Cross-species compatible adeno-associated virus compositions and methods of use thereof

Abstract

The present disclosure provides adeno-associated virus (AAV) vectors, pharmaceutical compositions, methods for producing, and methods for delivering such to subjects, which contain co-evolved capsid variant proteins.The present disclosure relates to modified capsid proteins from adeno-associated virus (AAV), and viral capsids and viral vectors containing the same.In particular, the present disclosure relates to modified AAV capsid proteins and capsids containing the same that can be incorporated into viral vectors to enable expression in any cell or tissue type of mammalian subjects.

Description

[Technical Field]

[0001] U.S. Federal Funding Statement This invention was made with government support under U.S. Federal Grant Nos. R01HL089221 and UG3AR075336, both awarded by the National Institutes of Health. The U.S. Federal Government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 020,062, filed May 5, 2020, which is incorporated herein by reference in its entirety for all purposes.

[0003] Disclosure Areas The present disclosure relates to modified capsid proteins from adeno-associated viruses (AAVs), as well as viral capsids and viral vectors containing the same. In particular, the present disclosure relates to modified AAV capsid proteins and capsids containing the same that can be incorporated into viral vectors to enable expression in any cell or tissue type in a mammalian subject.

[0004] Incorporation by reference of electronically submitted sequence listings An electronic version of the Sequence Listing is submitted herewith, the contents of which are incorporated by reference in their entirety. The electronic file is 611 kilobytes in size and has the file name 21-2006-WO_SequenceListing_ST25.txt. [Background technology]

[0005] background Adeno-associated virus (AAV) vectors have become the primary platform for gene therapy agents for the treatment of various diseases. While the use of AAV-based gene therapy agents has been clinically successful, limitations and challenges remain associated with the use of this gene delivery platform. For example, the efficacy of gene therapy agents using vectors (viral or non-viral) can be reduced due to the subject's immune response to the gene-carrying vector. Additionally, the route of administration must be optimized to ensure delivery to one or more target tissues in a subject. This is particularly true for treating disorders of the central nervous system (CNS) and peripheral nervous system (PNS). The blood-brain barrier can prevent AAV-based therapeutic agents from reaching the CNS when administered systemically, and direct administration to CNS tissues can require invasive surgery. Furthermore, the high doses of AAV-based therapeutic agents required to induce sufficient transduction of target CNS and PNS tissues increase the risk of side effects and / or unwanted immune responses. Additionally, the requirement to produce high doses of AAV imposes a manufacturing burden.

[0006] Although each known AAV serotype has specific tissue tropism, there are some tissues (such as kidney) that cannot be easily targeted using these AAVs.In addition, for a given dose, AAV transduction in systemic organs such as heart, liver or lung can vary significantly between various model organisms (such as dogs, pigs, non-human primates) used in clinical development and between human subjects.This also makes it difficult to accurately test AAV-based therapeutic agents in animal models before use in humans.

[0007] As the range of applications of AAV gene transfer expands, including the development of gene therapy agents for CNS and / or PNS disorders, there remains a need in the art to address differences in AAV tropism across different species. These differences often result in nonlinear vector dose-biodistribution relationships when scaling from small to large animal models—subsequently affecting clinical translation. Thus, there is an unmet need in the art to develop AAV gene delivery platforms that are more broadly translatable across multiple species. Additionally, there is a need to develop AAV-based gene therapy agents that can selectively and specifically target tissues of interest, including tissues that have been difficult to target using known AAV serotypes. Summary of the Invention [Means for solving the problem]

[0008] BRIEF SUMMARY OF THE DISCLOSURE The present disclosure provides methods and compositions comprising, at least in part, adeno-associated virus (AAV) capsid proteins that contain one or more amino acid substitutions that introduce one or more improved functionalities into AAV vectors containing these modified capsid proteins, such as, but not limited to, the ability to escape host antibodies, selective affinity, and / or higher transduction efficiency.

[0009] Aspects of the present disclosure provide recombinant AAV vectors comprising an AAV capsid protein variant as disclosed herein. In some embodiments, the recombinant AAV vectors herein may comprise an AAV capsid protein variant, wherein the capsid protein variant comprises a peptide having the sequence of any one of SEQ ID NOs: 2-19. In some embodiments, the recombinant AAV vectors herein comprise an AAV capsid protein variant, wherein the capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, in which amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20-28. In some embodiments, the recombinant AAV vectors herein may comprise an AAV capsid protein variant, wherein the capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, in which amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0010] In some embodiments, the recombinant AAV vector herein comprises an AAV capsid protein variant, wherein the capsid protein variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20 to 28, and wherein amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29 to 37.

[0011] In some embodiments, the recombinant AAV vectors herein may comprise an AAV capsid protein variant, wherein the capsid protein variant has the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having at least 90% or at least 95% identity thereto. In some embodiments, the recombinant AAV vectors herein comprise an AAV capsid protein variant, wherein the capsid protein variant has the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having 1 to 10, 11 to 20, 20 to 30, or 30 to 50 amino acid substitutions thereto.

[0012] Another aspect of the present disclosure provides an AAV capsid protein variant as disclosed herein. In some embodiments, the AAV capsid protein variant herein comprises a peptide having the sequence of any one of SEQ ID NOs: 2 to 19.

[0013] In some embodiments, the AAV capsid protein variants herein have at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20-28. In some embodiments, the AAV capsid protein variants herein have at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0014] In some embodiments, the AAV capsid protein variants herein may have at least 90% identity to the sequence of SEQ ID NO: 1, and amino acids corresponding to amino acids 452-458 of SEQ ID NO: 1 may be replaced with a peptide having the sequence of any one of SEQ ID NOs: 20-28, and amino acids corresponding to amino acids 586-592 of SEQ ID NO: 1 may be replaced with a peptide having the sequence of any one of SEQ ID NOs: 29-37.

[0015] In some embodiments, an AAV capsid protein variant herein may have the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having at least 90% or at least 95% identity thereto. In some embodiments, a capsid protein variant herein may have the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having 1-10, 11-20, 20-30, or 30-50 amino acid substitutions thereto.

[0016] Another aspect of the present disclosure provides a pharmaceutical composition comprising any of the AAV capsid protein variants and / or AAV vectors disclosed herein. In some embodiments, the pharmaceutical composition herein may further comprise at least one pharmaceutically acceptable carrier.

[0017] Another aspect of the present disclosure provides a method for introducing a recombinant AAV vector into a target cell. In some embodiments, the method for introducing a recombinant AAV vector into a target cell described herein may include contacting the target cell with any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein. In some embodiments, the method described herein can deliver one or more therapeutic heterologous molecules to a target cell in a subject, and the method includes administering any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein to the subject. In some embodiments, any of the recombinant AAV vectors (e.g., ccAAV) and / or pharmaceutical compositions disclosed herein can be administered to the subject by intramuscular injection, intravenous injection, intracoronary injection, intraarterial injection, or any combination thereof.

[0018] Another aspect of the present disclosure provides a method for evolving novel adeno-associated virus strains, comprising passaging an AAV capsid library across multiple mammalian species. In some embodiments, the methods herein may utilize an AAV capsid library containing AAV capsids packaging different genomes encoding mutated capsid gene sequences. In some embodiments, the methods herein may administer the AAV capsid library herein to Mus Musculus (mice), Sus scrofa (pigs), Canis Familiaris (dogs), non-human primates (Macaca), or Homo sapiens (humans), and any combination or repeated cycle thereof. In some embodiments, the methods herein may enrich for adeno-associated virus (AAV) capsid protein sequences herein by passaging the AAV capsid library according to the methods disclosed herein. In some embodiments, the methods herein can enrich for sequences encoding capsid protein variants herein by extracting AAV capsid protein variants from cells collected from or derived from the group consisting of spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, the methods herein can produce AAV capsid protein variants as disclosed herein with improved gene transfer efficiency in any mammalian species selected from the group consisting of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), or Homo sapiens (human), and any combination or repeated cycle thereof.In some embodiments, the methods herein are capable of producing AAV capsid protein variants as disclosed herein with improved gene transfer efficiency in any of the following cell types or tissues: spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, the methods herein are capable of producing AAV capsid protein variants as disclosed herein with improved immune response in any of the following cell types or tissues: spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, the methods herein are capable of producing AAV capsid protein variants as disclosed herein with improved affinity in any of the following cell types or tissues: spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0019] Aspects of the present disclosure provide kits that may include any of the compositions or AAV vectors disclosed herein and at least one container.

[0020] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0021] [Figure 1A]Figures 1A and 1B illustrate bubble plots showing the analysis of library diversity, directed evolution, and enrichment of AAVs containing capsid proteins with novel peptide substitutions according to certain embodiments herein. The parental library (Figure 1A) and the evolved library from three cycles (Figure 1B) were subjected to high-throughput sequencing using the Illumina MiSeq platform. After analysis with a custom Perl script, the enriched amino acid sequences were plotted. Each bubble represents a distinct capsid protein amino acid sequence, and the bubble radius is proportional to the number of reads for that variant in the respective library. The y-axis represents the percentage of total reads from the sequencing run. The data are spread along the x-axis for ease of visualization. The percent reduction of unique clones (96.5%) directly indicates that a large number of "misfit" sequences were removed after the first and / or second evolutionary rounds. Primary isolates were selected for further analysis. As shown in Figure 1B, next-generation sequencing revealed that the capsid protein of AAVcc47 was the most enriched amino acid sequence (i.e., clone) in the AAV VR4 library after three cycles of evolution in three different species. [Figure 1B]Figures 1A and 1B illustrate bubble plots showing the analysis of library diversity, directed evolution, and enrichment of AAVs containing capsid proteins with novel peptide substitutions according to certain embodiments herein. The parental library (Figure 1A) and the evolved library from three cycles (Figure 1B) were subjected to high-throughput sequencing using the Illumina MiSeq platform. After analysis with a custom Perl script, the enriched amino acid sequences were plotted. Each bubble represents a distinct capsid protein amino acid sequence, and the bubble radius is proportional to the number of reads for that variant in the respective library. The y-axis represents the percentage of total reads from the sequencing run. The data are spread along the x-axis for ease of visualization. The percent reduction of unique clones (96.5%) directly indicates that a large number of "misfit" sequences were removed after the first and / or second evolutionary rounds. Primary isolates were selected for further analysis. As shown in Figure 1B, next-generation sequencing revealed that the capsid protein of AAVcc47 was the most enriched amino acid sequence (i.e., clone) in the AAV VR4 library after three cycles of evolution in three different species.

[0022] [Figure 2] Figures 2A-2D illustrate mCherry reporter gene expression in the hearts of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing mCherry expression in cardiac vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 2A) or AAV.cc47 (Figure 2B). Figure 2C shows a graph depicting the corrected total cell fluorescence of a series of multiple images. Figure 2D shows a graph depicting the biodistribution of vectors in the hearts of infected mice.

[0023] [Figure 3]Figures 3A-3C illustrate mCherry reporter gene expression in skeletal muscle of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing mCherry expression in skeletal muscle vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 3A) or AAV.cc47 (Figure 3B). Figure 3C shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images.

[0024] [Figure 4] Figures 4A-4D illustrate mCherry reporter gene expression in the livers of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing mCherry expression in liver vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 4A) or AAV.cc47 (Figure 4B). Figure 4C shows a graph depicting the corrected total cell fluorescence of a series of multiple images. Figure 4D shows a graph depicting the biodistribution of vectors in the livers of infected mice.

[0025] [Figure 5] Figures 5A-5C illustrate mCherry reporter gene expression in the kidneys of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing mCherry expression in kidney vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 5A) or AAV.cc47 (Figure 5B). Figure 5C shows a graph depicting the corrected total cell fluorescence of a series of multiple images.

[0026] [Figure 6]Figures 6A-6D illustrate GFP reporter gene expression in the hearts of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing GFP expression in cardiac vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 6A), AAV.cc81 (Figure 6B), or AAV.cc84 (Figure 6C). Figure 6D shows a graph depicting the corrected total cell fluorescence of a series of multiple images.

[0027] [Figure 7] Figures 7A-7C illustrate GFP reporter gene expression in skeletal muscle of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing GFP expression in skeletal muscle vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 7A) or AAV.cc81 (Figure 7B). Figure 7C shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images.

[0028] [Figure 8] Figures 8A-8D illustrate GFP reporter gene expression in the liver of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing GFP expression in liver vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (Figure 8A), AAV.cc481 (Figure 8B), or AAV.cc84 (Figure 8C). Figure 8D shows a graph depicting the corrected total cell fluorescence of a series of multiple images.

[0029] [Figure 9] 9A-9C illustrate GFP reporter gene expression in the kidneys of C57 / B6 mice according to certain embodiments herein. Representative fluorescence microscopy images showing GFP expression in kidney vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 (FIG. 9A) or AAV.cc81 (FIG. 9B). FIG. 9C shows a graph depicting the corrected total cell fluorescence of a series of multiple images.

[0030] [Figure 10] Figures 10A-10E illustrate fluorescent reporter expression as assessed by immunohistochemistry (IHC) in brain regions of C57 / B6 mice according to certain embodiments herein. Figure 10A depicts a brain region from a mock-infected mouse, while Figure 10B depicts a brain region from a mouse infected with an AAV9 vector, Figure 10C depicts a brain region from a mouse infected with AAV.cc47, Figure 10D depicts a brain region from a mouse infected with AAV.cc81, and Figure 10E depicts a brain region from a mouse infected with AAV.cc84. Brain regions shown include Ctx = cerebral cortex, Hc = hippocampus, Cb = cerebellum, Th = thalamus, Str = striatum, and mb = mushroom body.

[0031] [Figure 11] 11A-11G illustrate AVV.cc47 transduction assessed by immunohistochemistry (IHC) in pig brain regions according to certain embodiments herein. FIG. 11A depicts IHC staining for mCherry in the pig frontal cortex. FIG. 11B depicts IHC staining for mCherry in the pig parietal cortex. FIG. 11C depicts IHC staining for mCherry in the pig parietal thalamus. FIG. 11D depicts IHC staining for mCherry in the pig occipital cortex. FIG. 11E depicts IHC staining for mCherry in the pig brainstem. FIG. 11F depicts IHC staining for mCherry in the pig cerebellum. FIG. 11G depicts IHC staining for mCherry in the pig midbrain.

