of the disclosed nucleic acid molecule, one or more amino acid substitutions may include substitutions other than GVSLGGG (SEQ ID NO: 50).

[0149] In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which each of the positions 452–458 may have an amino acid substitution. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein in which one or more of the positions 452–458 may have an amino acid substitution, or two or more of the positions 452–458 may have an amino acid substitution, or three or more of the positions 452–458 may have an amino acid substitution, or four or more of the positions 452–458 may have an amino acid substitution, or five or more of the positions 452–458 may have an amino acid substitution, or six or more of the positions 452–458 may have an amino acid substitution, or seven or more of the positions 452–458 may have an amino acid substitution.

[0150] A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in any one of SEQ ID NOs: 24 to 43 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in SEQ ID NOs: 31, 35, or 43 is disclosed herein. A nucleic acid molecule comprising a nucleic acid sequence encoding an AAV capsid protein containing the sequence shown in any one of SEQ ID NOs: 53 to 61 is disclosed herein.

[0151] A nucleic acid molecule comprising the sequence shown in Sequence ID No. 02 is disclosed herein, wherein the nucleotides at positions 1354 to 1374 contain seven amino acids, and one or more of these seven amino acids are substituted. A nucleic acid molecule comprising the sequence shown in any one of Sequence ID No. 171 to No. 190 is disclosed herein. A nucleic acid molecule comprising the sequence shown in any one of Sequence ID No. 171 to No. 190 is disclosed herein, wherein the encoded AAV capsid protein can improve and / or enhance gene transfer into one or more kidney cells or kidney-derived cell types compared to wild-type capsid protein. A nucleic acid molecule comprising the sequence shown in any one of Sequence ID No. 171 to No. 190 is disclosed herein, wherein the encoded AAV capsid protein can show improved and / or enhanced gene transfer into any region or part of the kidney. Disclosed herein are nucleic acid molecules comprising the sequence shown in any one of SEQ ID NOs: 171 to 190, wherein the encoded AAV capsid protein may exhibit improved dose-response correlation.

[0152] In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein that can be used to improve and / or enhance gene transfer into one or more renal cells, kidney-derived cell types, and / or kidney-associated cell types compared to a wild-type AAV capsid protein. In some embodiments, the disclosed nucleic acid molecule can be used to result in widespread transduction into one or more renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0153] In some embodiments, the disclosed nucleic acid molecule can be used to transduce one or more kidney cells or kidney-derived cell types more efficiently than nucleic acid molecules encoding wild-type capsid proteins. In some embodiments, the increase and / or improvement in transduction efficiency may include 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, an 80% increase and / or improvement, a 90% increase and / or improvement, a 100% increase and / or improvement, or an increase and / or improvement greater than 100%.

[0154] In some embodiments, the disclosed nucleic acid molecules can be used to improve and / or enhance gene transfer to any region or portion of the kidney. In some embodiments, the disclosed nucleic acid molecules may exhibit improved dose-response correlation (e.g., thereby improved efficiency).

[0155] For example, in one embodiment, the disclosed wild-type capsid protein may include the sequence shown in SEQ ID NO: 01. In one embodiment, the disclosed wild-type capsid protein may include the sequences shown 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 some embodiments, the disclosed renal cells or kidney-derived cell types may include renal epithelial cells and / or renal endothelial cell types. In some embodiments, the disclosed renal cells or kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0157] In some embodiments, the disclosed region or portion of the kidney may include the adrenal gland, cortex, medulla, trabecula, pyramidal region, pelvis, major calyces, minor calyces, papillae, ureter, or any combination thereof. [Table 3-1] [Table 3-2]

[0158] In some embodiments, the disclosed nucleic acid molecule may comprise a nucleotide sequence of one or more regulatory elements. For example, the disclosed regulatory element may comprise a promoter operably ligated to the disclosed nucleic acid molecule, wherein the promoter drives the expression of the disclosed capsid protein, the disclosed encoded polypeptide, the disclosed encoded therapeutic agent, or any of them.

[0159] In some embodiments, the disclosed nucleic acid molecule may comprise a sequence of at least one of polypeptides, glycopeptides, polysaccharides, glycolipids, lipids, or nucleic acid polymers, or a combination thereof. In some embodiments, the disclosed nucleic acid molecule may comprise a sequence of at least one therapeutic agent. In some embodiments, the disclosed therapeutic agent may be an oligonucleotide therapeutic agent. In some embodiments, the disclosed oligonucleotide therapeutic agent may be single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide nucleic acid (PNA), or analogs or conjugates thereof. In some embodiments, the disclosed therapeutic agent may be an ASO or RNAi. In some embodiments, the disclosed nucleic acid-based molecule may comprise one or more modifications at any applicable position. In some embodiments, the disclosed therapeutic agent may comprise a CRISPR-based endonuclease (e.g., Cas9). In some embodiments, the disclosed CRISPR-based endonuclease may be derived from a CRISPR / Cas type I, type II, or type III system.

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

[0161] In some embodiments, the disclosed nucleic acid molecules can be used to reduce and / or decrease one or more symptoms associated with and / or related to kidney disease and / or kidney impairment in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to prevent the development of undesirable physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to inhibit, i.e., prevent the development of, physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to mitigate, i.e., induce disease regression in a subject.

[0162] In some embodiments, the disclosed renal diseases or disorders include Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittellmann syndrome, Liddle syndrome, Lowe syndrome, nephronophthys (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof.

[0163] In some embodiments, the disclosed nucleic acid molecules can be used to improve renal function in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to reduce the risk of acute and / or chronic renal failure in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to reduce the risk of kidney infection in a subject. In some embodiments, the disclosed nucleic acid molecules can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in a subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed nucleic acid molecules can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0164] In some embodiments, the disclosed nucleic acid molecules can be used to treat subjects in need of them. In some embodiments, the disclosed nucleic acid molecules can be used in a method of delivering gene therapy to subjects in need of it. In some embodiments, subjects in need of it may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0165] In some embodiments, the gene therapy product may comprise a polypeptide, RNA molecule, or other gene product that, when expressed in target cells, produces a desired therapeutic effect. In some embodiments, the gene therapy product may comprise a substitute for a non-functional gene that is absent or mutated. In some embodiments, the disclosed payload nucleic acid may encode a transgene having a beneficial or desirable gene product.

[0166] In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein that may be incorporated into the disclosed AAV capsid. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein that can exhibit improved tropism to one or more cell types and / or one or more tissue types (e.g., one or more disclosed kidney cells, kidney-derived cell types, and / or kidney-associated cell types). In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein that, when introduced into one or more cell types and / or one or more tissue types, can exhibit improved transduction efficiency and / or properties. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid variant that can efficiently transduce one or more disclosed kidney cells, kidney-derived cell types, and / or kidney-associated cell types. In some embodiments, the disclosed nucleic acid molecule may encode an AAV capsid protein that can exhibit superior performance to parental wild-type AAV to one or more disclosed kidney cells, kidney-derived cell types, and / or kidney-associated cell types. In some embodiments, the disclosed nucleic acid molecules 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, retrograde ureteral infusion, renal artery administration, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ophthalmic administration, intraocular administration, ear drops, intrauterine administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable administrations such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration of the disclosed nucleic acid molecules may be continuous or intermittent. In some embodiments, the disclosed nucleic acid molecules can be administered by one or more ex vivo methods, such as an ex vivo perfusion protocol. In some embodiments, an ex vivo perfusion protocol using the disclosed nucleic acid molecules can be used in conjunction with a kidney (or portion thereof) obtained for a subject. In some embodiments, a kidney can be obtained from a donor subject and subjected to an ex vivo perfusion protocol using the disclosed nucleic acid molecules before implantation into a subject requiring it. In one embodiment, a kidney can be obtained from an object in need, subjected to an ex vivo perfusion protocol using the disclosed nucleic acid molecule, and then returned to the object in need. In another embodiment, the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule can be applied to other relevant tissues in the object in need.

[0167] In some embodiments, the disclosed nucleic acid molecules can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts. In some embodiments, the disclosed nucleic acid molecules can be used in a method to improve the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof before implantation into a subject requiring it). In some embodiments, the disclosed nucleic acid molecules can be used in a method to reduce the risk of graft-versus-host disease (GVHD) occurring after transplantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof before implantation into a subject requiring it). In some embodiments, the disclosed nucleic acid molecules can be used in a method for reducing the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it).In some embodiments, the disclosed nucleic acid molecules can be used in a method to enhance the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation and return to a subject in need). In some embodiments, the disclosed nucleic acid molecules can be used to extend and / or improve the life expectancy of a subject.

[0168] 3.Cells Cells containing the disclosed nucleic acid molecule are disclosed herein. Cells containing the disclosed AAV having a capsid containing a capsid protein are disclosed herein. Cells containing the disclosed capsid having a disclosed variant are disclosed herein. Cells containing the disclosed viral vector are disclosed herein. Cells containing AAV vector particles are disclosed herein. Producing cells capable of producing AAV having a disclosed AAV capsid variant are disclosed herein. Cells used to carry out the disclosed method and / or produce the effect thereof are disclosed herein. Cells used to carry out the disclosed method for directional evolution of the AAV capsid protein and / or produce the effect thereof are disclosed herein. Cells used to carry out the disclosed method for generating AAV particles and / or produce the effect thereof are disclosed herein. Cells used to carry out the disclosed method for delivering a payload and / or produce the effect thereof are disclosed herein. Cells used to carry out the disclosed method for treating a subject and / or produce the effect thereof are disclosed herein.

[0169] 4. Vectors and Particles A vector containing a disclosed nucleic acid molecule is disclosed herein. A vector containing a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein is disclosed herein.

[0170] Disclosed herein is an AAV vector comprising a nucleic acid molecule containing a target gene and a nucleic acid sequence encoding an adeno-associated virus (AAV) capsid protein, wherein positions 452–458 of the AAV capsid protein contain an amino acid sequence that is at least 85% identical to any one of SEQ ID NOs. 04–23, and positions 452–458 of the AAV capsid protein are numbered with reference to SEQ ID NOs. 01. In some embodiments of the disclosed AAV vector, the encoded AAV capsid protein may contain 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 NOs. 24–43.

[0171] AAV vectors comprising a disclosed nucleic acid molecule encoding a disclosed AAV capsid protein are disclosed herein. AAV vectors comprising at least one heterologous nucleic acid are disclosed herein. AAV vectors comprising a vector genome are disclosed herein. In some embodiments, the disclosed vector genome may comprise a first reverse-end repeat (ITR) and a second ITR. In some embodiments, the disclosed vector genome may comprise a nucleic acid sequence encoding a transgene or payload between the first and second ITRs. AAV vectors comprising at least one heterologous nucleic acid and at least one reverse-end repeat (ITR) are disclosed herein. In some embodiments, for example, the at least one ITR may be an AAV2 ITR.

[0172] AAV vectors containing heterologous nucleic acids for therapeutic proteins and / or therapeutic RNAs are disclosed herein. AAV vectors containing heterologous nucleic acids for treating subjects with renal disease and / or renal impairment are disclosed herein. AAV vectors containing nucleic acid sequences encoding transgenes are disclosed herein. AAV vectors containing nucleic acid sequences encoding transgenes for treating subjects with renal disease and / or renal impairment are disclosed herein.

[0173] AAV particles comprising an AAV capsid containing a disclosed AAV capsid protein are disclosed herein. AAV particles comprising (i) an AAV capsid containing at least one disclosed AAV capsid protein and (ii) a vector genome are disclosed herein.

[0174] AAV particles for use in the disclosed methods are disclosed herein. AAV particles for use in the disclosed methods of delivering a payload or processing a subject are disclosed herein.

[0175] In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having a sequence shown in SEQ ID NO: 03. In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 03.