[0032] [Figure 12]12A-12G illustrate AVV.cc84 transduction assessed by immunohistochemistry (IHC) in pig brain regions according to certain embodiments herein. FIG. 12A depicts IHC staining for GFP in the pig frontal cortex. FIG. 12B depicts IHC staining for GFP in the pig parietal cortex. FIG. 12C depicts IHC staining for GFP in the pig parietal thalamus. FIG. 12D depicts IHC staining for GFP in the pig occipital cortex. FIG. 12E depicts IHC staining for GFP in the pig brainstem. FIG. 12F depicts IHC staining for GFP in the pig cerebellum. FIG. 12G depicts IHC staining for GFP in the pig midbrain.

[0033] [Figure 13] 13A-13F illustrate AAV.cc47 and AVV.cc84 transduction in pig spinal cords according to certain embodiments herein. Sections of pig spinal cords were subjected to IHC staining for AVV.cc47 (FIG. 13A) and AVV.cc84 (FIG. 13B) in the tissue. mCherry fluorescence was measured in the white matter (FIG. 13C) and gray matter (FIG. 13E) to assess for AVV.cc47. GFP fluorescence was measured in the white matter (FIG. 13D) and gray matter (FIG. 13F) to assess for the presence of AVV.cc84.

[0034] [Figure 14] 14A-14F illustrate AAV.cc47 and AVV.cc84 transduction in pig heart and liver according to certain embodiments herein. AVV.cc47 transduction was assessed by IHC staining for mCherry in the pig left ventricle (FIG. 14A), pig right ventricle (FIG. 14B), and liver (FIG. 14C). AVV.cc84 transduction was assessed by IHC staining for GFP in the pig left ventricle (FIG. 14D), pig right ventricle (FIG. 14E), and liver (FIG. 14F).

[0035] [Figure 15]Figures 15A-15E illustrate AAV9 and AAV.cc47 transduction in the heart and liver of non-human primates according to certain embodiments herein. AVV9 transduction was assessed by IHC staining for mCherry in the liver (Figure 15A) and heart (Figure 15C) of non-human primates. AVV.cc47 transduction was assessed by IHC staining for mCherry in the liver (Figure 15B) and heart (Figure 15D) of non-human primates. Figure 15E shows the biodistribution of recombinant AAV in non-human primates.

[0036] [Figure 16] Figures 16A-16D illustrate AAV9, AAV.cc47, and AAV.cc84 transduction in non-human primate brains according to certain embodiments herein. In the non-human primate brains, AAV9 transduction was assessed by IHC staining for mCherry (Figure 16B), AAV.cc47 transduction was assessed by IHC staining for mCherry (Figure 16C), and AAV.cc84 transduction was assessed by IHC staining for GFP (Figure 16D). Figure 16A shows a brain slice from a sham-injected control non-human primate.

[0037] [Figure 17] Figures 17A-17E illustrate validation of AAVcc47 cardiac transduction according to certain embodiments herein. Figure 17A shows human iPSC cardiomyocytes transduced with AAV9 or cc47 packaging GFP driven by the Cbh promoter. Figure 17B shows quantification of the GFP+ area percent in multiple images from (Figure 17A). Figure 17C shows IV-injected AAV9 or AAVcc47 packaging CBh:GFP in a human cardiac patch mouse model. Figure 17D shows fluorescent imaging of the cardiac patch. Figure 17E shows iv-administered AAV9 and AAVcc47 delivering GFP under the control of an injury-inducible promoter after myocardial infarction. Immunofluorescence for troponin T (red) and GFP (green).

[0038] [Figure 18] Figures 18A-18E illustrate representative images of intrinsic tdTomato fluorescence in the hearts of mice following iv administration of sham (Figure 18A), AAV9 (Figure 18B), AVV.cc47 (Figure 18C), and AVV.cc84 (Figure 18D) in accordance with certain embodiments herein. Figure 18E shows the biodistribution of recombinant AAV in the hearts of mice.

[0039] [Figure 19] Figures 19A-19E illustrate representative images of intrinsic tdTomato fluorescence in the liver of mice following iv administration of mock (Figure 19A), AAV9 (Figure 19B), AVV.cc47 (Figure 19C), and AVV.cc84 (Figure 19D) in accordance with certain embodiments herein. Figure 19E shows the biodistribution of recombinant AAV in the liver of mice.

[0040] [Figure 20] Figures 20A-20E illustrate representative images of intrinsic tdTomato fluorescence in the lungs of mice following iv administration of mock (Figure 20A), AAV9 (Figure 20B), AVV.cc47 (Figure 20C), and AVV.cc84 (Figure 20D) in accordance with certain embodiments herein. Figure 20E shows the biodistribution of recombinant AAV in the liver of mice.

[0041] [Figure 21]Figures 21A-21D illustrate CRISPR / Cas9 gene editing with ccAAV vectors according to certain embodiments herein. Figure 21A shows the dual-vector strategy used herein, using one vector with a truncated CB promoter driving SaCas9 and a U6 promoter driving one sgRNA, and a second vector of the same design carrying a second sgRNA. Figure 21B shows intrinsic tdTomato fluorescence in the liver and heart of Ai9 mice after administration of AAV9 or cc47 at a dose of 2e12vg / kg. Figure 21C shows gene editing efficiency, determined by counting the total number of tdTomato+ cells and dividing by the total number of DAPI+ cells. Figure 21D shows a PCR editing assay, with the unedited band (**1160bp) and edited band (*270bp) marked. p**<0.01.

[0042] [Figure 22] Figures 22A-22C illustrate validation of CRISPR / Cas9 gene editing with ccAAV vectors according to certain embodiments herein. Figure 22A shows an Ai9 liver that was sectioned and imaged for native TdTomato expression. Figure 22B shows a graph depicting quantification of gene editing efficiency by counting the total number of TdTomato+ cells and normalizing to the total number of Dapi+ cells. Figure 22C shows an Ai9 heart that was sectioned and imaged for native TdTomato expression. Both tissues were cryosectioned into 14 μm thick sections.

[0043] [Figure 23]Figures 23A-23F illustrate quantification of CRISPR / Cas9 by measuring fluorescence intensity according to certain embodiments herein. Fluorescence intensity was measured from multiple images to quantify native TdTomato expression in Ai9 mice injected with either AAV9 or AAV.cc47 vectors. Figure 23A shows a graph of corrected total cell fluorescence in the livers of all injected Ai9 mice. Figure 23B shows a graph of corrected total cell fluorescence in the livers of all injected female Ai9 mice. Figure 23C shows a graph of corrected total cell fluorescence in the hearts of all injected Ai9 mice. Figure 23D shows a graph of corrected total cell fluorescence in the hearts of all injected female Ai9 mice. Figure 23E shows a graph of corrected total cell fluorescence in the livers of all injected male Ai9 mice. Figure 23F shows a graph of corrected total cell fluorescence in the hearts of all injected male Ai9 mice. P-value *<0.05; ns = non-significant.

[0044] [Figure 24] Figures 24A and 24B illustrate the quantification of CRISPR / Cas9 by measuring relative PCR band intensity according to certain embodiments herein. Figure 24A shows a graph of PCR band intensity (relative to mock unedited bands) for PCR bands resulting from PCR editing assays of liver tissue from Ai9 mice injected with either AAV9 vectors or AAV.cc47 vectors. Figure 24B shows a graph of PCR band intensity (relative to mock unedited bands) for PCR bands resulting from PCR editing assays of heart tissue from Ai9 mice injected with either AAV9 vectors or AAV.cc47 vectors.

[0045] [Figure 25]Figures 25A and 25B illustrate the quantification of CRISPR / Cas9 gene editing efficiency in liver and heart according to certain embodiments herein. Figure 25A shows a graph of the percentage of gene editing efficiency in liver tissue from Ai9 mice injected with either AAV9 vector or AAV.cc47 vector. Figure 25B shows a graph of the percentage of gene editing efficiency in heart tissue from Ai9 mice injected with either AAV9 vector or AAV.cc47 vector.

[0046] [Figure 26] Figures 26A and 26B illustrate mCherry reporter gene expression in the heart of a C57 / B6 mouse in accordance with certain embodiments herein. Figure 26A shows a representative fluorescent microscope image showing mCherry expression in a cardiac vibratome section 24 hours after fixation with 4% PFA in a mouse infected with AAV9 or AAV.cc44. Figure 26B shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images. Figures 26C and 26D illustrate mCherry reporter gene expression in the skeletal muscle of a C57 / B6 mouse in accordance with certain embodiments herein. Figure 26C shows a representative fluorescent microscope image showing mCherry expression in a skeletal muscle vibratome section 24 hours after fixation with 4% PFA in a mouse infected with AAV9 or AAV.cc44. Figure 26D shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images.

[0047] [Figure 27]Figures 27A and 27B illustrate mCherry reporter gene expression in the liver of a C57 / B6 mouse in accordance with certain embodiments herein. Figure 27A shows a representative fluorescent microscope image showing mCherry expression in liver vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44. Figure 27B shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images. Figures 27C and 27D illustrate mCherry reporter gene expression in the kidney of a C57 / B6 mouse in accordance with certain embodiments herein. Figure 27C shows a representative fluorescent microscope image showing mCherry expression in kidney vibratome sections 24 hours after fixation with 4% PFA in mice infected with AAV9 or AAV.cc44. Figure 27D shows a graph depicting the corrected whole-cell fluorescence of a series of multiple images.

[0048] [Figure 28] Figures 28A-28C illustrate fluorescent reporter expression as assessed by immunohistochemistry (IHC) in brain regions of C57 / B6 mice according to certain embodiments herein. Figure 28A depicts a brain region from a mock-infected mouse, while Figure 28B depicts a brain region from a mouse infected with an AAV9 vector, and Figure 28C depicts a brain region from a mouse infected with AAV.cc44. Brain regions shown include Ctx = cerebral cortex, Hc = hippocampus, Cb = cerebellum, Th = thalamus, Str = striatum, and mb = mushroom body.

[0049] [Figure 29]Figures 29A and 29B illustrate schematic diagrams of AAV vectors used for administration according to certain embodiments herein. Figure 29A depicts a complete capsid with variable region 4 (VR4) highlighted on the capsid surface (top panel) and recombinant capsid proteins produced as vectors packaging CBh-mCherry (AAV.cc47 and AAV.cc44) (bottom panel). Figure 29B depicts a complete capsid with variable region 8 (VR8) highlighted on the capsid surface (top panel) and recombinant capsid proteins produced as vectors packaging CBh-eGFP (AAV.cc81 and AAV.cc84) (bottom panel).

[0050] [Figure 30] Figures 30A-30F illustrate representative images of mCherry or eGFP expressed in mouse brains upon intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein. Figure 30A depicts a mouse whole brain and selected brain regions after ICV injection of an AAV9 vector (mCherry). Figure 30B depicts a mouse whole brain and selected brain regions after ICV injection of an AAV.cc44 vector (mCherry). Figure 30C depicts a mouse whole brain and selected brain regions after ICV injection of an AAV.cc47 vector (mCherry). Figure 30D depicts a mouse whole brain and selected brain regions after ICV injection of an AAV9 vector (eGFP). Figure 30E depicts a mouse whole brain and selected brain regions after ICV injection of an AAV.cc81 vector (eGFP). Figure 30F depicts the whole brain of a mouse after ICV injection of the AAV.cc84 vector (eGFP), and selected brain regions. Brain regions shown include Ctx = cerebral cortex, Hc = hippocampus, and Cb = cerebellum.

[0051] [Figure 31-1]Figures 31A-31E illustrate representative images and graphs of eGFP expressed in mouse brains upon intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein. Figure 31A depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV9 vectors (eGFP). Images from all three stains were merged to show colocalization. Figure 31B depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV.cc84 vectors (eGFP). Images from all three stains were merged to show colocalization. The number of neurons with both eGFP and Neuron staining was quantified in the cerebellum (CB, Figure 31C), hippocampus (HC, Figure 31D), and cerebral cortex (CTX, Figure 31E).

[0052] [Figure 31-2] Figures 31A-31E illustrate representative images and graphs of eGFP expressed in mouse brains upon intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein. Figure 31A depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV9 vectors (eGFP). Images from all three stains were merged to show colocalization. Figure 31B depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV.cc84 vectors (eGFP). Images from all three stains were merged to show colocalization. The number of neurons with both eGFP and Neuron staining was quantified in the cerebellum (CB, Figure 31C), hippocampus (HC, Figure 31D), and cerebral cortex (CTX, Figure 31E).

[0053] [Figure 32-1]Figures 32A-32E illustrate representative images and graphs of mCherry expressed in mouse brains upon intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein. Figure 32A depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, mCherry, and Neuron in brain tissue harvested and processed after ICV injection of AAV9 vectors (mCherry). Images from all three stains were merged to show colocalization. Figure 32B depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV.cc47 vectors (mCherry). Images from all three stains were merged to show colocalization. The number of neurons with both mCherry and Neuron staining was quantified in the cerebellum (CB, Figure 32C), hippocampus (HC, Figure 32D), and cerebral cortex (CTX, Figure 32E).

[0054] [Figure 32-2] Figures 32A-32E illustrate representative images and graphs of mCherry expressed in mouse brains upon intracerebroventricular (ICV) injection of AAV vectors according to certain embodiments herein. Figure 32A depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, mCherry, and Neuron in brain tissue harvested and processed after ICV injection of AAV9 vectors (mCherry). Images from all three stains were merged to show colocalization. Figure 32B depicts images from selected brain regions after immunofluorescence (IF) staining for DAPI, eGFP, and Neuron in brain tissue harvested and processed after ICV injection of AAV.cc47 vectors (mCherry). Images from all three stains were merged to show colocalization. The number of neurons with both mCherry and Neuron staining was quantified in the cerebellum (CB, Figure 32C), hippocampus (HC, Figure 32D), and cerebral cortex (CTX, Figure 32E). DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description Adeno-associated virus (AAV) vectors have become the primary platform for delivering therapeutic genes. Unfortunately, AAV-based gene therapy agents often fall short of their desired efficacy due to, for example, difficulties in optimizing the administration route to target cells or tissues of interest and the subject's immune response to the vector carrying the therapeutic gene (e.g., a transgene of interest). Pre-existing host antibodies, generated during natural encounters with AAV or recombinant AAV vectors, hinder initial and repeated administration of AAV vectors as vaccines and / or for gene therapy. Serological studies reveal high antibody prevalence in human populations worldwide, with approximately 67% of people possessing antibodies against AAV1, 72% against AAV2, and approximately 40% against AAV5 through AAV9. In gene therapy agents, pre-existing antibodies in subjects pose a problem because certain clinical scenarios involving gene silencing or tissue degeneration require multiple AAV vector administrations to sustain long-term transgene expression.

[0056] Although each known AAV serotype has a specific tissue tropism, there are some tissues (such as kidney) that cannot be easily targeted using these AAVs.The delivery of therapeutic genes using AAV vectors to treat disorders of the central nervous system (CNS) and peripheral nervous system (PNS) is particularly difficult because the blood-brain barrier can prevent AAV-based therapeutic agents from reaching the desired target.AAV transduction in systemic organs such as the heart, liver, or lung can vary significantly for a given dose among various model organisms (such as dogs, pigs, non-human primates) used in clinical development, and among human subjects.

[0057] To avoid these problems, recombinant AAV vectors that escape antibody recognition and / or selectively target CNS tissues are needed. The embodiments provided in the present disclosure will be useful for a) expanding the eligible cohort of patients suitable for AAV-based gene therapy agents, and b) enabling multiple repeated administrations of AAV-based gene therapy vectors. In addition, there is a need to develop AAV-based gene therapy agents that can selectively and specifically target tissues of interest, including tissues that are / are difficult to target using known AAV serotypes, such as the kidney.

[0058] The present disclosure is based at least in part on the novel discovery that the capsid antigenicity and functional properties, such as affinity and transduction, of AAV capsids and capsid proteins overlap in structural context and can be modified to provide improved functionality.Based on this disclosure, the AAV capsid proteins disclosed herein and adeno-associated virus (AAV) vectors comprising AAV capsid proteins can be co-evolved to induce cross-species compatibility, which is a potentially useful attribute that allows reliable translation of a given AAV from non-human disease models (e.g., rodents, non-human primates) to human use.Based on this, the present disclosure provides cross-species compatible AAV capsid proteins and AAV vectors comprising the AAV capsid proteins herein, as well as methods for their production and use. As used herein, "cross-species compatible AAV" can refer to an AAV vector comprising AAV capsid protein variants with mutated and / or substituted amino acid sequences that are co-evolved for cross-species compatibility. I. Definition

[0059] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to preferred embodiments and specific language will be used to describe the same, it being understood that no limitation on the scope of the disclosure is thereby intended, and that changes and further modifications on the disclosure as illustrated herein are contemplated as would normally occur to one skilled in the art to which the disclosure pertains.

[0060] As used herein, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and may include more than one element.

[0061] "About" is used to provide flexibility to the endpoints of a numerical range by stating that a given value may be "slightly above" or "slightly below" the endpoint without affecting the desired result. The term "about" in conjunction with a numerical value means that the numerical value may be plus or minus 5% or less of that numerical value.

[0062] Throughout this specification, unless the context requires otherwise, the words "comprise" and "include" and variations (e.g., "comprises," "comprising," "includes," "including") will be interpreted to imply the inclusion of a stated component, feature, element or step, or group of components, features, elements or steps, but not the exclusion of any other integer or step, or group of integers or steps.

[0063] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative ("or").

[0064] Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features set forth herein may be excluded or eliminated. By way of example, where a composite is described herein as comprising components A, B, and C, it is specifically contemplated that any or combination of A, B, or C, singly or in any combination, may be eliminated and discarded.

[0065] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are specifically recited herein. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between and including the lowest and highest values ​​recited should be considered to be specifically recited in this disclosure.

[0066] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other AAV now known or later discovered. See, e.g., Bernard N. Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Several AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Morris et al., (2004) Virology 33:375-383; and Table 1).

[0067] The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of native terminal repeats (TRs), Rep proteins, and capsid subunits, are known in the art.Such sequences can be found in literature or in public databases such as GenBank.For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02 275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579, the disclosures of which are incorporated herein by reference for their teaching of parvovirus and AAV nucleic acid and amino acid sequences. See also Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0068] The terms "heterologous nucleotide sequence" and "heterologous nucleic acid" are used interchangeably herein and refer to a sequence that does not naturally occur in a virus. Generally, a heterologous nucleic acid comprises an open reading frame that encodes a polypeptide or untranslated RNA of interest (e.g., for delivery to a cell or subject).

[0069] "Polynucleotide," as used herein, refers to a sequence of nucleotide bases, which may be RNA, DNA, or a DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is either a single-stranded or double-stranded DNA sequence.

[0070] As used herein, the term "peptide" refers to a short amino acid sequence. The term peptide can be used to refer to a portion or region of an AAV capsid amino acid sequence. A peptide can be a peptide that naturally occurs in a native AAV capsid, or a peptide that does not naturally occur in a native AAV capsid. A naturally occurring AAV peptide in an AAV capsid can be replaced with a non-naturally occurring peptide. For example, a non-naturally occurring peptide can be incorporated into an AAV capsid by substitution, such that a naturally occurring peptide is replaced with a non-naturally occurring peptide, to obtain a modified capsid. As used herein, the term "polypeptide" encompasses both peptides and proteins, unless otherwise specified.

[0071] As used herein, the term "amino acid" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids. Alternatively, an amino acid herein may be a modified amino acid residue and / or an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). Naturally occurring levorotatory (L-) amino acids are shown in Table 2. [Table 2]

[0072] Alternatively, the amino acid can be a modified amino acid residue (non-limiting examples are shown in Table 3) and / or can be an amino acid that is modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation). [Table 3-1] [Table 3-2]

[0073] Additionally, non-naturally occurring amino acids can be "unnatural" amino acids as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.

[0074] As used herein, the terms "viral vector," "vector," or "gene delivery vector" refer to a viral (e.g., AAV) particle that functions as a nucleic acid delivery vehicle and contains a vector genome (e.g., viral DNA [vDNA]) packaged within the virion. Alternatively, in some contexts, the term "vector" may be used to refer to the vector genome / vDNA alone.

[0075] As used herein, a "rAAV vector genome" or "rAAV genome" refers to an AAV genome (i.e., vDNA) containing one or more heterologous nucleic acid sequences. rAAV vectors generally require only the terminal repeats (TRs) in cis to generate virus. All other viral sequences are not essential and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). Typically, rAAV vector genomes will retain only one or more TR sequences to maximize the size of the transgene that can be efficiently packaged by the vector. Structural and nonstructural protein coding sequences may also be provided in trans (e.g., from a vector such as a plasmid or by stably integrating sequences into packaging cells). In embodiments of the invention, the rAAV vector genome comprises at least one TR sequence (e.g., an AAV TR sequence), and optionally two TRs (e.g., two AAV TRs), typically at the 5' and 3' ends of the vector genome, which will be adjacent to the heterologous nucleic acid, but need not be contiguous with the heterologous nucleic acid. The TRs may be the same as or different from one another.

[0076] The term "terminal repeat" or "TR" includes any viral or synthetic terminal repeat sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function, such as replication, viral packaging, integration, and / or proviral rescue). The TR can be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., an SV40 hairpin that serves as an SV40 origin of replication), can be used as the TR and can be further modified by truncation, substitution, deletion, insertion, and / or addition. Furthermore, the TR can be partially or completely synthetic, for example, a "double D sequence" as described in U.S. Patent No. 5,478,745 to Samulski et al.

[0077] An "AAV terminal repeat" or "AAV TR" can be from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or any other AAV now known or later discovered (see, e.g., Table 1). An AAV terminal repeat need not have the native terminal repeat sequence (e.g., the native AAV TR sequence may be altered by insertions, deletions, truncations, and / or missense mutations), so long as it mediates the desired function, such as replication, viral packaging, integration, and / or proviral rescue.

[0078] AAV vectors typically comprise a protein-based capsid and a nucleic acid encapsulated in the capsid. The nucleic acid can be, for example, a vector genome containing a transgene flanked by inverted terminal repeats. An AAV "capsid" is a roughly spherical protein shell containing individual "capsid proteins" or "subunits." An AAV capsid typically contains approximately 60 capsid protein subunits that are associated and arranged in a T=1 icosahedral symmetry. When an AAV vector is described herein as comprising an AAV capsid protein, it is understood that the AAV vector comprises a capsid, and that the capsid comprises one or more AAV capsid proteins (i.e., subunits). Also described herein are "viral particles" or "virus-like particles," which refer to capsids that do not contain any vector genome or nucleic acid, including a transgene.

[0079] The viral vectors of the present disclosure may further be "targeted" viral vectors (e.g., with directed tropism) and / or "hybrid" parvoviruses (i.e., where the viral TR and viral capsid are from different parvoviruses) as described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619.

[0080] The viral vector of the present disclosure may also be a double parvovirus particle as described in International Patent Publication WO01 / 92551 (the disclosure of which is incorporated herein by reference in its entirety). Thus, in some embodiments, a double-stranded (double-stranded) genome can be packaged in the viral capsid of the present invention. Furthermore, the viral capsid or genome element may contain other modifications, including insertions, deletions, and / or substitutions.

[0081] The term "self-complementary AAV" or "scAAV" refers to a recombinant AAV vector that forms dimeric inverted repeat DNA molecules that spontaneously anneal, resulting in faster and more robust transgene expression compared to conventional single-stranded (ss) AAV genomes. See, for example, McCarty, DM, et al., Gene Therapy 8, 1248-1254 (2001). Unlike conventional ssAAV, scAAV can avoid second-strand synthesis, a rate-limiting step in gene expression. Furthermore, double-stranded scAAV is less prone to DNA degradation after viral transduction, thereby increasing the copy number of stable episomes. Notably, scAAV can typically retain only a genome of approximately 2.4 kb, half the size of conventional AAV vectors. In some embodiments, the AAV vectors described herein are self-complementary AAV.

[0082] A "therapeutic polypeptide" or "therapeutic protein" is a polypeptide or protein that can alleviate, relieve, prevent, delay, and / or stabilize symptoms that occur as a result of the absence or deficiency of a protein in a cell or subject, and / or otherwise provide a benefit to the subject, e.g., an anti-cancer effect or improved graft survival.

[0083] The terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean to lessen the severity of, at least partially improve, or stabilize the subject's condition, and / or to achieve some alleviation, reduction, decrease, or stabilization of at least one clinical symptom, and / or to slow the progression of the disease or disorder.

[0084] The terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of, and / or reducing the severity of, a disease, disorder, and / or clinical symptom in a subject compared to what would occur in the absence of the methods of the present invention. Prevention can be complete prevention, e.g., the complete absence of a disease, disorder, and / or clinical symptom. Prevention can also be partial prevention, such that the appearance and / or severity of the onset of a disease, disorder, and / or clinical symptom in a subject is less than what would occur in the absence of the present invention.

[0085] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to both human and non-human animals. The term "non-human animal" of the present disclosure includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In some embodiments, the subject includes a human. In other embodiments, the subject includes a human in need of one or more gene therapy agents.

[0086] " Therapeutic effective amount " as used herein is the amount that is sufficient to bring about some improvement or benefit to the subject.In other words, " therapeutic effective amount " is the amount that will bring about some relief, alleviation, reduction or stabilization of at least one clinical symptom in the subject.It will be understood by those skilled in the art that the therapeutic effect does not need to be complete or curative, as long as some benefit is brought about to the subject.

[0087] A "prophylactically effective" amount, as used herein, is an amount that is sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, and / or to reduce the severity and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, compared to that which would occur in the absence of the method of the present invention. It will be understood by those skilled in the art that the level of prevention need not be complete, provided that some benefit is provided to the subject.

[0088] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. II. Cross-species compatibility AAV

[0089] Adeno-associated virus (AAV), a member of the parvovirus family, is a small, non-enveloped virus. Wild-type AAV is composed of an icosahedral protein capsid that encloses a single-stranded DNA genome. In wild-type AAV, inverted terminal repeats (ITRs) flank the coding nucleotide sequences (e.g., polynucleotides) for nonstructural proteins (encoded by the Rep gene) and structural proteins (encoded by the capsid gene or Cap gene). The Rep gene encodes nonstructural proteins that regulate functions, including replication, of the AAV genome. The Cap gene encodes the structural proteins VP1, VP2, and / or VP3, which assemble to form the capsid.

[0090] The present disclosure provides recombinant AAV capsid proteins (VP1, VP2, and / or VP3) that contain amino acid sequence modifications (e.g., substitutions) compared to the wild-type capsid proteins, as well as AAV capsids and AAV vectors that contain the modified AAV capsid proteins. The inventors have discovered that the modifications disclosed herein can confer one or more desirable properties to viral vectors that contain the modified AAV capsid protein variants herein, including, but not limited to, the ability to evade neutralizing antibodies and / or the ability to specifically and selectively target cells or tissues of interest. Thus, the present disclosure addresses some of the limitations associated with conventional AAV vectors.

[0091] In certain embodiments, the AAV vector herein can be engineered to contain one or more capsid protein variants. In some embodiments, the AAV vector herein can be a cross-species compatible vector, i.e., "ccAAV". In some embodiments, the AAV vector (e.g., ccAAV) can be engineered to contain at least one or more amino acid substitutions, and one or more substitutions can modify one or more antigenic sites on the AAV capsid protein. The modification of one or more antigenic sites can result in the inhibition of antibody binding to one or more antigenic sites and / or the inhibition of neutralization of the infectivity of virus particles containing the capsid protein variants herein.

[0092] Thus, in some embodiments herein, the present disclosure provides adeno-associated virus (AAV) capsid protein variants comprising one or more amino acid modifications (e.g., substitutions and / or deletions), wherein the one or more modifications modify one or more antigenic sites on the AAV capsid protein. In some embodiments, the modification of one or more antigenic sites can result in inhibition of antibody binding to one or more antigenic sites and / or inhibition of neutralization of infectivity of virus particles comprising the AAV capsid protein. In some embodiments, the modified antigenic site can prevent antibody binding, recognition, or neutralization of the AAV capsid. In some embodiments, the antibody can be IgG (including IgG1, IgG2a, IgG2b, and IgG3), IgM, IgE, or IgA. In some embodiments, the modified antigenic site can prevent binding, recognition, or neutralization of the AAV capsid by antibodies from a different animal species, where the animal is a human, dog, pig, cow, non-human primate, rodent, cat, or horse.