[0176] In some embodiments of the disclosed AAV particles, 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 some embodiments, positions 452-458 of the AAV capsid protein may contain the sequence shown in any one of SEQ ID NOs: 04-23. In some embodiments, positions 452-458 of the AAV capsid protein may contain the sequence shown in any one of SEQ ID NOs: 191-8873. In some embodiments, positions 452-458 of the AAV capsid protein may contain a sequence that is at least 85% identical to any one of SEQ ID NOs: 04-23. In some embodiments, positions 452-458 of the AAV capsid protein may contain a sequence that is at least 85% identical to any one of SEQ ID NOs: 191-8873. In some embodiments, positions 452–458 of the AAV capsid protein may contain the sequence shown in SEQ ID NO: 11, SEQ ID NO: 15, or SEQ ID NO: 23. In some embodiments, positions 452–458 of the AAV capsid may contain shortened substitutions, 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 some embodiments of the disclosed AAV particles, the AAV capsid is 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; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or the substitution at position 458 is any amino acid other than Q.

[0178] In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having the sequence shown in any one of SEQ ID NOs: 24 to 43. In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having the sequence shown in any one of SEQ ID NOs: 53 to 61. In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having the sequence shown in SEQ ID NOs: 31 or 35. In some embodiments of the disclosed AAV capsid, one or more amino acid substitutions in the AAV capsid protein may include substitutions other than GVSLGGG (SEQ ID NO: 50).

[0179] In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having one or more substitutions compared to the sequence shown 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 one embodiment of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having the sequence shown in SEQ ID NO: 65 or 66, SEQ ID NO: 70 or 71, SEQ ID NO: 75 or 76, SEQ ID NO: 80 or 81, SEQ ID NO: 85 or 86, SEQ ID NO: 90 or 91, SEQ ID NO: 95 or 96, SEQ ID NO: 100 or 101, SEQ ID NO: 105 or 106, SEQ ID NO: 110 or 111, SEQ ID NO: 115 or 116, SEQ ID NO: 120 or 121, SEQ ID NO: 125 or 126, SEQ ID NO: 130 or 131, SEQ ID NO: 135 or 136 or SEQ ID NO: 140 or 141.

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

[0182] In some embodiments of the disclosed AAV particles, the AAV capsid comprises an AAV capsid protein having a sequence that is at least 85% identical to the sequence shown 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, and having one or more substitutions in variable region IV (VR-IV).

[0183] In some embodiments, the nucleic acid sequence or heterologous nucleic acid encoding the disclosed transgene may be operably ligated to one or more transcriptional regulatory elements. In some embodiments, one or more transcriptional regulatory elements may increase the transcription and / or expression of the transgene or heterologous nucleic acid.

[0184] In some embodiments, the nucleic acid sequence or heterogeneous nucleic acid (i.e., the gene of interest) encoding the disclosed transgene may be operably ligated to a promoter. In some embodiments, the disclosed transcriptional regulatory element may include a ubiquitous promoter operably ligated to the disclosed transgene or heterogeneous nucleic acid, which drives the expression of the disclosed transgene or heterogeneous nucleic acid. In some embodiments, the disclosed transcriptional regulatory element may include a ubiquitous promoter operably ligated to the disclosed transgene or heterogeneous nucleic acid, and a tissue-specific promoter drives the expression of the disclosed transgene or heterogeneous nucleic acid. In some embodiments, the tissue-specific promoter may be a kidney-specific promoter and / or a kidney-specific enhancer. In some embodiments, the disclosed transcriptional regulatory element may include a kidney-specific promoter and / or a kidney-specific enhancer.

[0185] In some embodiments, the disclosed kidney-specific promoters may include the GOT promoter, SGLT2 promoter, PEPCK promoter, KAP promoter, KAP promoter including A GT intron, THP promoter, A QP-2 promoter, promoter of the B1 subunit of vacuolar proton ATPase, Hox-B7 promoter, Ksp-cadherin promoter, PAX-8 promoter, promoter, 11-beta-HSD2 promoter, renin promoter, nephrin promoter, podosin promoter, tenascin-C promoter, Osr-2 promoter, or any combination thereof. In some embodiments, the disclosed kidney-specific promoters may include human homologs of the GOT promoter, SGLT2 promoter, PEPCK promoter, KAP promoter, KAP promoter including A GT intron, THP promoter, A QP-2 promoter, promoter of the B1 subunit of vacuolar proton ATPase, Hox-B7 promoter, Ksp-cadherin promoter, PAX-8 promoter, promoter, 11-beta-HSD2 promoter, renin promoter, nephrin promoter, podosin promoter, tenascin-C promoter, Osr-2 promoter, or any combination thereof. In some embodiments, the disclosed kidney-specific promoters may include active fragments of the GOT promoter, SGLT2 promoter, PEPCK promoter, KAP promoter, KAP promoter including A GT intron, THP promoter, A QP-2 promoter, promoter of the B1 subunit of vacuolar proton ATPase, Hox-B7 promoter, Ksp-cadherin promoter, PAX-8 promoter, promoter, 11-beta-HSD2 promoter, renin promoter, nephrin promoter, podosin promoter, tenascin-C promoter, Osr-2 promoter, or any combination thereof.

[0186] In some embodiments, the disclosed transcriptional regulatory elements may include podocyte-specific transcriptional regulatory elements. In some embodiments, the disclosed transcriptional regulatory elements may include minimal NPHS1 promoters and / or minimal NPHS2 promoters.

[0187] In some embodiments, the disclosed payload may encode a therapeutic RNA or therapeutic protein. In some embodiments, the disclosed transgene or heteronucleotide may encode a therapeutic RNA or therapeutic protein. In some embodiments, the therapeutic RNA is circular RNA (cirRNA). In some embodiments, the disclosed therapeutic RNA may be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA. In some embodiments, the disclosed transgene or disclosed payload may encode a missing, defective, and / or mutated protein or enzyme. In some embodiments, the disclosed heteronucleotide may encode a missing, defective, and / or mutated protein or enzyme. In some embodiments, the disclosed missing, defective, and / or mutated proteins or enzymes may be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. In some embodiments, the disclosed transgenes or heterologous nucleic acids may be encoded by apolipoprotein L1, fibrocystin, myosin heavy chain 9, nephrocystin 1, polycystin 1, polycystin 2, or any combination thereof. In some embodiments, the disclosed transgene or disclosed heteronucleotide may encode NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0188] In some embodiments, the disclosed transgene or disclosed heterologous nucleic acid may encode a gene editing molecule. In some embodiments, the disclosed gene editing molecule may include a nuclease or a single guide RNA (sgRNA).

[0189] In some embodiments, the disclosed AAV particles or AAV vectors can be used to improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to AAV particles having wild-type capsid protein. In some embodiments, the disclosed AAV particles or AAV vectors can be used to induce widespread transduction of one or more renal cells or kidney-derived cell types. In some embodiments, the disclosed AAV particles or AAV vectors can be used to transduce one or more renal cells or kidney-derived cell types more efficiently than AAV particles or AAV vectors having wild-type capsid protein.

[0190] In some embodiments, an increase and / or improvement in transduction efficiency may include an increase and / or improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an increase and / or improvement of more than 100%.

[0191] In some embodiments, the disclosed AAV particles or the disclosed AAV vectors can be used to improve and / or enhance gene transfer into any region or portion of the kidney. In some embodiments of the disclosed AAV particles or the disclosed AAV vectors, the improvement and / or enhancement of gene transfer into renal cells or kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0192] In some embodiments, the disclosed AAV particles or disclosed AAV vectors may exhibit improved dose-response correlation (e.g., thereby improving efficiency).

[0193] In some embodiments, the disclosed AAV particles or AAV vectors can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In some embodiments, the disclosed AAV particles or AAV vectors can be used to improve the quality of life of a subject by at least 50% compared to the quality of life of a subject before treatment.

[0194] In some embodiments, the disclosed AAV particles or AAV vectors can be used to reduce and / or decrease one or more symptoms associated with and / or related to renal disease and / or renal impairment in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to prevent the development of undesirable physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to inhibit, i.e., prevent the development of, physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to mitigate, i.e., induce disease regression in a subject.

[0195] In some embodiments, the disclosed AAV particles or AAV vectors can be used to improve renal function in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to reduce the risk of acute and / or chronic renal failure in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to reduce the risk of kidney infection in a subject. In some embodiments, the disclosed AAV particles or AAV vectors can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in a subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed AAV particles or AAV vectors can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0196] In some embodiments, the disclosed AAV particles or AAV vectors can be used to treat subjects in need of them. In some embodiments, the disclosed AAV particles or AAV vectors can be used in a method of delivering gene therapy to subjects in need of it. In some embodiments, subjects in need of it may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittellmann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0197] In some embodiments, the disclosed AAV particles or disclosed AAV vectors, AAV capsid proteins may be incorporated into the disclosed AAV capsid. In some embodiments, the disclosed AAV particles or disclosed AAV vectors, AAV capsid proteins may exhibit improved tropism to one or more cell types and / or one or more tissue types (e.g., one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types). In some embodiments, the disclosed AAV particles or disclosed AAV vectors may exhibit improved transduction efficiency and / or properties when introduced into one or more cell types and / or one or more tissue types. In some embodiments, the disclosed AAV particles or disclosed AAV vectors can efficiently transduce one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types. In some embodiments, the disclosed AAV particles or disclosed AAV vectors may exhibit superior performance to parental wild-type AAV to one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0198] In some embodiments, the disclosed AAV particles or AAV vectors may be AAV1, AAV2, AAV3 (including 3a and 3b), AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11, AAV12, AAV13, AAVrh39, AAVrh43, or AAVcy.7. In some embodiments, the disclosed AAV vectors may be bovine AAV, goat AAV, dog AAV, horse AAV, sheep AAV, bird AAV, primate AAV, or non-primate AAV. In some embodiments, the disclosed AAV vectors may 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 some embodiments, the disclosed AAV particles and / or disclosed AAV vectors 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, retrograde ureteral infusion, renal artery administration, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ophthalmic administration, intra-otal administration, ear drops, intrauterine administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration, including injectable administrations such as intravenous administration, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration of the disclosed AAV particles or disclosed AAV vectors may be continuous or intermittent. In some embodiments, the disclosed AAV particles or disclosed AAV vectors can be administered by one or more ex vivo methods, such as an ex vivo perfusion protocol. In some embodiments, an ex vivo perfusion protocol using the disclosed AAV particles and / or disclosed AAV vectors can be used in conjunction with a kidney (or portion thereof) obtained for the subject. In one embodiment, a kidney can be obtained from a donor subject and subjected to an ex vivo perfusion protocol using the disclosed AAV particles and / or the disclosed AAV vector before implantation in a subject requiring it. In another embodiment, a kidney can be obtained from a subject requiring it, subjected to an ex vivo perfusion protocol using the disclosed AAV particles and / or the disclosed AAV vector, and then returned to the subject requiring it. In another embodiment, the disclosed ex vivo perfusion protocol using the disclosed AAV particles and / or the disclosed AAV vector can be applied to other relevant tissues in the subject requiring it.

[0200] In some embodiments, the disclosed AAV particles and / or the disclosed AAV vectors can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts. In some embodiments, the disclosed AAV particles and / or the disclosed AAV vectors can be used in a method to improve the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed AAV particles and / or the disclosed AAV vectors can be used in a method to reduce the risk of graft-versus-host disease (GVHD) occurring after implantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed AAV particles and / or the disclosed AAV vectors can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it).In some embodiments, the disclosed AAV particles and / or disclosed AAV vectors can be used in a way that enhances the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation and return to a subject in need). In some embodiments, the disclosed AAV particles and / or disclosed AAV vectors can be used to extend and / or improve the life expectancy of a subject.