[0093] In some embodiments, modification of one or more antigenic sites may result in the AAV vectors herein (e.g., ccAAV) exhibiting tropism for one or more cell types, one or more tissue types, or any combination thereof. As used herein, "tropism" refers to the preferential entry of a virus into a particular cell or tissue, optionally followed by expression (e.g., transcription, and optionally translation) of a sequence carried by the viral genome in the cell, e.g., expression of a heterologous nucleic acid of interest in the case of a recombinant virus. In some embodiments, modification of one or more antigenic sites may result in the AAV vectors herein exhibiting tropism for one or more cell types and / or tissues throughout the subject's body. In some aspects, modification of one or more antigenic sites may result in the AAV vectors herein exhibiting tropism for brain tissue, lung tissue, skeletal muscle tissue, heart tissue, liver tissue, kidney tissue, and / or pancreatic tissue. In some embodiments, modification of one or more antigenic sites can result in the AAV vector herein exhibiting affinity for one or more brain cells, one or more lung cells, one or more skeletal muscle cells, one or more heart cells, one or more liver cells, one or more kidney cells, and / or one or more pancreatic cells. In some embodiments, modification of one or more antigenic sites can result in the AAV vector herein exhibiting affinity for the kidney.

[0094] In some embodiments, one or more amino acid modifications (e.g., substitutions and / or deletions) in the capsid protein variants herein may be present in one or more antigenic footprints identified by peptide epitope mapping and / or cryo-electron microscopy studies of AAV-antibody complexes containing the AAV capsid protein. In some embodiments, one or more antigenic sites herein that may undergo one or more amino acid modifications may be common antigenic motifs (CAMs) as described in WO2017 / 058892, the entire contents of which are incorporated herein by reference.

[0095] In some embodiments, one or more antigenic sites herein that are amenable to one or more amino acid modifications may be present in the variable regions (VRs) of the AAV capsid protein. An AAV capsid contains 60 copies (total) of three VPs (VP1, VP2, and VP3), which are encoded by the cap gene and have overlapping sequences. Each VP may contain an eight-stranded β-barrel motif (βB-βI) and / or an α-helix (αA) conserved in autonomous parvovirus capsids. Structural variable regions (VRs) may be present within the surface loops connecting the β-strands, which cluster together to create local variation on the capsid surface. In some embodiments, one or more amino acid modifications herein that modify one or more antigenic sites within an AAV capsid protein variant herein may be in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-VI II, VR-IX, or any combination thereof. In some embodiments, one or more antigenic sites may be present within the HI loop of an AAV capsid protein variant herein.

[0096] In some embodiments, an AAV vector (e.g., ccAAV) herein may comprise (i) an AAV capsid protein variant disclosed herein and (ii) a cargo nucleic acid encapsidated by the capsid protein. In accordance with these embodiments, an AAV vector (e.g., ccAAV) comprising an AAV capsid protein variant described herein may have the following phenotypes: evasion of neutralizing antibodies, improved or maintained transduction efficiency, selective affinity for one or more cell types and / or tissue types, and any combination thereof.

[0097] In some embodiments, the AAV vectors disclosed herein exhibit at least about 2-fold (e.g., about 4-fold, about 5-fold, about 7-fold, about 10-fold, about 15-fold, about 16-fold, about 17-fold, about 18-fold, about 20-fold, about 25-fold, or about 30-fold, including all values ​​and subranges therebetween) greater transduction efficiency in heart, skeletal muscle, kidney, and brain neurons compared to parental AAV9. In some embodiments, the AAV vectors disclosed herein exhibit higher transduction efficiency than parental AAV9 in some tissue types (e.g., heart, skeletal muscle, kidney, brain neurons, etc.), and similar or reduced transduction efficiency compared to parental AAV9 in some cell types (e.g., glial cells).

[0098] The present disclosure provides AAVcc.47, which demonstrated approximately 15- to 18-fold greater transduction in the heart, skeletal muscle, and kidney compared to AAV9. AAVcc.47 transduction in neurons was greater than AAV9 in the brain, while glial cell transduction remained relatively unchanged. AAVcc.81 and AAVcc.84 increased transduction in the heart and skeletal muscle by 4-fold, but no significant increase in liver transduction was observed with either ccAAV compared to AAV9. Glial cell transduction was significantly reduced with ccAAV compared to AAV9, while neuronal affinity was slightly increased. Increased transduction efficiency with ccAAV may allow for lower dosing regimens of therapeutic vectors.

[0099] In some embodiments, the AAV capsid protein variants disclosed herein may contain at least one or more amino acid substitutions, and between about 1 amino acid residue and about 50 amino acid residues (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) may be substituted from the amino acid residues that make up the amino acid sequence of a naturally occurring capsid protein. According to some embodiments herein, the AAV capsid protein variants herein may have about 7 amino acid residues substituted from the amino acid residues that make up the amino acid sequence of a naturally occurring capsid protein.

[0100] In some embodiments, the AAV capsid protein variants disclosed herein may have an amino acid sequence similar to a naturally occurring capsid protein of about 85% (e.g., about 85%, 90%, 95%, 99%, 100%). As used herein, "naturally occurring" or "wild-type" means existing in nature without human modification. In some embodiments, the naturally occurring capsid proteins herein may be derived from a single species. Non-limiting examples of species from which the naturally occurring capsid proteins herein may originate include those from common organisms such as humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, or non-human primates (e.g., monkeys, chimpanzees, baboons, gorillas), birds, reptiles, worms, and fish. In some embodiments, the species from which the naturally occurring capsid proteins herein may originate may be Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), Homo sapiens (human), and any combination thereof. In some embodiments, AAV capsid protein variants having at least one amino acid substitution as disclosed herein are identified by GenBank accession numbers: NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF063497, AF063498, AF06349 ... 28704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579, and any combination thereof.

[0101] Methods for determining sequence similarity or identity between two or more amino acid sequences are known in the art. Sequence similarity or identity can be determined using standard techniques, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2, 482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48,443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85, 2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the BestFit sequence program described by Devereux et al., Nucl. Acid Res. 12, 387-395 (1984), or by inspection. Another suitable algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program, obtained from Altschul et al., Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2 uses several search parameters, which are set to default values ​​as necessary. The parameters are dynamic values, established by the program itself depending on the composition of the specific sequence and the composition of the particular database in which the sequence of interest is searched, but values ​​can be adjusted to increase sensitivity.Furthermore, an additional useful algorithm is gapped BLAST, as reported by Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. In this disclosure, unless otherwise indicated, percent identity is calculated using the Basic Local Alignment Search Tool (BLAST), available online at blast.ncbi.nlm.nih.gov / Blast.cgi. Those skilled in the art will understand that other algorithms can be substituted as appropriate.

[0102] In some embodiments, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from any one of the following serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAV10, AAV11, AAV12, AAVrh32.22, bovine AAV, avian AAV, and / or any other AAV now known or later identified. In some embodiments, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from a serotype with known tropism for one or more desired cell and / or tissue types. In some embodiments, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in an AAV capsid protein from a serotype with known affinity for one or more desired human cell and / or tissue types.

[0103] According to these embodiments, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein from a serotype that has affinity for the CNS and / or PNS. AAV can successfully target several different tissue and cell types within the CNS and PNS, including but not limited to neurons, astrocytes, oligodendrocytes, microglia, Müller glia, Schwann cells, and satellite cells. In some examples, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein of any AAV serotype (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9) that has affinity for astrocytes. In some other examples, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein of any AAV serotype (e.g., AAV8, AAV9) that has affinity for oligodendrocytes. In some examples, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein of any AAV serotype (e.g., AAV2, AAV5, AAV6, AAV8, AAV9) that has affinity for microglia. In some other examples, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein of any AAV serotype (e.g., AAV1, AAV2, AAV4, AAV6, AAV8, AAV9) that has affinity for Müller glia.In some examples, the AAV capsid protein variants disclosed herein may have at least one amino acid substitution that can replace any seven amino acids in the AAV capsid protein of any AAV serotype (e.g., AAV1, AAV2, AAV5, AAV6, AAV7, AAV8, AAV9) that has affinity for Schwann cells / satellite glia.

[0104] In some embodiments, the AAV capsid protein variants herein or fragments thereof may have an amino acid sequence with about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similarity to a naturally occurring VP1 capsid protein or fragment thereof. In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 262-268 (VP1 numbering) of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 370-379 (VP1 numbering) of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 451 to 459 (VP1 numbering) of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV.In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 472-473 (VP1 numbering) of AAV1, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 493-500 (VP1 numbering) of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 528 to 534 (VP1 numbering) of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 547 to 552 (VP1 numbering) of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV1, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV.In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 588 to 597 (VP1 numbering) of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., two) of amino acid residues 709-710 (VP1 numbering) of AAV1, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 716 to 722 (VP1 numbering) of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV.

[0105] In some embodiments, the capsid protein variants herein comprise one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 262-268 (VP1 numbering) of AAV1, one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 370-379 (VP1 numbering) of AAV1, one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 451-459 (VP1 numbering) of AAV1. , 2, 3, 4, 5, 6, or 7) of amino acid residues 472-473 (VP1 numbering) of AAV1; one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 493-500 (VP1 numbering) of AAV1; one or more (e.g., 2, 3, 4, 5, 6, or 7) of amino acid residues 528-534 (VP1 numbering) of AAV1. at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 547 to 552 (VP1 numbering) of AAV1, at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues 588 to 597 (VP1 numbering) of AAV1, at one or more (e.g., 2) amino acid residues 709 to 710 (VP1 numbering) of AAV1, at one or more (e.g., 2) amino acid residues 716 to 720 (VP1 numbering) of AAV1, 722 (VP1 numbering), or any combination of these in any combination, or the amino acid substitutions at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV or avian AAV.

[0106] In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of the amino acid residues within variable loop region IV (VR4) on the capsid surface of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of the amino acid residues within variable loop region VIII (VR8) on the capsid surface of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein comprise one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) on the capsid surface of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. The AAV may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the capsid surface of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV.

[0107] In some embodiments, the capsid protein variants herein may have at least 90% (e.g., about 90%, 95%, 99%, 100%) sequence identity to the native sequence of the AAV9 capsid (SEQ ID NO: 1). In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) on the capsid surface of AAV9, in any combination. In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) of the amino acid residues within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) on the capsid surface of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein may include substitutions in one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) on the capsid surface of AAV9, in any combination.

[0108] In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the capsid surface of AAV9, in any combination. In some embodiments, capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR8 (586-SAQAQAQ-592 (VP1 numbering); SEQ ID NO: 39) on the capsid surface of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR8 (586-SAQAQAQ-592 (VP1 numbering; SEQ ID NO: 39)) on the capsid surface of AAV9, in any combination.

[0109] In some embodiments, the capsid protein variants herein may include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region IV (VR4) and one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within variable loop region VIII (VR8) on the capsid surface of AAV9, in any combination. In some embodiments, the capsid protein variants herein comprise one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) on the capsid surface of AAV1, in any combination, or the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. The capsid surface of AAV1 may comprise an amino acid substitution at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR8 (586-SAQAQAQ-592 (VP1 numbering; SEQ ID NO: 39)) of AAV1, in any combination, or at the equivalent amino acid residues in AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV, or avian AAV. In some embodiments, the capsid protein variants herein may include substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR4 (452-NGSGQNQ-458 (VP1 numbering; SEQ ID NO: 38)) on the capsid surface of AAV9, in any combination, and amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within VR8 (586-SAQAQAQ-592 (VP1 numbering; SEQ ID NO: 39)) on the capsid surface of AAV9, in any combination.

[0110] In some embodiments, a capsid protein variant herein can have an amino acid sequence having about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similarity to a naturally occurring VP2 capsid protein or fragment thereof from any one of the serotypes described herein. In some embodiments, a capsid protein variant herein can include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within a naturally occurring VP2 capsid protein or fragment thereof from any one of the serotypes described herein, in any combination.

[0111] In some embodiments, a capsid protein variant herein can have an amino acid sequence having about 85% (e.g., about 85%, 90%, 95%, 99%, 100%) similarity to a naturally occurring VP3 capsid protein or fragment thereof from any one of the serotypes described herein. In some embodiments, a capsid protein variant herein can include amino acid substitutions at one or more (e.g., 2, 3, 4, 5, 6, or 7) amino acid residues within a naturally occurring VP3 capsid protein or fragment thereof from any one of the serotypes described herein, in any combination.

[0112] In some embodiments, the AAV vectors (e.g., ccAAV) herein may comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein. In accordance with these embodiments, the AAV vectors (e.g., ccAAV) herein may comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by the capsid protein, wherein the capsid protein is a native AAV9 capsid protein, comprising the sequence X at amino acids 452 to 458 (VP1 numbering) of (SEQ ID NO: 1). 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7(SEQ ID NO: 40), where the peptide is not present in the native AAV9 capsid protein sequence. In some embodiments, the AAV vectors herein comprise a peptide having the sequence X at amino acids 452 to 458 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1). 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40), wherein X 1 can be any amino acid other than N, and X 2 can be any amino acid other than G, and X 3 can be any amino acid other than S, and X 4 can be any amino acid other than G, and X 5 can be any amino acid other than Q, and X 6 can be any amino acid other than N, and / or X 7 can be any amino acid other than Q.

[0113] In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to EGGTVHA (SEQ ID NO: 20). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to FYGTDSA (SEQ ID NO: 21). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to HGQSASR (SEQ ID NO: 22). In some embodiments, capsid protein variants herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to DTPTNQA (SEQ ID NO: 23). In some embodiments, capsid protein variants herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to ITRQAYQ (SEQ ID NO: 24). In some embodiments, capsid protein variants herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to RMFKSNQ (SEQ ID NO: 25). In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to GVSLGGG (SEQ ID NO: 26).In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of a native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to KHFLQGE (SEQ ID NO: 27). In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of a native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to MGRERAG (SEQ ID NO: 28).