[0201] 5. Pharmaceutical preparations Pharmaceutical formulations comprising disclosed AAV particles or disclosed AAV vectors in a pharmaceutically acceptable carrier are disclosed herein. Pharmaceutical formulations comprising disclosed nucleic acid molecules in a pharmaceutically acceptable carrier are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising a nucleic acid sequence encoding a disclosed adeno-associated virus (AAV) capsid protein are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising a nucleic acid sequence encoding an AAV capsid protein comprising the sequence shown in Sequence ID No. 03 are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising a nucleic acid sequence encoding an AAV capsid protein comprising a sequence having at least 90% identity to the sequence shown in Sequence ID No. 03 are disclosed herein. Pharmaceutical formulations comprising nucleic acid molecules comprising a nucleic acid sequence encoding an AAV capsid protein comprising one or more amino acid substitutions at positions 452-458 compared to Sequence ID No. 01 are disclosed herein. AAV particles, AAV vectors, AAV capsid proteins, and nucleic acid molecules are disclosed herein.

[0202] In one embodiment, the disclosed pharmaceutical formulation is approximately 1 × 10 6DRP / mL ~ about 1×10 14 may contain DRP / mL. In certain embodiments, the disclosed pharmaceutical formulations may contain about 1×10 6 DRP / mL, 1×10 7 DRP / mL, 1×10 8 DRP / mL, 1×10 9 DRP / mL, 1×10 10 DRP / mL, 1×10 11 DRP / mL, 1×10 12 DRP / mL, 1×10 13 DRP / mL, or 1×10 14 DRP / mL.

[0203] In certain embodiments, the disclosed pharmaceutical formulations can be administered to a subject that needs it. In certain embodiments, the disclosed pharmaceutical formulations can be formulated for administration by 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 artery administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, vaginal administration, ophthalmic administration, otic administration, auricular administration, intrauterine administration, intracerebral administration, rectal administration, sublingual administration, buccal administration, and parenteral administration including injectable administration such as intravenous administration, intraarterial administration, intramuscular administration, and subcutaneous administration. Administration of the disclosed pharmaceutical formulations can be continuous or intermittent. In certain embodiments, the disclosed pharmaceutical formulations can be administered by one or more ex vivo methods, such as an ex vivo perfusion protocol. In certain embodiments, an ex vivo perfusion protocol can be used with a kidney (or a portion thereof) obtained for a subject. In certain embodiments, a kidney can be obtained from a donor subject and subjected to an ex vivo perfusion protocol using the disclosed pharmaceutical formulations prior to implantation into a subject that needs it. In certain embodiments, a kidney can be obtained from a subject that needs it, subjected to an ex vivo perfusion protocol, and then returned to the subject that needs it. In certain embodiments, the disclosed ex vivo perfusion protocol using the disclosed pharmaceutical formulations can be applied to other relevant tissues in a subject.

[0204] In some embodiments, the disclosed pharmaceutical formulation can be used in the disclosed manner. In some embodiments, the disclosed pharmaceutical formulation can be used in the disclosed manner for delivering a payload.

[0205] In some embodiments, the disclosed pharmaceutical formulation can be used to improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to AAV particles having wild-type capsid protein. In some embodiments, the disclosed pharmaceutical formulation can be used to induce widespread transduction of one or more renal cells or kidney-derived cell types. In some embodiments, the disclosed pharmaceutical formulation can be used to transduce one or more renal cells or kidney-derived cell types more efficiently than AAV particles or AAV vectors having wild-type capsid protein. In some embodiments, an increase and / or improvement in transduction efficiency may include an increase and / or improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an increase and / or improvement of more than 100%.

[0206] In certain aspects, the disclosed pharmaceutical formulations can be used to improve and / or enhance gene transfer to any region or part of the kidney. In certain aspects of the disclosed pharmaceutical formulations, improvement and / or enhancement of gene transfer to renal cells or kidney-derived cell types can include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, principal cells of the collecting duct, transitional cells of the collecting duct, intercalated cells of the collecting duct, or cells of the afferent arteriole, efferent arteriole, ascending vasa recta, descending vasa recta, proximal straight tubule and proximal convoluted tubule, thin descending limb of the loop of Henle, thin ascending limb and thick ascending limb, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule cells, or any combination thereof. In certain aspects, the disclosed pharmaceutical formulations can exhibit improved dose-response correlation (e.g., thereby improving efficiency).

[0207] In certain aspects, the disclosed pharmaceutical formulations can be used to improve and / or enhance the quality of life of a subject as compared to pre-treatment levels. In certain aspects, the disclosed pharmaceutical formulations can be used to improve the quality of life of a subject by at least 50% as compared to the pre-treatment quality of life of the subject.

[0208] In certain aspects, the disclosed pharmaceutical formulations can be used to attenuate and / or reduce one or more symptoms associated with and / or related to a renal disease and / or renal disorder in a subject. In certain aspects, the disclosed pharmaceutical formulations can be used to prevent an unwanted physiological change, disease, pathological condition, or disorder from occurring in a subject. In certain aspects, the disclosed pharmaceutical formulations can be used to inhibit, i.e., prevent the occurrence of, a physiological change, disease, pathological condition, or disorder in a subject. In certain aspects, the disclosed pharmaceutical formulations can be used to mitigate, i.e., cause regression of, a physiological change, disease, pathological condition, or disorder in a subject.

[0209] In some embodiments, the disclosed pharmaceutical formulation can be used to improve renal function in a subject. In some embodiments, the disclosed pharmaceutical formulation can be used to reduce the risk of acute and / or chronic renal failure in a subject. In some embodiments, the disclosed pharmaceutical formulation can be used to reduce the risk of kidney infection in a subject. In some embodiments, the disclosed pharmaceutical formulation can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in a subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed pharmaceutical formulation can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0210] In some embodiments, the disclosed pharmaceutical formulation can be used to treat subjects in need of it. In some embodiments, the disclosed pharmaceutical formulation can be used in a method of delivering gene therapy to subjects in need of it. In some embodiments, subjects in need of it may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0211] In some embodiments, the disclosed pharmaceutical formulation may have improved transduction efficiency and / or properties when introduced into one or more cell types and / or one or more tissue types. In some embodiments, the disclosed pharmaceutical formulation may efficiently transdose into one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types and / or exhibit superior performance to parental wild-type AAV in relation to one or more disclosed renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0212] In some embodiments, the disclosed pharmaceutical formulation can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts. In some embodiments, the disclosed pharmaceutical formulation can be used in a method to improve the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed pharmaceutical formulation can be used in a method to reduce the risk of graft-versus-host disease (GVHD) occurring after transplantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed pharmaceutical formulations can be used in a manner that reduces the risk of rejection of one or more parenchymal organ grafts (for example, (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it).In some embodiments, the disclosed pharmaceutical formulation can be used in a manner that enhances the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof, before implantation and return to a subject in need). In some embodiments, the disclosed pharmaceutical formulation can be used to extend and / or improve the life expectancy of a subject.

[0213] 6. Kit A kit comprising one or more disclosed compositions is disclosed herein. In some embodiments, the compositions of the disclosed kit may comprise one or more disclosed nucleic acid molecules, a disclosed plasmid, a disclosed AAV capsid protein, a disclosed AAV particle, a disclosed vector, a disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed wild-type AAV capsid protein, a disclosed wild-type AAV, or any combination thereof. In some embodiments, the disclosed kit may comprise a combination of one or more activators. In some embodiments, the disclosed kit may comprise at least two components that constitute the kit. Together, the components constitute a functional unit for a given purpose (e.g., carrying out directional evolution of an AAV capsid protein, generating AAV particles, or delivering a payload). Individual member components may be physically packaged together or separately. For example, a disclosed kit containing instructions for use of the kit may physically include instructions for use of other individual member components. Alternatively, instructions may be supplied as a separate member component in paper form, or in electronic form that can be supplied on a computer-readable memory device or downloaded from an internet website, or as a recorded presentation. In some embodiments, the disclosed kit for use in the disclosed method may comprise one or more containers holding a label or accompanying document with the disclosed composition (i.e., the disclosed nucleic acid molecule, the disclosed plasmid, the disclosed AAV capsid protein, the disclosed AAV particles, the disclosed vector, the disclosed AAV vector, the disclosed pharmaceutical formulation, the disclosed wild-type AAV capsid protein, the disclosed wild-type AAV, or any combination thereof) and instructions for use.

[0214] In some embodiments, the disclosed kit may contain one or more additional activators (e.g., excipients, buffers, active activators, bioactive activators, pharmaceutically active activators, immunotherapy agents, clinically approved activators, or combinations thereof). In some embodiments, one or more active activators may treat, inhibit, and / or improve one or more co-existing diseases in the subject. In some embodiments, one or more active activators may treat, inhibit, and / or improve diseases or disorders (e.g., renal disease and / or renal impairment), infections, symptoms, complications, or combinations thereof. In some embodiments, suitable containers may include, for example, bottles, vials, syringes, blister packs, etc. Containers may be formed from various materials such as glass or plastic. The container may be capable of holding the disclosed composition (e.g., the disclosed nucleic acid molecule, the disclosed plasmid, the disclosed AAV capsid protein, the disclosed AAV particles, the disclosed vector, the disclosed AAV vector, the disclosed pharmaceutical formulation, the disclosed wild-type AAV capsid protein, the disclosed wild-type AAV) or the disclosed pharmaceutical formulation, and may have a sterile access port (e.g., the container may be a vial with a stopper that can be punctured with an intravenous infusion bag or a subcutaneous needle). The label or accompanying information may indicate that the disclosed composition or a pharmaceutical formulation containing the disclosed composition can be used to treat, prevent, inhibit, and / or improve diseases and / or disorders (e.g., renal disease and / or renal impairment), infections, symptoms, complications, or combinations thereof. The disclosed kit may include additional components necessary for administration, such as other buffers, diluents, filters, needles, and syringes. The term “package insert” may refer to instructions that are customarily included with the market packaging of a therapeutic product, which include information relating to indications, use, dosage, administration, contraindications, and / or warnings relating to the use of such therapeutic product. In some embodiments, the disclosed kit may include some or all of the components necessary to carry out and / or perform one or more disclosed methods.In some embodiments, the disclosed kit can be used in a method for delivering a payload. In some embodiments, the disclosed kit can be used in a method for treating a subject.

[0215] In some embodiments, the disclosed kit can be used in a method for reducing the risk of rejection of one or more parenchymal organ grafts. In some embodiments, the disclosed kit can be used in a method for improving the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed kit can be used in a method for reducing the risk of graft-versus-host disease (GVHD) occurring after transplantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated with a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed kit can be used in a method to reduce the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed kit can be used in a method to enhance the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation into a subject requiring it, or (iii) a subject's own kidney treated by a disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, before implantation and return to a subject requiring it).In one embodiment, the disclosed kit can be used in a method for extending and / or improving the life expectancy of a subject.

[0216] C. Method 1. Method for generating and / or producing AAV particles A method for generating AAV particles is disclosed herein, comprising the steps of: delivering a nucleic acid molecule containing a nucleic acid sequence encoding an AAV capsid protein to one or more cells; culturing one or more cells; and recovering AAV particles from one or more producing cells. A method for generating AAV particles is disclosed herein, comprising the steps of: delivering three plasmids to one or more cells, wherein the first plasmid is a helper plasmid, the second plasmid is a RepCap plasmid, the second plasmid contains a nucleic acid molecule containing a nucleic acid sequence encoding an AAV capsid protein, and the third plasmid is a cis-plasmid or transfer plasmid; culturing one or more cells; and recovering AAV particles from one or more cells. A method for producing AAV particles is disclosed herein, comprising the steps of: preparing one or more cells containing a disclosed AAV vector; and culturing one or more cells under conditions that enable the formation of AAV particles.