[0114] In some embodiments, the capsid protein variants herein may share at least about 85% (e.g., about 85%, 90%, 95%, 99%, or 100%) amino acid sequence similarity with any one of the sequences set forth in SEQ ID NOs: 2-10. According to some embodiments herein, the capsid protein variants herein comprise any one of the sequences set forth in SEQ ID NOs: 2-10. The amino acid sequences of the native AAV9 capsid protein, (SEQ ID NO: 1) and SEQ ID NOs: 2-10, are provided in Table 4 below.

[0115] In some embodiments, an AAV vector (e.g., ccAAV) herein may comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by a capsid protein, wherein the capsid protein comprises the sequence X at amino acids 586 to 592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1). 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125), wherein the peptide is not present in the native AAV9 capsid protein sequence. In some embodiments, the AAV vectors herein comprise a peptide having the sequence X at amino acids 586 to 592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1).1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125), wherein X 1 can be any amino acid other than S, and X 2 can be any amino acid other than A, and X 3 can be any amino acid other than Q, and X 4 can be any amino acid other than A, and X 5 can be any amino acid other than Q, and X 6 can be any amino acid other than A, and / or X 7 can be any amino acid other than Q.

[0116] In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to LNSSVPS (SEQ ID NO: 29). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to YMDHQVS (SEQ ID NO: 30). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of a native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to TSDSLVS (SEQ ID NO: 31). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to NAVGALS (SEQ ID NO: 32). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to MPISHHE (SEQ ID NO: 33). In some embodiments, a capsid protein variant herein may comprise a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein (SEQ ID NO: 1) may be substituted with amino acids corresponding to DSGARGA (SEQ ID NO: 34). In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to NVALALG (SEQ ID NO: 35).In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to GALRMGM (SEQ ID NO: 36). In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to LSGEGAV (SEQ ID NO: 37).

[0117] In some embodiments, the capsid protein variants herein may share at least about 85% (e.g., about 85%, 90%, 95%, 99%, or 100%) amino acid sequence similarity with any one of the sequences set forth in SEQ ID NOs: 11-19. According to some embodiments herein, the capsid protein variants herein comprise any one of the sequences set forth in SEQ ID NOs: 11-19. The amino acid sequences of the native AAV9 capsid protein, (SEQ ID NO: 1) and SEQ ID NOs: 11-19, are provided in Table 4 below.

[0118] In some embodiments, an AAV vector (e.g., ccAAV) herein may comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by a capsid protein, wherein the capsid protein comprises the sequence X at amino acids 452 to 458 (VP1 numbering) and at amino acids 586 to 592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1). 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40), which peptide is not present in the native AAV9 capsid protein sequence.

[0119] In some embodiments, an AAV vector (e.g., ccAAV) herein may comprise (i) an AAV9 capsid protein variant and (ii) a cargo nucleic acid encapsidated by a capsid protein, wherein the capsid protein comprises the sequence X at amino acids 586-592 (VP1 numbering) of a native AAV9 capsid protein, (SEQ ID NO: 1), and the sequence X at amino acids 586-592 (VP1 numbering). 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125), which peptide is not present in the native AAV9 capsid protein sequence.

[0120] In some aspects, the AAV vectors herein comprise: (1) a native AAV9 capsid protein, (SEQ ID NO: 1), at amino acids 452 to 458 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), supplemented with the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 40) (wherein, X 1 can be any amino acid other than N, and X 2 can be any amino acid other than G, and X 3 can be any amino acid other than S, and X 4 can be any amino acid other than G, and X 5 can be any amino acid other than Q, and X 6 can be any amino acid other than N, and / or X 7 can be any amino acid other than Q, and (2) amino acids 586 to 592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), are supplemented with the sequence X 1 -X 2 -X 3 -X 4 -X 5 -X 6 -X 7 (SEQ ID NO: 125) (wherein, X 1can be any amino acid other than S, and X 2 can be any amino acid other than A, and X 3 can be any amino acid other than Q, and X 4 can be any amino acid other than A, and X 5 can be any amino acid other than Q, and X 6 can be any amino acid other than A, and / or X 7 The present invention may include AAV9 capsid protein variants comprising a peptide having

[0121] In some embodiments, a capsid protein variant herein may include a peptide in which amino acids corresponding to amino acids 452-458 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to any one of SEQ ID NOs: 20-28, and amino acids corresponding to amino acids 586-592 (VP1 numbering) of the native AAV9 capsid protein, (SEQ ID NO: 1), may be substituted with amino acids corresponding to any one of SEQ ID NOs: 29-37.

[0122] In some embodiments, the capsid protein variants herein may share at least about 85% (e.g., about 85%, 90%, 95%, 99%, or 100%) amino acid sequence similarity with any one of the sequences set forth in SEQ ID NOs: 46-123. According to some embodiments herein, the capsid protein variants herein comprise any one of the sequences set forth in SEQ ID NOs: 46-123. The amino acid sequences of the native AAV9 capsid protein, (SEQ ID NO: 1) and SEQ ID NOs: 46-123, are provided in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3]

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

Table 4-16

Table 4-17

Table 4-18

Table 4-19

Table 4-20

[0123] X 1 From X 7 In embodiments where none of the amino acid residues identified as up to 452N have been substituted, the amino acid residue at the unsubstituted position may be the wild-type amino acid residue of a reference amino acid sequence (e.g., AAV9 (SEQ ID NO: 1)). In some embodiments, the capsid protein variants herein may have amino acid substitutions at residues 452N, 453G, 454S, 455G, 456Q, 457N, and / or 458Q of SEQ ID NO: 1 (AAV9 capsid protein; VP1 numbering) in any combination. In some embodiments, the capsid protein variants herein may have amino acid substitutions at residues 586S, 587A, 588Q, 589A, 590Q, 591A, and / or 592Q of SEQ ID NO: 1 (AAV9 capsid protein; VP1 numbering) in any combination.

[0124] In some embodiments, the capsid protein variants of the present disclosure can be produced by modifying the capsid protein of any currently known or later discovered AAV capsid protein. Furthermore, the AAV capsid protein to be modified in accordance with the present disclosure can be, but is not limited to, a naturally occurring AAV capsid protein (e.g., AAV2, AAV3a or 3b, AAV4, AAV5, AAV8, AAV9, AAV10, or AAV11 capsid protein, or any of the AAVs listed in Table 1). It will be understood by those skilled in the art that various manipulations of AAV capsid proteins are known in the art, and that the present invention is not limited to modification of naturally occurring AAV capsid proteins. For example, the capsid protein to be modified may already have one or more alterations compared to a naturally occurring AAV (e.g., derived from a naturally occurring AAV capsid protein, such as AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or any other AAV now known or later discovered). Such AAV capsid proteins are also within the scope of the present disclosure.

[0125] Some aspects of the present disclosure provide a viral capsid that can have one or more of any of the capsid protein variants disclosed herein.In some embodiments, the viral capsid herein can be a parvovirus capsid, and the parvovirus capsid can further be an autonomous parvovirus capsid or a dependent virus capsid.Optionally, the viral capsid herein can be an AAV capsid.In some embodiments, the AAV capsid of the present disclosure can be AAV1, AAV2, AAV3a, AAV3b, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAVrh32.33, bovine AAV capsid, avian AAV capsid, and / or any other AAV currently known or later identified.

[0126] In some embodiments, the modified viral capsids herein can be used as capsid vehicles. In some embodiments, molecules can be packaged by the modified viral capsids herein and transferred into cells, and the molecules can include heterologous DNA, RNA, polypeptides, small organic molecules, metals, or combinations thereof. A heterologous molecule is defined herein as one that is not naturally found during AAV infection, e.g., not encoded by the wild-type AAV genome. Furthermore, therapeutically useful molecules for use herein can be associated with the outside of the chimeric viral capsid for transfer of the molecules into one or more host target cells. Such associated molecules can include DNA, RNA, small organic molecules, metals, carbohydrates, lipids, and / or polypeptides. In some embodiments, therapeutically useful molecules herein can be covalently linked (i.e., conjugated or chemically coupled) to capsid proteins. Methods for covalently linking molecules are known to those skilled in the art.

[0127] In some embodiments, the modified viral capsids herein can be used to raise antibodies against the capsid protein variants disclosed herein. As a further alternative, a foreign amino acid sequence can be inserted into the modified viral capsid for antigen presentation to cells, for example, for administration to a subject to raise an immune response against the foreign amino acid sequence.

[0128] In some embodiments, the modified viral capsid herein may be a targeted viral capsid that includes a targeting sequence (e.g., substituted or inserted into the viral capsid) that can direct the viral capsid to interact with a cell surface molecule present on a desired target tissue (see, e.g., International Patent Publication WO 00 / 28004 and Hauck et al., (2003) J. Virology 77:2768-2774; Shi et al., Human Gene Therapy 17:353-361 (2006) [describing the insertion of the integrin receptor binding motif RGD at positions 520 and / or 584 of the AAV capsid subunit]; and U.S. Patent No. 7,314,912 [describing the insertion of a P1 peptide containing an RGD motif after amino acids 447, 534, 573, and 587 of the AAV2 capsid subunit]). Other positions within the AAV capsid subunit that tolerate insertions are known in the art (eg, positions 449 and 588 described by Grifman et al., Molecular Therapy 3:964-975 (2001)).

[0129] As one example, the viral capsids of the present disclosure may have relatively inefficient affinity for certain target tissues of interest (e.g., liver, skeletal muscle, heart, diaphragm muscle, kidney, brain, stomach, intestine, skin, endothelial cells, and / or lung). Targeting sequences can be advantageously incorporated into these low-transduction vectors to confer desired affinity, and optionally selective affinity for specific tissues, to the viral capsid. AAV capsid proteins, capsids, and vectors containing targeting sequences are described, for example, in International Patent Publication WO 00 / 28004. As another example, one or more non-naturally occurring amino acids, such as those described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006), can be incorporated into the orthogonal site of an AAV capsid subunit of the present invention as a means of redirecting the low-transduction vector to a desired target tissue. These unnatural amino acids can be advantageously used to chemically link molecules of interest to AAV capsid proteins, including, but not limited to, glycans (mannose—dendritic cell targeting); RGD, bombesin, or neuropeptides for targeted delivery to specific cancer cell types; RNA aptamers or peptides selected from phage display targeted to specific cell surface receptors, such as growth factor receptors, integrins, etc. Methods for chemically modifying amino acids are known in the art (see, for example, Greg T. Hermanson, Bioconjugate Techniques, 1999). st edition, Academic Press, 1996).

[0130] In some embodiments, the targeting sequence can be a viral capsid sequence (e.g., an autonomous parvovirus capsid sequence, an AAV capsid sequence, or any other viral capsid sequence) that directs infection to a specific cell type.

[0131] As another non-limiting example, a heparin-binding domain (e.g., a respiratory syncytial virus heparin-binding domain) can be inserted or substituted into a capsid subunit that does not normally bind HS receptors (e.g., AAV9) to confer heparin binding to the resulting mutant. In another non-limiting example, the globoside receptor-binding domain of the B19 capsid can be substituted into an AAV capsid protein of the invention to target viral capsids or viral vectors containing same to erythroid cells.

[0132] In some embodiments, the exogenous targeting sequence for use herein may be any amino acid sequence encoding a peptide that alters the affinity of a viral capsid or viral vector, including a modified AAV capsid protein. In some embodiments, the targeting peptide or protein may be naturally occurring or alternatively, completely or partially synthetic. In some examples, the targeting sequence may include ligands and other peptides that bind to cell surface receptors and glycoproteins, such as RGD peptide sequences, bradykinin, hormones, peptide growth factors (e.g., epidermal growth factor, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, insulin-like growth factor I and II, etc.), cytokines, melanocyte-stimulating hormones (e.g., α, β, or γ), neuropeptides, and endorphins, and fragments thereof that retain the ability to target cells to their cognate receptors. Other illustrative peptides and proteins include substance P, keratinocyte growth factor, neuropeptide Y, gastrin-releasing peptide, interleukin 2, hen egg white lysozyme, erythropoietin, gonadoliberin, corticostatin, β-endorphin, leucine enkephalin, limorphin, α-neo-enkephalin, angiotensin, pneumadin, vasoactive intestinal peptide, neurotensin, motilin, and fragments thereof as described above. Furthermore, as a further alternative, a binding domain from a toxin (e.g., tetanus toxin or a snake venom, such as α-bungarotoxin) can be substituted into the capsid protein as a targeting sequence. In some other embodiments, AAV capsid proteins can be modified by incorporation into the AAV capsid protein by substitution of "non-classical" import / export signal peptides (e.g., fibroblast growth factor-1 and -2, interleukin-1, HIV-1 Tat protein, herpesvirus VP22 protein, etc.) as described by Cleves (Current Biology 7:R318 (1997)).Also encompassed are peptide motifs that direct uptake by specific cells, for example, the FVFLP (SEQ ID NO: 41) peptide motif induces uptake by liver cells. In some embodiments, targeting sequences for use herein may be peptides that can be used for chemical coupling to another molecule that targets entry into a cell (e.g., may contain arginine and / or lysine residues that can be chemically coupled via their R groups).

[0133] In some embodiments, the capsid protein variants, viral capsids and / or AAV vectors (e.g., ccAVV) disclosed herein can have equivalent or improved transduction efficiency compared to the transduction efficiency of the AAV serotype from which the capsid protein variants, viral capsids and / or vectors are derived. In some embodiments, the capsid protein variants, viral capsids and / or vectors (e.g., ccAVV) disclosed herein can have reduced transduction efficiency compared to the transduction efficiency of the AAV serotype from which the capsid protein variants, viral capsids and / or vectors are derived. In some embodiments, the capsid protein variants, viral capsids and / or vectors (e.g., ccAVV) disclosed herein can have equivalent or improved affinity compared to the affinity of the AAV serotype from which the capsid protein variants, viral capsids and / or vectors are derived. In some embodiments, the capsid protein variants, viral capsids and / or vectors (e.g., ccAVV) disclosed herein can have altered or different tropism compared to the tropism of the AAV serotype from which the capsid protein variants, viral capsids and / or vectors are derived. In some embodiments, the capsid protein variants, viral capsids and / or vectors (e.g., ccAVV) disclosed herein have, or can be engineered to have, tropism for brain tissue. In some embodiments, the capsid protein variants, viral capsids and / or AAV vectors (e.g., ccAVV) disclosed herein can generate an attenuated immunological response compared to the immunological response of the AAV serotype from which the capsid protein variants, viral capsids and / or vectors are derived.In some embodiments, a subject may be administered multiple doses (e.g., about 2, about 3, about 4, about 5, about 10, about 15, about 20, about 40 times, or as many times as necessary to observe one or more desired responses) of a capsid protein variant, viral capsid and / or AAV vector (e.g., ccAVV) disclosed herein relative to the number of doses that may be administered using the AAV serotype from which the capsid protein variant, viral capsid and / or vector is derived. (A) Capsid and ccAAV manipulation

[0134] In some embodiments, rational engineering and / or mutation methods can be used to identify capsid protein variants of the AAV vectors (e.g., ccAAV) disclosed herein. In some embodiments, the methods herein can be used to produce AAV vectors that escape neutralizing antibodies. In some embodiments, the methods herein can be used to produce AAV vectors with improved gene transfer efficiency. In some embodiments, the methods herein can be used to produce AAV vectors with improved gene transfer efficiency in more than one mammalian species. In some embodiments, the methods herein can be used to produce AAV vectors that specifically target cells or tissues of interest (e.g., kidney cells).

[0135] In some embodiments, the recombinant AAVs described herein have improved gene transfer efficiency in one or more mammalian species compared to recombinant AAVs having otherwise identical capsid proteins except for lacking one or more amino acid substitutions. In some embodiments, the improved gene transfer efficiency exists / occurs in another of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), or Homo sapiens (human). In some embodiments, the improved gene transfer efficiency occurs in one or more of the following cell types or tissues: spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver. In some embodiments, the improved gene transfer efficiency occurs in kidney cells or kidney tissue.

[0136] Aspects of the present disclosure provide methods for producing AAV vectors as disclosed herein. In some embodiments, the method may include one or more of the following steps: a) identifying contact amino acid residues that form a three-dimensional antigen footprint on the AAV capsid protein; b) generating a library of AAV capsid proteins containing amino acid substitutions of the contact amino acid residues identified in (a); c) producing AAV particles containing capsid proteins from the library of AAV capsid proteins in (b); d) contacting the AAV particles in (c) with cells under conditions that allow infection and replication; e) selecting AAV particles that have completed at least one infection cycle and can replicate to a titer similar to that of control AAV particles; f) contacting the AAV particles selected in (e) with a neutralizing antibody and cells under conditions that allow infection and replication; and g) selecting AAV particles that are not neutralized by the neutralizing antibody in (f). Non-limiting examples of methods for identifying contact amino acid residues include peptide epitope mapping and / or cryo-electron microscopy. Those skilled in the art will appreciate that there are a constantly evolving variety of methods and protocols (e.g., rational design, barcoding, directed evolution, computational discovery) that can be used to generate libraries of AAV capsid proteins. Any method of generating libraries of AAV capsid proteins known in the art or to be discovered that is suitable for use herein can be used and / or optimized for use in accordance with the methods disclosed herein.

[0137] In some embodiments, generating a library of AAV capsid proteins containing amino acid substitutions of contact amino acid residues identified in the AAV capsid protein can result in a parental AAV capsid protein library. In some embodiments, the method of producing a ccAAV vector herein can include administering the parental AAV capsid protein library to a mammal. In some embodiments, administering the parental AAV capsid protein library to a mammal can be systemic administration to the mammal. In some embodiments, the parental AAV capsid protein library can be administered to a mammal having a Mus Musculus (mouse) species, a Sus scrofa (pig) species, a Canis Familiaris (dog) species, a non-human primate (Macaca) species, or a Homo sapiens (human) species. In some embodiments, capsid proteins can be enriched by collecting cells and / or tissues from the mammal after administration of the parental AAV capsid protein library. In some embodiments, capsid proteins can be enriched by collecting them from cells and / or tissues from a mammal after administration of the parental AAV capsid protein library, including cells and / or tissues from the spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, capsid proteins can be collected from the mammal about 1 day to about 1 month (e.g., about 1 day, 5 days, 1 week, 2 weeks, 3 weeks, 1 month) after administration of the parental AAV capsid protein library. In some embodiments, capsid proteins collected from the mammal after administration of the parental AAV capsid protein library can be used to generate another AAV capsid protein library, referred to as an evolved AAV capsid protein library.

[0138] In some embodiments, the evolved AAV capsid protein library can be administered to a mammal having a Mus Musculus (mouse) species, Sus scrofa (pig) species, Canis Familiaris (dog) species, non-human primate (Macaca) species, or Homo sapiens (human) species, provided that these species are not the same as the species to which the parent AAV capsid protein library was administered. In some embodiments, capsid proteins can be enriched by collecting them from cells and / or tissues from the mammal after administration of the evolved AAV capsid protein library. In some embodiments, capsid proteins can be enriched by collecting them from cells and / or tissues from the mammal after administration of the evolved AAV capsid protein library, including spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. In some embodiments, capsid proteins can be collected and identified from the mammal after administration of the evolved AAV capsid protein library. In some embodiments, capsid proteins can be collected and identified from the mammal about 1 day to about 1 month (e.g., about 1 day, 5 days, 1 week, 2 weeks, 3 weeks, 1 month) after administration of the evolved AAV capsid protein library. In some embodiments, the identified capsid proteins collected from the mammal after administration of the evolved AAV capsid protein library can be used to generate an additional, second evolved AAV capsid protein library. In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal having the species Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primate (Macaca), or Homo sapiens (human), provided that these species are not the same as the species to which the first evolved AAV capsid protein library was administered.In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal having the species Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primate (Macaca), or Homo sapiens (human), provided that the species is not the same as the species to which the first evolved AAV capsid protein library was administered, and provided that the species is the same as the species to which the parental AAV capsid protein library was administered. In some embodiments, the second evolved AAV capsid protein library can be administered to a mammal having the species Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primate (Macaca), or Homo sapiens (human), provided that these species are not the same as the species to which the first evolved AAV capsid protein library was administered and that these species are not the same as the species to which the parental AAV capsid protein library was administered.

[0139] In some embodiments, each generation of an evolved library can be referred to as a "cycle" of coevolving an AAV capsid protein library. In some embodiments, the methods herein for coevolving an AAV capsid protein library can include about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) cycles. In some embodiments, each cycle as disclosed herein can be performed in a different species than the preceding cycle. In some examples, the methods herein for coevolving an AAV capsid protein library can include one cycle in mice, a second cycle in pigs, a third cycle in mice, a fourth cycle in pigs, etc. In some examples, the methods herein for coevolving an AAV capsid protein library can include one cycle in mice, a second cycle in non-human primates, a third cycle in mice, a fourth cycle in non-human primates, etc. In some examples, the methods herein for co-evolving an AAV capsid protein library may include one cycle in a mouse, a second cycle in a pig, a third cycle in a non-human primate, a fourth cycle in a mouse, etc. In some examples, the methods herein for co-evolving an AAV capsid protein library may include one cycle in a pig, a second cycle in a mouse, a third cycle in a non-human primate (e.g., a monkey), etc.

[0140] In some embodiments, a method for evolving a novel adeno-associated virus strain comprises passaging an AAV library across multiple mammalian species, wherein the AAV library comprises a plurality of recombinant AAV vectors, each recombinant AAV vector comprising a capsid protein variant comprising one or more amino acid mutations relative to a wild-type AAV capsid protein. In some embodiments, each recombinant AAV vector in the AAV library comprises one or more amino acid mutations relative to a wild-type AAV9 capsid protein (SEQ ID NO: 1). In some embodiments, the one or more amino acid mutations are in a region corresponding to amino acids 452-458 of SEQ ID NO: 1, or 586-592 of SEQ ID NO: 1, or mutations are found in both the regions corresponding to amino acids 452-458 and 586-592 of SEQ ID NO: 1.

[0141] In some embodiments, a method for evolving a novel AAV strain includes administering a first AAV library to a first mammalian species. AAVs from the first AAV library present in one or more target tissues of the first mammalian species can then be sequenced and used to generate a second AAV library. The second AAV library can then be administered to a second mammalian species, where the first and second mammalian species are different. AAVs from the second AAV library present in one or more target tissues of the second mammalian species can then be sequenced. In some embodiments, the first and second mammalian species are each independently selected from the group consisting of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), and Homo sapiens (human). These steps can then be repeated with a third, fourth, fifth, sixth, etc. species. In some embodiments, the one or more target tissues of the first mammalian species, the second mammalian species (or any subsequent species) are selected from spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. (B) AAV vector

[0142] In certain embodiments, the present disclosure provides an AAV vector comprising one or more of the capsid protein variants disclosed herein. As used herein, "vector" refers to any molecule or moiety that transports, transduces, or otherwise acts as a carrier for heterologous molecules. A "viral vector" is a vector that contains one or more polynucleotide regions encoding or containing a desired payload molecule, such as a transgene, a polynucleotide encoding a polypeptide or multiple polypeptides, or a regulatory nucleic acid. The viral vector of the present invention can be produced recombinantly using methods known in the art. Such techniques are fully described in the literature, for example, in Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed. 1984); Methods in Molecular Biology, Humana Press; and Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1989) Academic Press.

[0143] In some embodiments, the AAV viral particles disclosed herein can have a vector genome for expressing one or more of the capsid protein variants disclosed herein. In some embodiments, the vector genome of an AAV vector can be derived from the wild-type genome of a virus, such as AAV, by using molecular methods to remove the wild-type genome from the virus (e.g., AAV) and replace it with a non-native nucleic acid, such as a heterologous polynucleotide sequence (e.g., a coding sequence for a transgene of interest). Typically, in the case of an AAV vector, one or both inverted terminal repeat (ITR) sequences of the wild-type AAV genome are retained in the AAV vector, while other portions of the wild-type viral genome between the retained ITRs are replaced with non-native sequences, such as heterologous polynucleotide sequences. The vector genome disclosed herein can include backbone elements derived from the AAV genome, a coding sequence for a capsid protein variant disclosed herein, and a suitable promoter operably linked to the coding sequence. In some examples, the vector genome disclosed herein can further include regulatory sequences that regulate the expression and / or secretion of the encoded protein. Examples include, but are not limited to, an enhancer, a polyadenylation signal site, an internal ribosome entry site (IRES), a sequence encoding a protein transduction domain (PTD), a microRNA-target site, or a combination thereof.

[0144] In some instances, the vector genome described herein can be single-stranded.In other instances, the vector genome disclosed herein can be double-stranded.For example, the vector genome described herein can be the self-complementary AAV vector genome, which can comprise double-stranded portions therein.

[0145] (1) AAV-backbone element

[0146] In some embodiments, the vector genome disclosed herein can have one or more AAV genome-derived backbone elements, which refer to the minimum AAV genome elements required for the biological activity of AAV vector.For example, the AAV genome-derived backbone elements can include the packaging site for assembling the vector into an AAV virus particle, one or more of the capsid protein variants disclosed herein, and the elements required for the replication of the vector and / or the expression of the transgene coding sequence contained therein in host cells.

[0147] In some examples, the vector genome backbone disclosed herein may comprise at least one inverted terminal repeat (ITR) sequence. In some examples, the vector genome backbone herein may comprise two ITR sequences. In some examples, one ITR sequence may be 5' of the polynucleotide sequence encoding the transgene. In some examples, one ITR sequence may be 3' of the polynucleotide sequence encoding the transgene. In some examples, the polynucleotide sequence encoding the transgene herein may be flanked on both sides by ITR sequences. Thus, in some embodiments, the vector genome comprises a transgene located between a first ITR and a second ITR.

[0148] In some embodiments, the vector genome herein may contain sequences or components derived from at least one distinct AAV serotype. In some examples, the AAV vector genome backbone disclosed herein may contain at least an ITR sequence from one distinct AAV serotype. In some examples, the AAV vector genome backbone disclosed herein may contain at least an ITR sequence from one distinct human AAV serotype. Such human AAV may be derived from any known serotype, for example, any one of serotypes 1 to 11. In some examples, the AAV serotypes used herein have tropism for the central nervous system (CNS), heart tissue, skeletal muscle, and / or liver tissue. In some examples, the AAV vector genome backbone disclosed herein may have an ITR sequence from serotype AAV9.

[0149] In some embodiments, the AAV vector herein may be a pseudotyped AAV vector (i.e., comprise sequences or components derived from at least two distinct AAV serotypes). In some embodiments, the pseudotyped AAV vector herein may comprise an AAV genome backbone derived from one AAV serotype and capsid proteins derived at least in part from a distinct AAV serotype. In some examples, the pseudotyped AAV vector herein may have an AAV2 vector genome backbone and capsid proteins derived from an AAV serotype (e.g., AAV1, AAV2, AAV4, AAV5, AAV8, or AAV9) that has tropism for cardiac tissue.

[0150] To analyze the success of viral vector-mediated gene transfer, it can be important to be able to monitor both the distribution of the vector and the effectiveness of vector-mediated gene expression. This can be achieved by subcloning a reporter gene into the vector genome backbone. In some examples, the AAV vector genome backbone disclosed herein can contain a reporter gene. Several reporter genes are commonly used for this purpose, including, but not limited to, fluorescent proteins of various colors (including green fluorescent protein (GFP) and red fluorescent protein (RFP)), E. coli β-galactosidase (LacZ), and various forms of luciferase (Luc). In some examples, the AAV vector backbone disclosed herein can contain GFP.

[0151] The vector constructs disclosed herein can be prepared using known techniques (see, for example, Current Protocols in Molecular Biology, Ausubel, F. et al., eds., Wiley and Sons, New York 1995). The fragment length can be selected so that the recombinant genome does not exceed the packaging capacity of the AAV particle. If necessary, a "stuffer" DNA sequence can be added to the construct to maintain a standard AAV genome size for comparison. Such fragments can be derived from such non-viral sources, such as lacZ, or other genes known and available to those skilled in the art.

[0152] (2) Self-complementary AAV viral vector

[0153] In some embodiments, the AAV vector disclosed herein can be a self-complementary AAV (scAAV) vector. Self-complementary AAV (scAAV) vectors contain complementary sequences, which can spontaneously anneal (fold to form a double-stranded genome) upon entry into infected cells, thus eliminating the need to use the cell's DNA replication mechanism to convert single-stranded DNA vectors. AAVs herein that have a self-complementary genome can rapidly form double-stranded DNA molecules through their partial complementary sequences (for example, the complementary coding and non-coding strands of the transgene coding sequence).