[0217] In some embodiments, the disclosed method may include the step of expressing the disclosed encoded AAV capsid protein. In some embodiments, the disclosed method may include the step of culturing one or more cells in a culture medium. In some embodiments, the disclosed method may include the step of recovering AAV particles. In some embodiments, the disclosed method may include the step of purifying the recovered AAV particles. In some embodiments, the disclosed method may include the step of using the purified AAV particles for gene therapy. In some embodiments, the disclosed method may include the step of delivering a helper plasmid to one or more cells. In some embodiments, the disclosed method may include the step of delivering a cis-plasmid or transfer plasmid encoding the gene or transgene of interest to one or more cells. In some embodiments, the disclosed secreted AAV particles may contain the gene or transgene of interest. In some embodiments, the disclosed secreted AAV particles may contain one or more base-editing components and / or one or more gRNAs.

[0218] In some embodiments, the disclosed method may include a step of recovering AAV particles and / or AAV particles by lysing the host cells of the production culture or by recovering the consumed medium from the production culture. Suitable methods for lysing cells are known to those skilled in the art and include multiple freeze / thaw cycles, sonication, microsolution manipulation, and treatment with chemicals (e.g., surfactants and / or proteases), or any combination thereof.

[0219] In some embodiments, the disclosed method may further include a step of purifying AAV particles. In some embodiments, “purification” may include preparing AAV particles that lack at least some of other components that may also be present when the AAV particles are naturally occurring or when they are first prepared. In some embodiments, isolated AAV particles may be prepared using a purification technique that enriches AAV particles from a source mixture (e.g., a culture lysate or a productive culture supernatant). In some embodiments of the disclosed method, AAV particles may be isolated or purified using one or more of the following purification steps: equilibrium centrifugation; flow-through anion exchange filtration; tangential flow filtration (TFF) for concentrating AAV particles; AAV capture by apatite chromatography; thermal inactivation of helper viruses; AAV capture by hydrophobic interaction chromatography; buffer exchange by size exclusion chromatography (SEC); nanofiltration; and AAV capture by anion exchange chromatography, cation exchange chromatography, affinity chromatography, or any combination thereof. In some embodiments, enrichment can be measured by various methods known in the art, including by the proportion of DNA-degrading enzyme-resistant particles (DRPs) or genome copies (gc) in the solution, or by infectivity. In some embodiments, enrichment can be measured in relation to a second potentially interfering substance present in the source mixture (e.g., contaminants including helper viruses, culture medium components, and other contaminants in the produced culture or process). In some embodiments, a disclosed method for generating and / or producing AAV particles may include verifying the purity and / or functionality of the AAV particles.

[0220] Disclosed herein is a method for generating AAV capsid proteins, comprising the step of carrying out multiple rounds of evolution in one or more kidney or kidney-related models derived from one or more species. In some embodiments of the disclosed method, the step of generating an initial library of capsid proteins may include using saturation mutagenesis on variable region IV (corresponding to amino acids 425-458) of SEQ ID NO: 01. In some embodiments, the disclosed method may further include the step of packaging the initial library of capsid proteins into an AAV vector using triple plasmid transfection. Triple plasmid transfection is known in the art and has been discussed above. In some embodiments, the first round of evolution disclosed may include intravenous administration of an AAV vector containing the initial variant capsid library to mice. In some embodiments, the second round of evolution disclosed may include intravenous administration of an AAV vector containing the variant capsid library generated in the first round of evolution to pigs. In one embodiment, the disclosed third round of evolution may include transduction of the variant capsid library generated in the second round of evolution into differentiated human kidney organoids. In one embodiment, the disclosed fourth round of evolution may include ex vivo perfusion of the variant capsid library generated in the third round of evolution into non-human primate kidneys.

[0221] In some embodiments, the disclosed method may further include the step of generating an initial library of capsid proteins identified by sequential evolution. In some embodiments, the disclosed parental capsid protein or the disclosed wild-type capsid protein may include sequences shown 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 some embodiments, the disclosed method may further include a step of assessing the sequence diversity of variant capsid libraries generated in one or more rounds of evolution. In some embodiments, the disclosed method may further include a step of calculating the occupancy percentage and enrichment ratio of each evolved variant capsid library compared to the parent capsid library. In some embodiments, the disclosed method may further include a step of identifying one or more candidate capsid proteins by ordering their amino acid sequences based on the occupancy percentage and enrichment ratio. In some embodiments, the disclosed method may further include a step of characterizing one or more candidate capsid proteins.

[0223] In some embodiments, one or more disclosed species may include Mus musculus (mouse), Sus scrofa (pig), non-human primates (Macaca), or Homo sapiens (human), or any combination thereof.

[0224] In one embodiment, the disclosed method may include generating a disclosed AAV capsid protein that includes, for example, the sequence shown in SEQ ID NOs. 24 to 43. In another embodiment, the disclosed method may include generating a disclosed AAV capsid protein that includes, for example, the sequence shown in SEQ ID NOs. 53 to 61.

[0225] In certain aspects, the disclosed method may include a step of generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 to 458 compared to SEQ ID NO: 01, wherein the sequence at positions 452 to 458 may include a sequence shown in any one of SEQ ID NOs: 04 to SEQ ID NO: 23 or SEQ ID NOs: 191 to SEQ ID NO: 8873. In certain aspects, the disclosed method may include a step of generating an AAV capsid protein comprising one or more amino acid substitutions at positions 452 to 458 compared to SEQ ID NO: 01, wherein the sequence at positions 452 to 458 may include a sequence that is at least 85% identical to any one of SEQ ID NOs: 04 to SEQ ID NO: 23 or SEQ ID NOs: 191 to SEQ ID NO: 8873.

[0226] 2. Method for delivering a payload, transgene, or heterologous nucleic acid Disclosed herein is a method for delivering a payload, comprising contacting one or more target cells with the disclosed AAV particles and expressing the encoded payload.

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

[0228] Disclosed herein is a method for delivering a payload, comprising contacting one or more target cells in a subject that needs it with the disclosed AAV particles and expressing the encoded payload.

[0229] A method for delivering a payload is disclosed herein, comprising the steps of: contacting one or more target cells in an object requiring such delivery with a therapeutically effective amount of disclosed AAV particles 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 some embodiments, the disclosed payload may include nucleic acids capsidated to AAV particles. For example, in some embodiments of the disclosed method, the disclosed payload may encode therapeutic RNA (e.g., antisense oligonucleotides, ribozymes, siRNA, shRNA, or miRNA) or therapeutic proteins. In some embodiments, the therapeutic RNA is circular RNA (cirRNA). In some embodiments, the disclosed payload nucleic acid may encode polypeptides, inhibitory RNA, antibodies or antibody reagents, oligonucleotides, or miRNAs. In some embodiments, the disclosed payload may encode messenger RNA (mRNA) that can be encoded by the disclosed payload. In some embodiments, the disclosed payload may encode gene therapy products. Gene therapy products may include polypeptides, RNA molecules, or other gene products that, when expressed in target cells, produce a desired therapeutic effect. In some embodiments, gene therapy products may include substitutes for non-functional genes that are absent or mutated. In some embodiments, the disclosed payload nucleic acid may encode transgenes that have beneficial or desirable gene products. In some embodiments, the disclosed transgene or heterologous nucleic acid may encode a therapeutic RNA or therapeutic protein. In some embodiments, the disclosed therapeutic RNA may be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.

[0231] In some embodiments, the disclosed payload may encode one or more base-editing components and / or one or more gRNAs targeting a region to be edited. In some embodiments, the disclosed transgene or heteronucleotide may encode a gene-editing molecule. In some embodiments, the disclosed gene-editing molecule may include a nuclease or a single guide RNA (sgRNA). In some embodiments, the disclosed transgene or heteronucleotide may encode a missing, defective, and / or mutated protein or enzyme. In some embodiments, the disclosed transgene or heteronucleotide may encode a missing, defective, and / or mutated protein or enzyme.

[0232] In some embodiments, the disclosed missing, defective, and / or mutated proteins or enzymes may be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. In some embodiments, the disclosed transgenes or heterologous nucleic acids may be encoded by apolipoprotein L1, fibrocystin, myosin heavy chain 9, nephrocystin 1, polycystin 1, polycystin 2, or any combination thereof.

[0233] In some embodiments, the disclosed method may improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to AAV particles having wild-type capsid protein. In some embodiments, the disclosed method can be used to result in widespread transduction of one or more renal cells or kidney-derived cell types. In some embodiments, the disclosed method can be used to transduce one or more renal cells or kidney-derived cell types more efficiently than AAV particles or AAV vectors having wild-type capsid protein. In some embodiments, the increase and / or improvement in transduction efficiency may include an increase and / or improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an increase and / or improvement of more than 100%.

[0234] In some embodiments, the disclosed method can be used to improve and / or enhance gene transfer to any targeted region or part of the kidney. In some embodiments of the disclosed method, the improvement and / or enhancement of gene transfer to target renal cells or target kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0235] In some embodiments, the disclosed method can be used to treat subjects in need of it. In some embodiments, the disclosed method can be used in a method for delivering gene therapy to subjects in need of it.

[0236] In some embodiments, the disclosed method can be used to improve and / or enhance the quality of life of a subject compared to a pre-treatment level. In some embodiments, the disclosed method can be used to improve the quality of life of a subject by at least 50% compared to a pre-treatment level. In some embodiments, the disclosed method can be used to attenuate and / or reduce one or more symptoms associated with and / or related to renal disease and / or renal impairment of a subject. In some embodiments, the disclosed method can be used to prevent the development of undesirable physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed method can be used to inhibit, i.e., prevent the development of, physiological changes, diseases, pathological conditions, or disorders in a subject. In some embodiments, the disclosed method can be used to mitigate, i.e., induce disease regression of, physiological changes, diseases, pathological conditions, or disorders in a subject.

[0237] In some embodiments, the disclosed method can be used to improve renal function in a subject. In some embodiments, the disclosed method can be used to reduce the risk of acute and / or chronic renal failure in a subject. In some embodiments, the disclosed method can be used to reduce the risk of kidney infection in a subject. In some embodiments, the disclosed method can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in a subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed method can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types.

[0238] In some embodiments, the disclosed method can be used to treat subjects that require it. In some embodiments, the subjects may be of any age and may be male or female. In some embodiments of the disclosed method, the subjects may be treatment-inexperienced. In some embodiments, the subjects may have received treatment prior to the contact step and / or administration step. In some embodiments, the subjects may require a kidney transplant, or the subjects may have already received a kidney transplant. In some embodiments, the subjects requiring it may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0239] In some embodiments, the disclosed method can be used in a method for delivering gene therapy to a target that needs it. In some embodiments, the contact step enables the expression of a payload in one or more target cells. In some embodiments, the contact step enables the expression of a transgene or heterologous nucleic acid in one or more target cells.

[0240] In some embodiments of the disclosed method for delivering a payload, the disclosed AAV particles may comprise the disclosed AAV capsid protein. In some embodiments, the disclosed AAV capsid protein may comprise any AAV capsid protein disclosed herein.

[0241] In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁻⁶ 10 vg / kg ~ approx. 2×10 14This may include a range of vg / kg. In some embodiments, for example, the disclosed AAV particles or disclosed vectors may be approximately 1 × 10⁻⁶ 11 ~Approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 12 ~Approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 13 ~about 6×10 13 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or disclosed AAV vectors are at least about 1 × 10⁻¹⁶ 10 , at least about 5 × 10 10 , at least about 1 × 10 11 , at least about 5 × 10 11 , at least about 1 × 10 12 , at least about 5 × 10 12 , at least about 1 × 10 13 , at least about 5 × 10 13 , or at least about 1 × 10 14 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or disclosed AAV vectors are approximately 1 × 10⁻⁶ 10 Below, about 5×10 10 Below, approximately 1×10 11 Below, about 5×10 11 Below, approximately 1×10 12 Below, about 5×10 12 Below, approximately 1×10 13 Below, about 5×10 13 The following, or approximately 1 × 10 14 The following doses can be administered: vg / kg. In one embodiment, the disclosed AAV particles or disclosed AAV vectors are approximately 1 × 10⁻⁶ 12 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or the disclosed vector are approximately 1 × 10⁻⁶ 11 It can be administered in doses of vg / kg. In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁶ in total per subject. 12 vg~Total per target approximately 1 x 10 17This may include the range of vg. In some embodiments, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁻¹⁶ in total per subject. 12 vg, total per target approximately 1 x 10 13 vg, total per target approximately 1 x 10 14 vg, total per target approximately 1 x 10 15 vg, total per target approximately 1 x 10 16 vg, or approximately 1 × 10⁻¹⁰ per target. 17 The range may include vg. In some embodiments, the disclosed AAV particles or vectors may be administered as a single dose or as multiple doses (e.g., two, three, four, five, six, seven, eight, nine, or ten doses) as needed for the desired therapeutic outcome. In some embodiments, the therapeutically effective dose of the disclosed AAV particles or vectors may include a range determined by those skilled in the art.

[0242] In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10¹⁶ per subject in total. 12 vg~Total per target approximately 1 x 10 17 This may include the range of vg. In some embodiments, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁻¹⁶ in total per subject. 12 vg, total per target approximately 1 x 10 13 vg, total per target approximately 1 x 10 14 vg, total per target approximately 1 x 10 15 vg, total per target approximately 1 x 10 16 vg, or approximately 1 × 10⁻¹⁰ per target. 17 This may include the range of vg. In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector can be delivered by retrograde ureteral infusion and / or renal artery administration, totaling approximately 1 × 10⁶ per subject. 12 vg~Total per target approximately 1 x 10 17 This may include the range of vg.

[0243] In some embodiments of the disclosed methods, restoring the activity and / or functionality of a deficient, defective, and / or mutated protein or enzyme (e.g., NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) may include restoring 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as the pre-treatment level. In some embodiments, the amount of recovery may be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% above an existing level, such as the pre-treatment level. In some embodiments, recovery may be measured against a control or reference level (e.g., determined using one or more subjects that do not have the missing, defective, and / or mutated protein or enzyme). In some embodiments, recovery may be partial or incomplete. In some embodiments, recovery may be complete or near-complete, and therefore the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0244] In some embodiments of the disclosed methods, techniques for monitoring, measuring, and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular functionality may include qualitative (or subjective) and quantitative (and objective) means. These means are known to those skilled in the art. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed above.

[0245] In some embodiments of the disclosed methods, the step of contacting cells may include methods known in the art. For example, the contact step may include administering one or more disclosed enzymes or recombinant enzymes, one or more 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 to a target.

[0246] In some embodiments, administration may include retrograde ureteral infusion, renal artery administration, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, intrahepatic artery, hepatic portal vein (HPV), or intrauterine administration. In some embodiments, the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may 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 immunomodulators, and / or gene editing systems. In some embodiments, the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may be administered by LNP administration. In some embodiments, the disclosed compositions, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, and / or disclosed AAV vectors can be administered to a subject in parallel and / or sequentially via multiple routes of administration. For example, in some embodiments, administration of the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may include IV administration. In some embodiments, multiple routes of administration, including retrograde ureteral infusion and / or arterial routes, may be used to administer the subject in the disclosed method. In some embodiments, a first route of administration, which may be the same as or different from a second and / or subsequent route of administration, may be used in the disclosed method.

[0247] In some embodiments, an ex vivo perfusion protocol can be used in the disclosed method. In some embodiments, an ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof can be used in conjunction with a kidney (or portion thereof) obtained for a subject. In some embodiments, a kidney can be obtained from a donor subject and subjected to an ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof before implantation into a subject requiring it. In some embodiments, a kidney can be obtained from a subject requiring it, subjected to an ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof, and then returned to the subject requiring it. In some embodiments, the disclosed ex vivo perfusion protocol using the disclosed nucleic acid molecules, disclosed AAV particles, disclosed AAV vectors, disclosed pharmaceutical formulations, or any combination thereof can be applied to other relevant tissues in a subject requiring it.

[0248] In some embodiments, a disclosed method for delivering a payload may further include the step of administering a therapeutic agent to a subject in a therapeutically effective dose. The therapeutic agent may be any disclosed agonist that produces a desired clinical outcome.

[0249] In some embodiments, the disclosed therapeutic agent may be an enzyme or recombinant enzyme. In some embodiments, a therapeutically effective dose of the disclosed replacement enzyme or recombinant enzyme may contain approximately 0.01 mg to approximately 100 mg per kg of body weight. In some embodiments, the disclosed enzyme or recombinant enzyme may be therapeutically effective when the dose contains approximately 0.01 mg, approximately 1 mg, approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, approximately 45 mg, approximately 50 mg, approximately 55 mg, approximately 60 mg, approximately 65 mg, approximately 70 mg, approximately 75 mg, approximately 80 mg, approximately 85 mg, approximately 90 mg, approximately 95 mg, or approximately 100 mg per kg of body weight.

[0250] In some embodiments of the disclosed method for delivering a payload, the administration step can treat a subject in need thereof. In some embodiments, treating a subject may involve administering one or more additional treatments to the subject once or more times. In some embodiments, the disclosed method for delivering a payload may further include a step of monitoring the subject for adverse effects. In some embodiments, if no adverse effects are present, the method may further include a step of continuing treatment of the subject. In some embodiments, if adverse effects are present, the method may further include a step of modifying the treatment step. Methods for monitoring the well-being of a subject may include both subjective and objective criteria (as discussed above). Such methods are known to those skilled in the art.

[0251] In some embodiments, a disclosed method for delivering a payload may further include administering to a subject a therapeutically effective dose of an agonist capable of correcting one or more aspects of a dysregulated metabolic or enzymatic pathway. In some embodiments, such an agonist may include an enzyme for enzyme replacement therapy. In some embodiments, the disclosed enzyme can replace any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway. In some embodiments, the disclosed method may include replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway.

[0252] In some embodiments, the disclosed method for delivering a payload may further include the step of administering one or more immune modulators. In some embodiments, the disclosed immune modulators may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In some embodiments, the disclosed immune modulators may be bortezomib or SVP-rapamycin. In some embodiments, the disclosed immune modulators may be tacrolimus. In some embodiments, the disclosed immune modulators may be, for example, methotrexate, administered in low to high doses over a short period. In some embodiments, the disclosed immune modulators may be administered in doses of approximately 0.1 mg to approximately 0.6 mg per kg of body weight. In some embodiments, the disclosed immune modulators may be administered in doses of approximately 0.4 mg per kg of body weight. In some embodiments, the disclosed immunomodulator may be administered at an approximate daily dose of 0.4 mg per kg of body weight for 3 to 5 cycles or more, with each cycle lasting up to 3 days. In some embodiments, the disclosed immunomodulator may be administered at an approximate daily dose of 0.4 mg per kg of body weight for a minimum of 3 cycles, with each cycle lasting 3 days. In some embodiments, the appropriate number of cycles can be determined by those skilled in the art. In some embodiments, the disclosed immunomodulator may be administered multiple times as needed to achieve the desired clinical effect.

[0253] In some embodiments, a disclosed method for improving and / or enhancing the efficacy and / or expression of a transgene may further include the step of administering one or more immunosuppressants. In some embodiments, the immunosuppressants may be, but are not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or combinations thereof. In some embodiments, the disclosed method may include administering one or more immunosuppressants more than once. In some embodiments, the disclosed method may include administering one or more immunosuppressants repeatedly over time. In some embodiments, the disclosed method may include the step of administering compounds that target or modify antigen presentation or humoral or cellular or innate immune responses.

[0254] In some embodiments, the method of delivering the payload may further include the step of administering a compound that exerts a therapeutic effect on B cells and / or a compound that targets or modifies antigen presentation, humoral immune response, or cellular immune response. In some embodiments, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or a combination thereof. Treg infusion (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of adjuncting immune tolerance are also disclosed herein.

[0255] In some embodiments, the disclosed method may further include the step of administering lipid nanoparticles (LNPs). In some embodiments, the LNPs can be targeted to an organ (e.g., renal cells or kidney-derived cells). In some embodiments, the LNPs can be targeted to one or more parts or regions of the kidney. For example, in some embodiments, mRNA therapy using LNP encapsulation for systemic delivery to a target has the potential to restore the functionality and / or structural integrity of a deficient, faulty, and / or mutated protein or enzyme.

[0256] In some embodiments, the disclosed method for delivering a payload may further include plasmapheresis and immunosuppression. In some embodiments, the disclosed method may include using immunosuppression to reduce T cell, B cell, and / or plasma cell populations, thereby reducing innate immune responses, inflammatory responses, and antibody levels in general.

[0257] In some embodiments, the disclosed method may include repeating the disclosed administration step, for example, administering a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV particle or disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immunomodulator, a disclosed proteasome inhibitor, a disclosed immunosuppressant, a disclosed compound that exerts a therapeutic effect on B cells, and / or a disclosed compound that targets or modifies antigen presentation, humoral immune response, or cellular immune response.

[0258] In some embodiments, a disclosed method for delivering a payload may include a step of modifying one or more of the disclosed steps. For example, modifying one or more of the steps of the disclosed method may include modifying or changing one or more features or aspects of one or more steps of the disclosed method. For example, in some embodiments, the method can be modified by changing the amount of one or more disclosed enzymes or recombinants, one or more 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 to a subject of one or more disclosed enzymes or recombinants, one or more 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; or by changing the duration of administration to a subject of one or more disclosed enzymes or recombinants, one or more 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.

[0259] In some embodiments, the disclosed method can be modified by changing the amount of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or modify antigen presentation, humoral immune responses, or cellular immune responses administered to a subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or modify antigen presentation, humoral immune responses, or cellular immune responses administered to a subject.

[0260] In some embodiments, the disclosed methods may include parallel administration of one or more disclosed enzymes or recombinant enzymes, one or more 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 immunomodulators, one or more disclosed proteasome inhibitors, one or more disclosed immunosuppressants, one or more disclosed compounds that exert a therapeutic effect on B cells, one or more disclosed compounds that target or modify antigen presentation or humoral or cellular immune responses, or any combination thereof.

[0261] In some embodiments, the disclosed immunomodulator may be administered before or after the administration of the disclosed therapeutic agent. In some embodiments, the disclosed method for delivering the payload may further include the step of generating one or more disclosed enzymes or disclosed recombinant enzymes, one or more 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 one embodiment, a disclosed method for delivering a payload may further include a step of generating disclosed AAV particles or disclosed AAV vectors. In one embodiment, the step of generating disclosed AAV particles or disclosed viral vectors may include generating AAV particles or AAV particles or AAV vectors or recombinant AAV (e.g., those disclosed herein).

[0263] In some embodiments, a disclosed method for delivering a payload further includes a step of gene editing one or more related genes (e.g., a missing, defective, and / or mutated protein or enzyme), wherein the editing includes, but is not limited to, a single gene knockout, simultaneous loss-of-function screening of multiple genes, gene knock-in, or a combination thereof.

[0264] In some embodiments of the disclosed method, the payload may comprise one or more base-editing components and one or more sgRNAs targeting the region to be edited.

[0265] In some embodiments, the disclosed method for delivering a payload may further include the step of administering an oligonucleotide therapeutic agent. The disclosed oligonucleotide therapeutic agent may include single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide nucleic acid (PNA), or analogs or conjugates thereof. In some embodiments, the disclosed oligonucleotide therapeutic agent may be an ASO or RNAi. In some embodiments, the disclosed oligonucleotide therapeutic agent may include one or more modifications at any applicable site. In some embodiments, the disclosed oligonucleotide therapeutic agent may include a CRISPR-based endonuclease. In some embodiments, the disclosed endonuclease may be Cas9. In some embodiments, the disclosed Cas9 may be derived from Staphylococcus aureus or Streptococcus pyogenes.

[0266] In some embodiments, a disclosed method for delivering a payload may further include the step of generating and / or verifying one or more disclosed enzymes or disclosed recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof.