[0154] In some embodiments, the scAAV viral vectors disclosed herein may comprise a first heterologous polynucleotide sequence and a second heterologous polynucleotide sequence capable of forming intrastrand base pairs. In some examples, the first heterologous polynucleotide sequence and the second heterologous polynucleotide sequence are linked by a sequence that promotes intrastrand base pairing, for example, to form a hairpin DNA structure. In some examples, the dimeric structure of the scAAV vector upon entry into a cell may be stabilized by mutation or deletion of one of the two terminal resolution sites (trs). Because the trs is a Rep binding site contained within each ITR, mutation or deletion of such a trs can prevent cleavage of the dimeric structure of the scAAV vector by the AAV Rep protein to form monomers. In some embodiments, the scAAV viral vectors disclosed herein may comprise a truncated 5' inverted terminal repeat (ITR), a truncated 3' ITR, or both. In some examples, the scAAV vectors disclosed herein may include a truncated 3' ITR in which the D region or a portion thereof (e.g., the terminal resolution sequence therein) may be deleted. Such a truncated 3' ITR may be located between the first and second heterologous polynucleotide sequences described above. (3) Promoter

[0155] In some embodiments, the AAV vectors disclosed herein contain additional elements necessary for expression, such as at least one suitable promoter that controls the expression of the transgene coding sequence. Such promoters can be universal, tissue-specific, strong, weak, regulated, chimeric, etc., to enable efficient and suitable production of proteins in infected tissues. The promoter can be homologous or heterologous to the encoded protein and includes cellular, viral, fungal, plant, or synthetic promoters. The most preferred promoters for use herein can be functional in human cells. Non-limiting examples of universal promoters include viral promoters, particularly CMV promoters, RSV promoters, SV40 promoters, etc., and cellular promoters, such as the PGK (phosphoglycerate kinase) promoter. In some embodiments, the viral promoter herein can be a CMV promoter, an SV40 promoter, or any combination thereof.

[0156] In some embodiments, the AAV vector disclosed herein may contain additional elements required for expression, such as at least one suitable promoter that controls the expression of transgene coding sequence after infection of suitable cells.Promoters suitable for use herein include AAV promoters, such as cytomegalovirus (CMV) promoters or chicken beta-actin / cytomegalovirus hybrid promoters (CAG), as well as endothelial cell-specific promoters, such as VE-cadherin promoters, and steroid promoters and metallothionein promoters.In some embodiments, the promoter used in the vector disclosed herein may be a CAG promoter.

[0157] In some embodiments, the transgene coding sequence of the present invention includes a tissue-specific promoter operably linked to the transgene coding sequence to be expressed. This can further increase the specificity of the vector according to the present disclosure for a tissue (e.g., brain, heart, muscle, liver). In some examples, the vectors disclosed herein may have a tissue-specific promoter whose activity in a specific tissue is at least about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold higher than in a non-specific tissue. In some examples, the tissue-specific promoter herein is a human tissue-specific promoter. In some examples, the expression cassette may also include an enhancer element to increase the expression level of the exogenous protein to be expressed. Furthermore, the expression cassette may further include a polyadenylation sequence, such as an SV40 polyadenylation sequence or a bovine growth hormone polyadenylation sequence. (4) Other regulatory elements for gene expression

[0158] In some embodiments, the AAV vectors disclosed herein may contain one or more conventional control elements operably linked to the transgene coding sequence to enable its transcription, translation, and / or expression in cells transfected with the plasmid vector or infected with the virus produced by the present invention. As used herein, "operably linked" sequences may include both expression control sequences contiguous with the transgene coding sequence and expression control sequences acting in trans or at a distance to control the transgene coding sequence. Expression control sequences may further include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (e.g., Kozak consensus sequences); sequences that improve protein stability; and, if desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including native, constitutive, inducible, and / or tissue-specific promoters, are known in the art and may be utilized herein.

[0159] In some embodiments, the AAV vectors disclosed herein may contain modified capsids containing non-viral or structurally modified proteins or peptides to alter the tropism of the vector. For example, the capsid may contain a ligand for a specific receptor, or a receptor for a specific ligand, to target the vector to cell types that express the receptor or ligand, respectively. (C) Serotype of AAV virus particles

[0160] In some embodiments, the AAV vectors disclosed herein can be prepared or derived from AAVs of various serotypes. The term "serotype" refers to a distinct distinction between AAVs, which have a capsid that is serologically distinct from other AAV serotypes. Serological distinctness is determined based on the lack of cross-reactivity between antibodies against that AAV compared to other AAVs. Cross-reactivity can be measured using methods known in the art. For example, cross-reactivity herein can be measured using a neutralizing antibody assay. For this assay, polyclonal sera against a specific AAV are generated in rabbits or other suitable animal models using adeno-associated viruses. In this assay, the sera generated against a specific AAV are then tested for their ability to neutralize either the same (homologous) AAV or a heterologous AAV. The dilution that achieves 50% neutralization is considered the neutralizing antibody titer. For two AAVs, if the quotient of the heterologous titer divided by the homologous titer is reciprocally less than 16, the two vectors are considered to be of the same serotype. Conversely, if the ratio of heterologous titer to homologous titer is reciprocally 16 or greater, the two AAVs are considered to be distinct serotypes.

[0161] In some embodiments, the AAV vector herein may be a mixture of at least two serotypes of AAV, or may be a mixture with other types of viruses to produce a chimeric (e.g., pseudotyped) AAV virus. In some embodiments, the AAV vector herein may be a human serotype AAV vector. Such human AAV may be derived from any known serotype, for example, any one of serotypes 1 to 11. (D) Method for producing AAV particles

[0162] In some embodiments, the AAV vector genome described herein can be packaged into viral particles, and these viral particles can be used to deliver the genome for the expression of transgene coding sequences in target cells.In some embodiments, the AAV vector genome disclosed herein can be packaged into particles by transient transfection, by using a producer cell line, by integrating viral traits into Ad-AAV hybrids, by using herpes virus systems, or by using baculovirus to produce in insect cells.

[0163] The method for generating packaging cells for use herein may include creating a cell line that stably expresses all of the components necessary for the production of AAV particles. For example, a plasmid (or multiple plasmids) containing a rAAV genome lacking the AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the cell's genome. The AAV genome is introduced into a bacterial plasmid by procedures such as GC tailing, adding a synthetic linker containing a restriction endonuclease cleavage site, or directly by blunt-end ligation. The packaging cell line is then infected with a helper virus such as adenovirus. The advantage of this method is that the cells are selectable and suitable for large-scale production of rAAV. An example of a suitable method herein is to use adenovirus or baculovirus instead of a plasmid to introduce the rAAV genome and / or the rep and cap genes into packaging cells. (E) Characteristics of AAV vectors and AAV particles

[0164] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have one or more improvements compared to naturally isolated AAV vectors. As used herein, "naturally isolated AAV vector" refers to a vector that does not contain one or more of the capsid protein variants disclosed herein. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have increased gene transfer efficiency in cells compared to naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have at least about a 2-fold to about a 50-fold (e.g., about a 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) increase in gene transfer efficiency in cells compared to naturally isolated AAV vectors.

[0165] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have increased gene transfer efficiency in cells and / or tissues of one or more mammalian species. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have increased gene transfer efficiency in cells and / or tissues of one or more of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), or Homo sapiens (human), and any combination thereof. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have increased gene transfer efficiency in cells and / or tissues of mammalian spinal cord (e.g., glial cells, neurons, endothelial cells), dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, liver tissue, and any combination thereof.

[0166] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have higher vector titers than naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may have vector titers that are at least about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) higher than naturally isolated AAV vectors.

[0167] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may be less susceptible to antibody-mediated neutralization than naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may be about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) less susceptible to antibody-mediated neutralization than naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein may be less susceptible to antibody-mediated neutralization than naturally isolated AAV vectors for at least about 1 hour to about 24 hours (e.g., about 1, 2, 4, 8, 12, 16, 20, 24 hours) after administration to a subject.

[0168] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles described herein may, after at least one administration to a subject herein, produce lower levels of anti-AAV antibodies compared to naturally isolated AAV vectors. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles described herein may, after at least one administration to a subject herein, produce about 2- to about 50-fold (e.g., about 2-, 4-, 6-, 8-, 10-, 20-, 30-, 40-, 50-fold) lower levels of anti-AAV antibodies compared to naturally isolated AAV vectors. In some embodiments, a gene therapy agent comprising an AAV vector (e.g., ccAAV) and / or AAV particles described herein can be administered to a subject herein about 2 to about 10 times (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10) without becoming susceptible to antibody-mediated neutralization.

[0169] In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be expressed in any cell or tissue type of more than one mammal. In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be expressed in any cell or tissue type of more than one mammal, including humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, or non-human primates (e.g., monkeys, chimpanzees, baboons, gorillas). In some embodiments, the AAV vectors (e.g., ccAAV) and / or AAV particles herein can be expressed in any cell or tissue type of humans, mice, dogs, and non-human primates. III. Pharmaceutical Compositions

[0170] In some embodiments, any of the AAV vectors (e.g., ccAAV), viral capsids, and / or AAV viral particles disclosed herein can be formulated to form pharmaceutical compositions.In some examples, the pharmaceutical compositions herein can further comprise pharmaceutically acceptable carriers, diluents, or excipients.Any of the pharmaceutical compositions to be used in the present method can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions.

[0171] A carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient of the composition, preferably capable of stabilizing the active ingredient, and not harmful to the subject being treated. For example, "pharmaceutically acceptable" can refer to the molecular entities and other components of the composition, including those that are physiologically acceptable and generally do not cause adverse reactions when administered to a mammal (e.g., a human). In some examples, the "pharmaceutically acceptable" carrier used in the pharmaceutical compositions disclosed herein may be approved by a federal or state regulatory agency for use in mammals, particularly humans, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia.

[0172] Pharmaceutically acceptable carriers, including buffers, are well known in the art and may include phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20 th See Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0173] In some embodiments, the pharmaceutical composition or formulation is for parenteral administration, such as intravenous, intraventricular, intracisternal, or intraparenchymal injection, or a combination thereof. Such pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, and mineral oil. Saline solutions and aqueous dextrose, polyethylene glycol (PEG), and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions disclosed herein can further comprise additional ingredients, such as preservatives, buffers, isotonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity-increasing agents, and the like. The pharmaceutical compositions described herein can be packaged in single-unit dosage or multi-dosage forms.

[0174] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Aqueous solutions may be suitably buffered (preferably to a pH of 3 to 9). The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0175] Pharmaceutical compositions used for in vivo administration should be sterile. This can be easily achieved, for example, by filtration through a sterile filtration membrane. Sterile injectable solutions are generally prepared by mixing the required amount of active substance (for example, AAV vector (e.g., ccAAV), viral capsid, and / or AAV viral particles) with a suitable solvent, optionally containing various other ingredients as listed above, followed by filtration sterilization. Generally, dispersions are prepared by incorporating sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which produces a powder of the active ingredient, plus any additional desired ingredients, from its solution, which has previously been sterile-filtered.

[0176] The pharmaceutical compositions disclosed herein can also contain other components such as diluents and adjuvants.Acceptable carriers, diluents and adjuvants are non-toxic to recipients, and preferably inert at the dosage and concentration used, and include buffers such as phosphates, citrates or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, Pluronic® or polyethylene glycol. IV.How to use

[0177] Any of the compositions described herein (e.g., AAV vectors (e.g., ccAAV), viral capsids, and / or AAV viral particles) can be used to alleviate and / or treat a disease or condition. Accordingly, in some aspects, the present disclosure provides methods for alleviating one or more symptoms and / or treating a disease or condition in a subject in need thereof with a composition disclosed herein, as well as pharmaceutical compositions comprising such compositions. In some embodiments, the subject of the methods herein may be a human subject. In some embodiments, the subject may be a subject who has not previously been exposed to wild-type AAV or a recombinant (rAAV) vector. In some embodiments, the subject may be a subject who has not previously been administered an rAAV vector. In some embodiments, the subject is a subject who has previously been administered an rAAV vector, such as an rAAV vector described herein. Subjects who have been exposed to or administered AAV or rAAV can be identified using methods known in the art, for example, by PCR detection of viral DNA or by measuring antibody titers against AAV or rAAV, either the capsid or transgene. In some embodiments, the subject may be a subject who has not received enzyme replacement therapy (e.g., by administration of an enzyme protein). Subjects who have received enzyme replacement therapy can be identified using methods known in the art, for example, by measuring antibody titers against the enzyme. However, in some embodiments, the subject has previously been treated with enzyme replacement therapy. In some embodiments, the subject has undergone one or more procedures (e.g., plasmapheresis, immunosuppression, enzyme degradation) to remove neutralizing antibodies (NAbs). In some embodiments, suitable subjects for the methods of use herein may not need to remove neutralizing antibodies (NAbs) before administration of any of the compositions described herein (e.g., AAV vectors (e.g., ccAAV), viral capsids, and / or AAV viral particles).