[0267] In some embodiments of the disclosed methods, the disclosed enzyme, the disclosed recombinant enzyme, the disclosed nucleic acid molecule, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof may be delivered and / or administered before, in parallel with, or after the delivery and / or administration of enzyme replacement therapy, protein replacement therapy, gene therapy, recombinant products, or any combination thereof.

[0268] In some embodiments, a disclosed method for delivering a payload may further include a step of reducing and / or minimizing vector-mediated immunotoxicity and / or the immunogenicity of the transgene (e.g., the ability to induce specific immunity). In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the efficacy of the recombinant product encoded by the transgene. In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the likelihood of re-administering one or more disclosed enzymes or recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof to a subject. In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the likelihood of re-administering gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof to a subject.

[0269] In some embodiments, the disclosed method may further include the step of administering one or more disclosed enzymes or recombinant enzymes, one or more disclosed nucleic acid molecules, one or more disclosed AAV vectors, one or more disclosed pharmaceutical formulations, or any combination thereof, once or more times.

[0270] In some embodiments, the disclosed method may further include a step of measuring and / or determining the pre-treatment level of one or more clinical and / or metabolic indicators of renal function in question (e.g., expression of NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) or evaluation criteria in question. In some embodiments, the disclosed method may further include a step of measuring and / or determining the level of one or more clinical and / or metabolic indicators in question one or more times.

[0271] In some embodiments, a disclosed method for delivering a payload, a transgene, or a heterologous nucleic acid may further include a step of reducing the risk of rejection of one or more parenchymal organ grafts. In some embodiments, a disclosed method for treating a subject may further include a step of improving the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, a disclosed method for delivering a payload, a transgene, or a heterologous nucleic acid may further include a step of reducing the risk of graft-versus-host disease (GVHD) occurring after implantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, the disclosed method for delivering the payload, the transgene, or the heterologous nucleic acid may further include steps to reduce the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it).In some embodiments, the disclosed method for delivering a payload, a transgene, or a heterologous nucleic acid may further include a step of enhancing the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation and return to a subject in need). In some embodiments, the disclosed method for treating a subject may extend and / or improve the subject's life expectancy.

[0272] 3. Method of treating the target A method for treating a subject is disclosed herein, comprising the step of administering a disclosed AAV particle or a disclosed AAV vector to a subject in need of such treatment in one or more therapeutic doses. A method for treating a subject is disclosed herein, comprising the step of administering a disclosed pharmaceutical formulation containing the disclosed AAV particle or a disclosed AAV vector to a subject in need of such treatment in one or more therapeutic doses. A method for treating a subject is disclosed herein, comprising the step of contacting one or more cells in the subject with a therapeutic dose of the disclosed AAV particle containing a payload. A method for treating a subject is disclosed herein, comprising the steps of contacting one or more cells in the subject with a therapeutic dose of the disclosed AAV particle containing (i) 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] A method for treating a subject is disclosed herein, comprising the steps of: contacting one or more cells in the subject in a therapeutically effective amount of disclosed AAV particles comprising (i) 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 contact step enables the expression of the encoded payload, the encoded gene of interest, or the encoded transgene in one or more cells.

[0274] In some embodiments, the subject may be of any age and may be male or female. In some embodiments of the disclosed method, the subject may be treatment-inexperienced. In some embodiments, the subject may have received treatment prior to the contact step and / or administration step. In some embodiments, the subject may require a kidney transplant, or may have already received a kidney transplant. In some embodiments, the subjects requiring it may have one or more renal diseases and / or disorders (e.g., Alport syndrome, Bartter syndrome types 1-4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittemann syndrome, Liddle syndrome, Lowe syndrome, nephronophthiriasis (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, tuberous sclerosis (TSC), or any combination thereof).

[0275] In some embodiments, the disclosed AAV particles may include the disclosed AAV capsid protein, such as those described above. For example, in some embodiments, the disclosed AAV particles used in a disclosed method for treating a subject may include an AAV capsid protein having a sequence shown in any one of SEQ ID NOs: 24 to 43.

[0276] In some embodiments, the disclosed AAV particles used in a disclosed method for treating a subject may include an AAV capsid protein containing the sequence shown in any one of SEQ ID NOs. 53 to 61. For example, the disclosed AAV particles used in a disclosed method for treating a subject may include an AAV capsid protein containing the sequence shown in SEQ ID NOs. 03. For example, the disclosed AAV particles used in a disclosed method for treating a subject may include an AAV capsid protein containing a sequence having at least 90% identity to the sequence shown in SEQ ID NOs. 03. For example, the disclosed AAV particles used in a disclosed method for treating a subject may include an AAV capsid protein containing one or more amino acid substitutions at positions 452 to 458 relative to SEQ ID NOs. 01. In some embodiments, the substitutions at positions 452 to 458 relative to SEQ ID NOs. 01 may include the sequences shown in any of SEQ ID NOs. 04 to 23 or SEQ ID NOs. 191 to 8873. In some embodiments, substitutions at positions 452-458 relative to SEQ ID NO: 01 may contain a sequence that is at least 85% identical to any one of SEQ ID NOs: 04-23 or SEQ ID NOs: 191-8873. In some embodiments, the disclosed AAV capsid protein may contain any AAV capsid protein disclosed herein.

[0277] In some embodiments, the disclosed payload may include nucleic acids capsid-formed on AAV particles. For example, in some embodiments of the disclosed method, the disclosed payload may encode a therapeutic RNA or therapeutic protein. In some embodiments, the disclosed payload nucleic acid may encode a polypeptide, inhibitory RNA, antibody or antibody reagent, oligonucleotide, or miRNA. In some embodiments, the disclosed payload may encode messenger RNA (mRNA) that can be encoded by the disclosed payload. In some embodiments, the disclosed payload may encode a gene therapy product. A gene therapy product may include a polypeptide, RNA molecule, or other gene product that, when expressed in target cells, produces a desired therapeutic effect. In some embodiments, a gene therapy product may include a substitute for a non-functional gene that is absent or mutated. In some embodiments, the disclosed payload nucleic acid may encode a transgene having a beneficial or desirable gene product. In some embodiments, the disclosed transgene or heterologous nucleic acid may encode a therapeutic RNA or therapeutic protein. In some embodiments, the disclosed therapeutic RNA may be an antisense oligonucleotide, a ribozyme, siRNA, shRNA, or miRNA.

[0278] In some embodiments, the disclosed payload may encode one or more base-editing components and / or one or more gRNAs targeting a region to be edited. In some embodiments, the disclosed transgene or heteronucleotide may encode a gene-editing molecule. In some embodiments, the disclosed gene-editing molecule may include a nuclease or a single guide RNA (sgRNA). In some embodiments, the disclosed transgene or heteronucleotide may encode a missing, defective, and / or mutated protein or enzyme. In some embodiments, the disclosed transgene or heteronucleotide may encode a missing, defective, and / or mutated protein or enzyme.

[0279] In some embodiments, the disclosed missing, defective, and / or mutated proteins or enzymes may be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof. In some embodiments, the disclosed transgenes or heterologous nucleic acids may be encoded by apolipoprotein L1, fibrocystin, myosin heavy chain 9, nephrocystin 1, polycystin 1, polycystin 2, or any combination thereof.

[0280] In some embodiments, the disclosed method for treating a subject may improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to AAV particles having wild-type capsid protein. In some embodiments, the disclosed method for treating a subject can be used to induce widespread transduction of one or more renal cells or kidney-derived cell types. In some embodiments, the disclosed method for treating a subject can be used to transduce one or more renal cells or kidney-derived cell types more efficiently than AAV particles or AAV vectors having wild-type capsid protein. In some embodiments, an increase and / or improvement in transduction efficiency may include an increase and / or improvement of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or an increase and / or improvement of more than 100%.

[0281] In some embodiments, a disclosed method for treating a subject can be used to improve and / or enhance gene transfer to any targeted region or part of the kidney. In some embodiments of the disclosed method, the improvement and / or enhancement of gene transfer to target renal cells or target kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0282] In some embodiments, the disclosed method can be used to treat a subject in need of it. In some embodiments, the disclosed method can be used in a method for delivering gene therapy to a subject in need of it. In some embodiments, the disclosed method for treating a subject can be used to improve and / or enhance the subject's quality of life compared to a pre-treatment level. In some embodiments, the disclosed method for treating a subject can be used to improve the subject's quality of life by at least 50% compared to the subject's quality of life before treatment.

[0283] In some embodiments, a disclosed method for treating a subject can be used to reduce and / or decrease one or more symptoms associated with and / or related to renal disease and / or renal impairment of the subject. In some embodiments, a disclosed method for treating a subject can be used to prevent the development of undesirable physiological changes, diseases, pathological conditions, or disorders in the subject. In some embodiments, a disclosed method for treating a subject can be used to inhibit, i.e., prevent the development of, physiological changes, diseases, pathological conditions, or disorders in the subject. In some embodiments, a disclosed method for treating a subject can be used to mitigate, i.e., induce disease regression in the subject.

[0284] In some embodiments, the disclosed method for treating a subject can be used to improve renal function in the subject. In some embodiments, the disclosed method for treating a subject can be used to reduce the risk of acute and / or chronic renal failure in the subject. In some embodiments, the disclosed method for treating a subject can be used to reduce the risk of kidney infection in the subject. In some embodiments, the disclosed method for treating a subject can be used to reduce the risk of inflammation occurring in one or more parts or regions of the kidney in the subject. For example, in some embodiments, inflammation may affect the renal filtration units (e.g., glomerulonephritis) and / or the renal tubules and surrounding structures (e.g., interstitial nephritis). In some embodiments, the disclosed method for treating a subject can be used to repair affected and / or dysfunctional renal cells, kidney-derived cell types, and / or kidney-associated cell types. In some embodiments, the disclosed method can be used to reduce the need for kidney transplantation in the subject and / or to reduce the risk of rejection of the transplanted kidney in the subject.

[0285] In some embodiments, a disclosed method for treating a subject can be used in a method for delivering gene therapy to a subject in need thereof. In some embodiments of the disclosed method, restoring the activity and / or functionality of a deficient, defective, and / or mutated protein or enzyme (e.g., NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) may include restoration of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount compared to an existing level, such as the pre-treatment level. In some embodiments, the amount of recovery may be 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 90-100% above an existing level, such as the pre-treatment level. In some embodiments, recovery may be measured against a control or reference level (e.g., determined using one or more subjects that do not have the missing, defective, and / or mutated protein or enzyme). In some embodiments, recovery may be partial or incomplete. In some embodiments, recovery may be complete or near-complete, and therefore the levels of expression, activity, and / or functionality are similar to wild-type or control levels.

[0286] In one embodiment, after the administration step, the payload is expressed in one or more target cells in the subject. In another embodiment, after the administration step, the transgene or heterologous nucleic acid is expressed in one or more target cells in the subject.

[0287] In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁻⁶ 10 vg / kg ~ approx. 2×10 14This may include a range of vg / kg. In one embodiment, for example, the disclosed AAV particles or disclosed vector may be about 1 × 10⁻⁶ 11 ~Approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 12 ~Approx. 8×10 13 vg / kg or approximately 1 × 10⁻⁶ 13 ~about 6×10 13 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or the disclosed AAV vector are distributed at least about 1 × 10⁻¹⁶ 10 , at least about 5 × 10 10 , at least about 1 × 10 11 , at least about 5 × 10 11 , at least about 1 × 10 12 , at least about 5 × 10 12 , at least about 1 × 10 13 , at least about 5 × 10 13 , or at least about 1 × 10 14 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or disclosed AAV vector are distributed at approximately 1 × 10⁻⁶ doses. 10 Below, about 5×10 10 Below, approximately 1×10 11 Below, about 5×10 11 Below, approximately 1×10 12 Below, about 5×10 12 Below, approximately 1×10 13 Below, about 5×10 13 The following, or approximately 1 × 10 14 The following doses of vg / kg may be administered. In one embodiment, the disclosed AAV particles or the disclosed AAV vector are administered at approximately 1 × 10⁻⁶ doses. 12 It can be administered in doses of vg / kg. In one embodiment, the disclosed AAV particles or the disclosed vector are distributed at approximately 1 × 10⁻⁶ doses. 11 It can be administered in doses of vg / kg. In one embodiment, the therapeutically effective dose of the disclosed AAV particles or disclosed AAV vector is approximately 1 × 10⁶ in total per subject. 12 vg~Total per target approximately 1 x 10 17 This may include the range of vg.

[0288] In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector can be in the range of about 1×10 12 vg, about 1×10 13 vg, about 1×10 14 vg, about 1×10 15 vg, about 1×10 16 vg, or about 1×10 17 vg per subject in total. In one aspect, the disclosed AAV particles or the disclosed vector can be administered as a single dose or as multiple doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses) as needed for the desired therapeutic result. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector can be in a range determined by one of ordinary skill in the art. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector can be in the range of about 1×10 12 vg to about 1×10 17 vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector can be about 1×10 12 vg, about 1×10 13 vg, about 1×10 14 vg, about 1×10 15 vg, about 1×10 16 vg, or about 1×10 17 vg per subject in total. In one aspect, the therapeutically effective amount of the disclosed AAV particles or the disclosed AAV vector can be in the range of about 1×10 12 vg to about 1×10 17 vg per subject in total.

[0289] In some embodiments of the disclosed methods, techniques for monitoring, measuring, and / or assessing the restoration of one or more aspects of cellular homeostasis and / or cellular functionality may include qualitative (or subjective) and quantitative (and objective) means. These means are known to those skilled in the art. For example, representative regulated variables and sensors relating to systemic homeostasis are discussed above.

[0290] In some embodiments of the disclosed methods, the step of contacting target cells may include methods known in the art. For example, the contact step may include administering one or more disclosed enzymes or recombinant enzymes, one or more 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 to the target.

[0291] In some embodiments, administration may include retrograde ureteral infusion, renal artery administration, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intra-CSF, intrathecal, intraventricular, intrahepatic, intrahepatic artery, hepatic portal vein (HPV), or intrauterine administration. In some embodiments, administration may include retrograde ureteral infusion and / or arterial routes. In some embodiments, the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may be administered in combination with RNAi, antisense oligonucleotides, miRNAs, one or more small molecules, one or more therapeutic agents, one or more proteasome inhibitors, one or more replacement enzymes, one or more immunomodulators, and / or gene editing systems. In some embodiments, the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may be administered by LNP administration. In some embodiments, the disclosed compositions, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, and / or disclosed AAV vectors can be administered to a subject in parallel and / or sequentially via multiple routes of administration. For example, in some embodiments, administration of the disclosed compositions, disclosed enzymes or disclosed recombinant enzymes, disclosed nucleic acid molecules, disclosed pharmaceutical formulations, disclosed AAV particles, and / or disclosed AAV vectors may include IV administration. In some embodiments, multiple routes of administration can be used to the subject in the disclosed method. In some embodiments, multiple routes of administration, including retrograde ureteral infusion and / or arterial routes, can be used to the subject in the disclosed method. In some embodiments, a first route of administration, which may be the same as or different from a second and / or subsequent route of administration, can be used in the disclosed method.

[0292] In some embodiments, an ex vivo perfusion protocol can be used in a disclosed method for treating a subject. In some embodiments, an ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof can be used in conjunction with a kidney (or portion thereof) obtained for a subject. In some embodiments, a kidney can be obtained from a donor subject and subjected to an ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof before implantation into a subject requiring it. In some embodiments, a kidney can be obtained from a subject requiring it, subjected to an ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, and then returned to the subject requiring it. In some embodiments, a disclosed ex vivo perfusion protocol using 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 a subject requiring it.

[0293] In some embodiments, a disclosed method for treating a subject may further include the step of administering a therapeutic agent to the subject in a therapeutically effective dose. The therapeutic agent may be any disclosed activator that produces a desired clinical outcome. In some embodiments, the disclosed therapeutic agent may be an enzyme or recombinant enzyme. In some embodiments, a therapeutically effective dose of the disclosed replacement enzyme or recombinant enzyme may contain approximately 0.01 mg to approximately 100 mg per kg of body weight.

[0294] In some embodiments, the disclosed method may include administering one or more additional treatments to a subject, one or more times. In some embodiments, the disclosed method for delivering a payload may further include a step of monitoring the subject for adverse reactions. In some embodiments, if no adverse reactions are present, the method may further include a step of continuing treatment of the subject. In some embodiments, if adverse reactions are present, the method may further include a step of modifying the treatment step. Methods for monitoring the well-being of a subject may include both subjective and objective criteria (as discussed above). Such methods are known to those skilled in the art.

[0295] In some embodiments, a disclosed method for treating a subject may further include administering to the subject a therapeutically effective dose of an agonist capable of correcting one or more aspects of a dysregulated metabolic or enzymatic pathway. In some embodiments, such an agonist may include an enzyme for enzyme replacement therapy. In some embodiments, any enzyme in a dysregulated or dysfunctional metabolic or enzymatic pathway can be replaced by the disclosed enzyme. In some embodiments, the disclosed method may include replacing one or more enzymes in a dysregulated or dysfunctional metabolic pathway. In some embodiments, the dysregulated metabolic or enzymatic pathway originates from or resides in one or more of the kidneys of the subject.

[0296] In some embodiments, the disclosed method for treating a subject may further include the step of administering one or more immunomodulators. In some embodiments, the disclosed immunomodulators may be methotrexate, rituximab, intravenous gamma globulin, or bortezomib, or a combination thereof. In some embodiments, the disclosed immunomodulators may be bortezomib or SVP-rapamycin. In some embodiments, the disclosed immunomodulators may be tacrolimus. In some embodiments, the disclosed immunomodulators may be, for example, methotrexate, administered in low to high doses over a short period. In some embodiments, the disclosed immunomodulators may be administered in doses of approximately 0.1 mg to approximately 0.6 mg per kg of body weight. In some embodiments, the disclosed immunomodulators may be administered in doses of approximately 0.4 mg per kg of body weight. In some embodiments, the disclosed immunomodulator can be administered at an approximate daily dose of 0.4 mg per kg of body weight for 3 to 5 cycles or more, with each cycle lasting up to 3 days. In some embodiments, the disclosed immunomodulator can be administered at an approximate daily dose of 0.4 mg per kg of body weight for a minimum of 3 cycles, with each cycle lasting 3 days. In some embodiments, the appropriate number of cycles can be determined by those skilled in the art. In some embodiments, the disclosed immunomodulator can be administered multiple times as needed to achieve the desired clinical effect.

[0297] In some embodiments, a disclosed method for treating a subject may further include the step of administering one or more immunosuppressants. In some embodiments, the immunosuppressants may be, but are not limited to, azathioprine, methotrexate, sirolimus, anti-thymocyte globulin (ATG), cyclosporine (CSP), mycophenolate mofetil (MMF), steroids, or combinations thereof. In some embodiments, a disclosed method may include administering one or more immunosuppressants more than once. In some embodiments, a disclosed method may include administering one or more immunosuppressants repeatedly over time. In some embodiments, a disclosed method may include administering to a subject a compound that targets or modifies antigen presentation or humoral or cellular or innate immune responses.

[0298] In some embodiments, a disclosed method for treating a subject may further include the step of administering a compound that exerts a therapeutic effect on B cells and / or a compound that targets or modifies antigen presentation, humoral immune response, or cellular immune response. In some embodiments, the disclosed compounds may be rituximab, methotrexate, intravenous gamma globulin, anti-CD4 antibody, anti-CD2, anti-FcRN antibody, BTK inhibitor, anti-IGF1R antibody, CD19 antibody (e.g., inebilizumab), anti-IL6 antibody (e.g., tocilizumab), antibody against CD40, IL2 mutant protein, or a combination thereof. Treg infusion (e.g., antigen-specific Treg cells against AAV) that can be administered as a means of adjuncting immune tolerance are also disclosed herein.

[0299] In some embodiments, a disclosed method for treating a subject may include repeating a disclosed administration step, for example, administering a disclosed enzyme, a disclosed recombinant enzyme, a disclosed nucleic acid molecule, a disclosed AAV particle or disclosed AAV vector, a disclosed pharmaceutical formulation, a disclosed therapeutic agent, a disclosed immunomodulator, a disclosed proteasome inhibitor, a disclosed immunosuppressant, a disclosed compound that exerts a therapeutic effect on B cells, a disclosed compound that targets or modifies antigen presentation or humoral or cellular immune responses, or any combination thereof.

[0300] In some embodiments, a disclosed method for treating a subject may include a step of modifying one or more of the disclosed steps. For example, a step of modifying one or more of the steps of a disclosed method may include modifying or changing one or more features or aspects of one or more steps of the disclosed method. For example, in some embodiments, a method may be modified by changing the amount of one or more disclosed enzymes or recombinants, one or more 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 to a subject of one or more disclosed enzymes or recombinants, one or more 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; or by changing the duration of administration to a subject of one or more disclosed enzymes or recombinants, one or more 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.

[0301] In some embodiments, a disclosed method for treating a subject can be modified by changing the amount of one or more disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or modify antigen presentation, humoral immune responses, or cellular immune responses administered to the subject, or by changing the frequency of administration of one or more of the disclosed therapeutic agents, disclosed immunomodulators, disclosed proteasome inhibitors, disclosed immunosuppressants, disclosed compounds that exert a therapeutic effect on B cells, and / or disclosed compounds that target or modify antigen presentation, humoral immune responses, or cellular immune responses administered to the subject.

[0302] For example, in one embodiment, a disclosed method for treating a subject may further include a step of generating disclosed AAV particles or disclosed AAV vectors. In one embodiment, the step of generating disclosed AAV particles or disclosed viral vectors may include generating AAV particles or AAV particles or AAV vectors or recombinant AAV (e.g., those disclosed herein) using a method disclosed herein.

[0303] In some embodiments, a disclosed method for treating a subject may further include the step of generating and / or verifying one or more 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 some embodiments, a disclosed method for treating a subject may further include a step of gene editing one or more relevant genes (e.g., a missing, defective, and / or mutated protein or enzyme), wherein the editing includes, but is not limited to, single-gene knockout, simultaneous loss-of-function screening of multiple genes, gene knock-in, or a combination thereof.

[0305] In some embodiments of the disclosed method, the payload may comprise one or more base-editing components and one or more sgRNAs targeting the region to be edited.

[0306] In some embodiments, a disclosed method for treating a subject may further include the step of administering an oligonucleotide therapeutic agent. The disclosed oligonucleotide therapeutic agent may include single-stranded or double-stranded DNA, iRNA, shRNA, siRNA, mRNA, non-coding RNA (ncRNA), antisense molecules, miRNA, morpholino, peptide nucleic acid (PNA), or analogs or conjugates thereof. In some embodiments, the disclosed oligonucleotide therapeutic agent may be an ASO or RNAi. In some embodiments, the disclosed oligonucleotide therapeutic agent may include one or more modifications at any applicable site. In some embodiments, the disclosed oligonucleotide therapeutic agent may include a CRISPR-based endonuclease. In some embodiments, the disclosed endonuclease may be Cas9. In some embodiments, the disclosed Cas9 may be derived from Staphylococcus aureus or Streptococcus pyogenes.

[0307] In some embodiments of the disclosed methods, the disclosed enzyme, the disclosed recombinant enzyme, the disclosed nucleic acid molecule, the disclosed AAV particle, the disclosed AAV vector, the disclosed pharmaceutical formulation, or any combination thereof may be delivered and / or administered before, in parallel with, or after the delivery and / or administration of enzyme replacement therapy, protein replacement therapy, gene therapy, recombinant product, or any combination thereof.

[0308] In some embodiments, the disclosed method for treating a subject may further include plasmapheresis and immunosuppression. In some embodiments, the disclosed method may include using immunosuppression to reduce T cell, B cell, and / or plasma cell populations, and generally to reduce innate immune responses, inflammatory responses, and antibody levels. In some embodiments, the antibody may be against one or more components of the disclosed AAV particles or the disclosed AAV vector, or against a product encoded by the disclosed transgene, heterologous nucleic acid, or payload.

[0309] In some embodiments, a disclosed method for treating a subject may further include a step of reducing and / or minimizing vector-mediated immunotoxicity and / or the immunogenicity of the transgene (e.g., the ability to induce specific immunity). In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the efficacy of the recombinant product encoded by the transgene. In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the likelihood of re-administering one or more disclosed enzymes or recombinant enzymes, one or more 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. In some embodiments, vector-mediated immunotoxicity and / or the immunogenicity of the transgene may reduce and / or reduce the likelihood of re-administering gene therapy, enzyme replacement therapy, protein replacement, or any combination thereof to a subject.

[0310] In some embodiments, the disclosed method may further include the step of administering one or more disclosed enzymes or recombinant enzymes, one or more 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, once or more times. In some embodiments, a disclosed method for treating a subject may further include a step of measuring and / or determining the pre-treatment level of one or more clinical and / or metabolic indicators of the subject's renal function (e.g., expression of NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, or DZIP1L) or evaluation criteria of the subject. In some embodiments, a disclosed method may further include a step of measuring and / or determining the level of one or more clinical and / or metabolic indicators of the subject one or more times.

[0311] In some embodiments, a disclosed method for treating a subject may further include a step of reducing the risk of rejection of one or more parenchymal organ grafts. In some embodiments, a disclosed method for treating a subject may further include a step of improving the survival rate of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, a disclosed method for treating a subject may further include a step of reducing the risk of graft-versus-host disease (GVHD) occurring after implantation of one or more parenchymal organs (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it). In some embodiments, a disclosed method for treating a subject may further include a step of reducing the risk of rejection of one or more parenchymal organ grafts (e.g., (i) a donor kidney, or (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject requiring it).In some embodiments, a disclosed method for treating a subject may further include a step to enhance the transplantation efficiency of one or more parenchymal organs (e.g., (i) a donor kidney, (ii) a donor kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation into a subject in need, or (iii) the subject's own kidney treated by a disclosed ex vivo perfusion protocol using a disclosed nucleic acid molecule, a disclosed AAV particle, a disclosed AAV vector, a disclosed pharmaceutical formulation, or any combination thereof, before implantation and return to a subject in need). In some embodiments, a disclosed method for treating a subject may extend and / or improve the subject's life expectancy.

[0312] D. Others An adeno-associated virus (AAV) capsid protein comprising the sequence shown in SEQ ID NO: 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising a sequence having at least 90% identity with the sequence shown in SEQ ID NO: 03 is disclosed herein. An adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452-458 compared to SEQ ID NO: 01 is disclosed herein. In one embodiment, positions 452-458 comprises the sequence shown in any one of SEQ ID NOs: 04-23 or SEQ ID NOs: 191-8873. In one embodiment, positions 452-458 comprises a sequence that is at least 85% identical to any one of SEQ ID NOs: 04-23 or SEQ ID NOs: 191-8873.

[0313] An adeno-associated virus (AAV) capsid protein comprising one or more amino acid substitutions at positions 452-458 of Sequence ID No. 01 is disclosed herein, wherein the substitution at position 452 is any amino acid other than N; the substitution at position 453 is any amino acid other than G; the substitution at position 454 is any amino acid other than S; the substitution at position 455 is any amino acid other than G; the substitution at position 456 is any amino acid other than Q; the substitution at position 457 is any amino acid other than N; and / or the substitution at position 458 is any amino acid other than Q.

[0314] An adeno-associated virus (AAV) capsid protein comprising the sequence shown in any one of sequence numbers 24 to 43 is disclosed herein.

[0315] In some embodiments, the disclosed variant can be used to improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to the wild-type capsid protein. In some embodiments, the disclosed renal cells or kidney-derived cell types may include renal epithelial cells and / or renal endothelial cell types.

[0316] In some embodiments, the disclosed renal cells or kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0317] In some embodiments, the disclosed variant can be used to improve and / or enhance gene transfer to any region or portion of the kidney. In some embodiments, the disclosed region or portion of the kidney may include the adrenal gland, cortex, medulla, trabecula, pyramidal region, pelvis, major calyces, minor calyces, papillae, ureter, or any combination thereof. In some embodiments, the disclosed AAV capsid protein can be used to improve the dose-response correlation (e.g., thereby improving efficiency).

[0318] AAV capsids containing the disclosed AAV capsid protein are disclosed herein. AAV vectors containing a vector genome capsid-encapsulated by or capsid-encapsulated by the disclosed AAV capsid containing the disclosed AAV capsid protein are disclosed herein. In some embodiments, the disclosed vector genome may include a first reverse-ended repeat (ITR) and a second ITR. In some embodiments, the disclosed vector genome may include a nucleic acid sequence encoding a transgene or payload between the first and second ITRs. In some embodiments, the disclosed nucleic acid sequence encoding the transgene, heteronucleotide, or payload may be operably ligated to a promoter. In some embodiments, the disclosed transgene, heteronucleotide, or payload may encode a therapeutic RNA or therapeutic protein. In some embodiments, the therapeutic RNA is circular RNA (cirRNA). In some embodiments, the disclosed therapeutic RNA may be an antisense oligonucleotide, ribozyme, siRNA, shRNA, or miRNA. In some embodiments, the disclosed transgene, heteronucleotide, or payload may encode a missing, defective, and / or mutated protein or enzyme. In some embodiments, the disclosed missing, defective, and / or mutated protein or enzyme may be encoded by NPHS1, NPHS2, PLCE1, CD2AP, LAMB2, NPHS2, ACTN4, TRPC6, WT1, LMX1B, SMARCAL1, COQ2, PDSS2, MTTL1, SCARB2, FN1, COL4A5, COL4A6, COL4A3, COL4A4, PKD1, PKD2, PKHD1, DZIP1L, or any combination thereof.

[0319] In some embodiments, the disclosed transgene, heteronucleotide, or payload may encode apolipoprotein L1, fibrocystin, myosin heavy chain 9, nephrocystin 1, polycystin 1, polycystin 2, or any combination thereof. In some embodiments, the disclosed transgene, heteronucleotide, or payload may encode a gene editing molecule. In some embodiments, the disclosed gene editing molecule may comprise a nuclease or a single guide RNA (sgRNA). In some embodiments, the disclosed AAV particles or AAV vectors may be used to improve and / or enhance gene transfer into one or more renal cells or kidney-derived cell types compared to AAV particles or AAV vectors having wild-type capsid protein. In some embodiments, the disclosed renal cells or kidney-derived cell types may comprise renal epithelial cells and / or renal endothelial cell types. In some embodiments, the disclosed renal cells or kidney-derived cell types may include glomerular endothelial cells, podocytes, mesangial cells, smooth muscle cells, pericytes, juxtaglomerular cells, peritubular capillary cells, collecting duct chief cells, collecting duct transition cells, collecting duct interstitial cells, or cells of the afferent duct, efferent duct, ascending straight vessel, descending straight vessel, proximal straight tubule and proximal convoluted tubule, thin descending limb, thin ascending limb and thick ascending limb of the loop of Henle, macula densa, distal convoluted tubule 1 or distal convoluted tubule 2, connecting tubule, or any combination thereof.

[0320] Disclosed herein are nucleic acid molecules comprising nucleic acid sequences encoding a disclosed adeno-associated virus (AAV) capsid protein. Disclosed herein are pharmaceutical formulations comprising a disclosed AAV particle or a disclosed AAV vector and at least one pharmaceutically acceptable carrier. Disclosed herein are methods for delivering a transgene or payload to target cells in a subject, comprising the step of administering t...

Claims

1. An adeno-associated virus (AAV) capsid protein, wherein the AAV capsid protein contains an amino acid sequence in which positions 452 to 458 are at least 85% identical to any one of sequence numbers 04 to 23, and the AAV capsid protein is numbered with reference to sequence number 01.

2. The AAV capsid protein according to 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 NOs: 24 to 43.

3. The AAV capsid protein according to claim 1, wherein positions 452 to 458 of the AAV capsid protein contain the amino acid sequence of SEQ ID NO:

11.

4. The AAV capsid protein according to claim 1, wherein positions 452 to 458 of the AAV capsid protein contain the amino acid sequence of SEQ ID NO:

15.

5. The AAV capsid protein according to claim 2, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:

31.

6. The AAV capsid protein according to claim 2, wherein the AAV capsid protein comprises the amino acid sequence of SEQ ID NO:

35.

7. The AAV capsid protein according to any one of claims 1 to 6, wherein the AAV capsid protein is a variant of the parent wild-type capsid protein.

8. The AAV capsid protein according to claim 7, wherein the parent wild-type capsid protein is the capsid protein of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAVrh10.

9. The AAV capsid protein according to claim 8, wherein the parent wild-type capsid protein is VP1 of AAV9.

10. The AAV capsid protein according to any one of claims 7 to 9, wherein the AAV capsid protein improves gene transfer and / or expression in one or more regions or parts of the kidney compared to the parental wild-type capsid protein.

11. The AAV capsid protein according to claim 10, wherein the gene transfer and / or expression is improved by 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 times, 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 according to claim 10 or 11, wherein the region or part of the kidney is the adrenal gland, cortex, medulla, trabecula, pyramidal cavity, pelvis, major calyces, minor calyces, papilla, or ureter.

13. The AAV capsid protein according to claim 12, wherein the region or portion of the kidney is a proximal tubule within the cortex.

14. A nucleic acid molecule comprising a nucleic acid sequence encoding the AAV capsid protein according to any one of claims 1 to 13.

15. (a) the gene of interest; and (b) The nucleic acid molecule according to claim 14 AAV vectors containing this.

16. The AAV vector according to claim 15, wherein the target gene encodes a therapeutic RNA or a therapeutic protein.

17. The AAV vector according to claim 16, wherein the therapeutic RNA is circular RNA (crRNA).

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

19. The AAV vector according to any one of claims 15 to 18, wherein the target gene is operably linked to a promoter.

20. The AAV vector according to any one of claims 15 to 19, further comprising a first reverse end repeat (ITR) and a second ITR.

21. A pharmaceutical formulation comprising an AAV vector according to any one of claims 15 to 20 and at least one pharmaceutically acceptable carrier.

22. A method for treating a subject having kidney disease or kidney impairment, the method comprising the step of administering to the subject a therapeutically effective amount of an AAV vector according to any one of claims 15 to 20 or a pharmaceutical preparation according to claim 21.

23. The method according to claim 22, wherein the renal disease or renal impairment is Alport syndrome, Bartter syndrome types 1 to 4, congenital nephrotic syndrome, cystinuria, cystine storage disease, Dent's disease types 1 and 2, distal tubular acidosis, Fabry disease, familial amyloidosis, Gittellmann syndrome, Liddle syndrome, Lowe syndrome, nephronophthria (NPHP), autosomal dominant polycystic kidney disease, autosomal recessive polycystic kidney disease, tubulointerstitial kidney disease, proximal tubular acidosis, thin basement membrane disease, tubulointerstitial kidney disease, or tuberous sclerosis (TSC).

24. The method according to claim 22 or claim 23, wherein the AAV vector according to any one of claims 15 to 20 or the pharmaceutical preparation according to claim 21 is administered by retrograde ureteral infusion.

25. The method according to claim 22 or 23, wherein the AAV vector according to any one of claims 15 to 20 or the pharmaceutical formulation according to claim 21 is administered by an arterial route.

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