[0178] In some embodiments, the subject has or is suspected of having a disease that can be treated with a gene therapy agent. Illustrative diseases or conditions that can be treated using the methods disclosed herein include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane conductance regulator) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeat sequences), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors) and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand; cytokines, including interferons;RNAi, including RNAi against VEGF or multidrug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma], diabetes (insulin), muscular dystrophies including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., a, beta, gamma], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides, e.g., I kappa B dominant mutant, sarcospan, utrophin, mini-utrophin, antisense or RNAi against splice sites in the dystrophin gene to induce exon skipping (see, e.g., WO / 2003 / 095647), U7 to induce exon skipping antisense to snRNA (see, e.g., WO / 2006 / 021724), and antibodies or antibody fragments to myostatin or myostatin propeptide) and Baker, Gaucher disease (glucocerebrosidase), Hurler disease (αL-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (as well as other diseases of the eye and retina;For example, PDGF for macular degeneration, and / or vasohibin or other VEGF inhibitors or other angiogenesis inhibitors to treat / prevent retinal damage, for example in type 1 diabetes), diseases of solid organs such as the brain (including Parkinson's disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin, and / or VEGF], glioblastoma [RNAi against endostatin, angiostatin, and / or VEGF]), liver, kidney, heart (congestive heart failure or peripheral arterial disease ( PAD) (e.g., protein phosphatase inhibitor I (II) and fragments thereof (e.g., IIC), serca2a, zinc finger proteins regulating the phospholamban gene, Barkct, P2-adrenergic receptor, p2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that result in knockdown of G protein-coupled receptor kinase type 2, e.g., truncated constitutively active bARKct; calsarcin; RNAi against phospholamban; phospholamban inhibitory molecules or dominant negative molecules, e.g., phospholamban S16E), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivering endothelial NO synthase, inducible NO synthase), improving cardiac transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle wasting ( Insulin-like growth factor I), kidney deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, e.g., IRAP and TNFα soluble receptor), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease; spinal cerebral ataxias including SCA1, SCA2, and SCA3; phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc.;

[0179] In some embodiments, the AAV vectors, compositions, and methods described herein can be used to treat kidney diseases or disorders, such as Alport syndrome, benign familial hematuria, polycystic kidney disease (e.g., type 1 or type 2), Von Lippel-Lindau disease, diabetes insipidus, familial hypocalciuric hypercalcemia, nephrolithiasis, hypophosphatemic rickets, Fabry disease, nephronophytis, or steroid-resistant nephrotic syndrome.

[0180] To carry out the methods disclosed herein, an effective amount of a composition (e.g., an AAV vector (e.g., ccAAV), viral capsid, and / or AAV viral particle) or a pharmaceutical composition comprising such a composition can be administered to a subject in need of treatment by a suitable route (e.g., intramuscular, intravenous, intracerebroventricular injection, intracisternal injection, intravitreal, subretinal, subconjunctival, retrobulbar, intracameral, suprachoroidal, intracoronary injection, intraarterial injection, and / or intraparenchymal injection) in a suitable amount as disclosed herein.

[0181] In certain embodiments, the present disclosure also provides a method for introducing one or more AAV vectors (e.g., ccAAV) into a cell, the method comprising contacting the cell with a composition disclosed herein. In some embodiments, the method herein may comprise delivering one or more AAV vectors (e.g., ccAAV) herein into a cell, the method comprising contacting the cell or layer with a viral vector, wherein the viral vector comprises an AAV capsid protein variant disclosed herein. In some embodiments of this method, the AAV vector (e.g., ccAAV) herein can deliver one or more heterologous molecules to a cell. In accordance with these embodiments, the AAV vector (e.g., ccAAV) herein can deliver one or more therapeutic heterologous molecules to a cell. In some examples, the one or more therapeutic heterologous molecules delivered to a cell using the methods herein can be a therapeutic protein, therapeutic DNA, and / or therapeutic RNA. In some embodiments, the therapeutic protein can be a monoclonal antibody or a fusion protein. In some embodiments, the therapeutic DNA and / or RNA may be an antisense oligonucleotide, siRNA, shRNA, mRNA, DNA oligonucleotide, or the like.

[0182] In certain embodiments, the present disclosure also provides methods for introducing an AAV vector (e.g., ccAAV) into CNS tissue, heart tissue, kidney tissue, liver tissue, skeletal muscle tissue, or any combination thereof, comprising contacting a cell with a viral vector and / or composition disclosed herein. In some embodiments, the AAV vectors herein can be delivered to a particular tissue by administering AAV particles having one or more AAV capsid protein variants disclosed herein that have enhanced affinity for CNS tissue, heart tissue, kidney tissue, liver tissue, skeletal muscle tissue, or any combination thereof.

[0183] In some embodiments, methods of administering at least one AAV vector (e.g., ccAAV), viral capsid, and / or AAV viral particle having one or more nucleic acid molecules described herein to a tissue substantially modulate the expression of at least one protein and / or gene compared to baseline. As used herein, "baseline" refers to the expression of at least one transgene (and the transgene's encoded product) before the AAV vector (e.g., ccAAV) described herein is administered. As used herein, "substantially modulate expression" refers to at least a one-fold change in expression (e.g., increased expression, decreased expression) compared to baseline. In some embodiments, methods of administering at least one AAV particle or AAV vector (e.g., ccAAV) having one or more AAV capsid protein variants disclosed herein to a tissue modulate the expression of at least one protein and / or gene by at least about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) compared to baseline. In some embodiments, methods of administering to a tissue at least one AAV particle or AAV vector having one or more AAV capsid protein variants disclosed herein modulate expression of at least one protein and / or gene by at least about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) compared to baseline when the at least one AAV particle or AAV vector (e.g., ccAAV) is delivered to CNS tissue, kidney tissue, heart tissue, liver tissue, skeletal muscle tissue, or any combination thereof.

[0184] In any of the methods disclosed herein, an effective amount of a composition described herein (e.g., an AAV vector, viral capsid, AAV particle, AAV genome, ccAAV) can be administered to a subject in need thereof to alleviate one or more symptoms associated with a disease and / or condition. "Effective amount," as used herein, refers to a dose of the disclosed composition that is sufficient to provide a therapeutic effect to a subject with a disease and / or condition. In some embodiments, an effective amount can be an amount that reduces at least one symptom of a disease or condition in a subject.

[0185] In some embodiments, methods of administering at least one AAV vector (e.g., ccAAV) as disclosed herein may have an increased gene transfer efficiency in cells compared to a naturally isolated AAV vector. In some embodiments, methods of administering at least one AAV vector as disclosed herein may have at least about a 2-fold to about a 50-fold (e.g., about a 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) increase in gene transfer efficiency in cells compared to a naturally isolated AAV vector. In some embodiments, methods of administering at least one AAV vector as disclosed herein may have an increased gene transfer efficiency in tissue compared to a naturally isolated AAV vector. In some embodiments, methods of administering at least one AAV vector as disclosed herein may have at least about a 2-fold to about a 50-fold (e.g., about a 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) increase in gene transfer efficiency in tissue compared to a naturally isolated AAV vector. In some embodiments, methods of administering at least one AAV vector as disclosed herein may have an increased efficiency of gene transfer in a subject compared to a naturally isolated AAV vector. In some embodiments, methods of administering at least one AAV vector as disclosed herein may have an increase in gene transfer efficiency in a subject of at least about 2-fold to about 50-fold (e.g., about 2-fold, 4-fold, 6-fold, 8-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold) compared to a naturally isolated AAV vector.

[0186] In some embodiments, the methods herein may include administering at least one AAV vector (e.g., ccAAV) to a subject at least once. In some embodiments, the methods herein may include administering at least one AAV particle and / or at least one AAV vector to a subject more than once. In some embodiments, the methods herein may include administering at least one AAV vector herein to a subject at least once to at least 10 times (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times). In some embodiments, the methods herein may include administering at least one AAV vector herein to a subject at least two times, at least three times, at least four times, or at least five times. In some embodiments, the methods herein may include administering at least one AAV vector herein to a subject once daily, every other day, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, once every year, or twice yearly. In some embodiments, the method herein can include administering at least one AAV vector herein to a subject as many times as necessary until a desired response is observed.In some examples, the desired response can be a reduction in at least one symptom of a disease and / or condition in a subject after administering a dose of an AAV vector herein compared to before administering the AAV vector.Those skilled in the art will understand that the dosage regimen can be optimized according to the disease / condition, the severity of the disease / condition, the characteristics of the subject (e.g., age, sex, weight), etc.

[0187] In some embodiments, the AAV vectors herein (e.g., ccAAV) can be used to deliver cre-recombinase. In some embodiments, the AAV vectors herein (e.g., ccAAV) can be used to deliver cre-recombinase to cause the conditional activation, conditional inactivation, activation, inactivation, or any combination thereof, of one or more genes in cells, tissues, and / or subjects. In accordance with some of these embodiments, the AAV vectors herein (e.g., ccAAV) deliver cre-recombinase to one or more specific cell and / or tissue types.

[0188] In some embodiments, AAV vectors (e.g., ccAAV) herein can be used to deliver the CRISPR-Cas system. The "CRISPR / Cas9" system or "CRISPR / Cas9-mediated gene editing" refers to a type II CRISPR / Cas system modified for genome editing / manipulation. This system typically consists of a "guide" RNA (gRNA) and a nonspecific CRISPR-associated endonuclease (Cas9). The term "guide RNA (gRNA)" is used interchangeably herein with "short guide RNA (sgRNA)" or "single-stranded guide RNA (sgRNA)." The sgRNA is a short synthetic RNA composed of a "scaffold" sequence required for Cas9 binding and a user-defined approximately 20-nucleotide "spacer" or "targeting" sequence that defines the genome target to be modified. The genome target of Cas9 can be altered by altering the targeting sequence present in the sgRNA.

[0189] In some embodiments, the AAV vector comprises a vector genome, and the vector genome encodes a gene editing molecule. In some embodiments, the gene editing molecule is a nuclease. In some embodiments, the nuclease is a Cas9 nuclease. In some embodiments, the nuclease is a Cas12a nuclease. In some embodiments, the gene editing molecule is an sgRNA. V. Kit

[0190] The present disclosure also provides kits for use in preparing any one of the compositions described herein (e.g., AAV vectors, AAV particles, AAV genomes, viral capsids, ccAAV), and kits having one or more therapeutic uses described herein. Kits for the uses described herein may include one or more containers further containing a composition described herein (e.g., AAV vectors, AAV particles, AAV genomes, viral capsids, ccAAV) formulated into a pharmaceutical composition.

[0191] In some embodiments, the kit may further include instructions for using the composition (e.g., AAV vector, AAV particle, AAV genome, viral capsid, ccAAV) in any of the methods described herein. The included instructions may include a description of administering the composition or a pharmaceutical composition containing such a composition to a subject to achieve a desired activity in the subject. The kit may further include a description of selecting a suitable subject for treatment based on identifying whether the subject is in need of treatment. The instructions for using the compositions described herein generally include information regarding the dosage, dosing schedule, and route of administration for the desired treatment.

[0192] The container can be a unit dose, bulk package (e.g., multi-dose package), or partial unit dose.The instructions provided in the kit of the present disclosure are typically written on a label or package insert.The label or package insert indicates that the pharmaceutical composition is used to treat, delay the onset of, and / or alleviate a disease or disorder in a subject.

[0193] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, etc. Packaging for use in combination with specific devices, such as inhalers, nasal administration devices, or injection devices, is also contemplated. The kits may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container may also have a sterile access port.

[0194] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture including the contents of the above-described kit. [Example]

[0195] While the present disclosure has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step(s), to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the present disclosure. Example 1 Cross-species evolution of AAV capsids

[0196] The method for generating co-evolved AAV capsid protein variants is as follows. The first step involved identifying conformational 3D antigenic epitopes on the AAV9 capsid surface using cryo-electron microscopy. An AAV9 library was then engineered by saturation mutagenesis of the identified amino acid residues within the surface loops. Specifically, amino acid residues within variable region IV (452-NGSGQNQ-458; SEQ ID NO: 38) and variable region VIII (586-SAQAQAQ-592; SEQ ID NO: 39) were selected for saturation mutagenesis and the generation of two distinct AAV libraries: a variable region IV (VR4) AAV parental library and a variable region VIII (VR8) AAV parental library. The selected residues within the antigenic motifs were subjected to mutagenesis using degenerate primers, each codon was replaced with the nucleotide NNK, and the gene fragments were joined together by Gibson assembly (a method based on sequence overlap). Specifically, oligonucleotides containing a 21-mer (NNRNNRNNRNNRNNRNNRNNR; SEQ ID NO: 124) for generating the AAV VR4 (variable region IV) and VR8 (variable region VIII) libraries, and homology arms to the AAV9 Cap gene, where "N" corresponds to any nucleotide (A, T, G, C) and "R" corresponds to either G or C to prevent premature stop codon generation in the capsid library, were synthesized by Integrated DNA Technologies.

[0197] The resulting capsid-encoding genes containing a degenerate library of mutant antigen motifs were cloned into the wild-type AAV genome to replace the original Cap-encoding DNA sequence, resulting in a plasmid library. Specifically, the plasmids contained genes encoding AAV2 Rep and AAV9 Cap flanked by AAV2 ITRs, and amino acids in the AAV9 Cap were mutated to stop codons to reduce contamination of the wild-type AAV9 plasmid.

[0198] The VR4 and VR8 parental plasmid libraries were then transfected into the HEK293 producer cell line along with an adenovirus helper plasmid to generate AAV VR4 capsid and AAV VR8 capsid parental libraries. Briefly, HEK293 cells were transfected at 70-80% confluence with polyethyleneimine and an equimolar ratio of the pTR-AAV9-library and the adenovirus helper plasmid pXX680. HuH7 (human hepatocellular carcinoma) cells were cultured to approximately 75% confluence and infected overnight with the AAV9 library at 5,000 viral genomes per cell. The next day, the culture medium was replaced with medium containing Ad5 at a multiplicity of infection (MOI) of 0.5. At 50-75% cytopathic effect, the supernatant was collected and incubated at 55°C for 30 minutes to inactivate Ad5. Deoxyribonuclease I-resistant viral genomes in the culture medium were quantified and served as inoculum for subsequent rounds of infection.

[0199] Cross-species in vivo AAV capsid screening. To select new AAV9 strains capable of evading neutralizing antibodies (NAbs), targeting the central nervous system (CNS), and / or acting more potently than naturally occurring AAV9, the AAV libraries prepared as described above were subjected to multiple rounds or "cycles" of infection in three different mammalian species. In the first cycle, approximately 3 x 10 AAV VR4 or VR8 capsid parental libraries prepared as described above were inoculated into 4-week-old piglets. 13 ~5×10 13The AAV was injected intravenously (iv) at 1000 ng / kg (viral genomes / kilogram). Six days after injection, the pigs were sacrificed, and viral DNA was extracted from various brain regions (cerebellum, frontal, temporal, parietal, occipital cortex, hippocampus, thalamus, and midbrain). Viral DNA was amplified by PCR using oligonucleotides targeting VR4 or VR8 flanking the DNA sequence and used to amplify AAV library sequences. Briefly, to amplify the evolved AAV library from this first cycle, DNase I-resistant viral genomes were isolated from harvested pig brain tissue and amplified for 10 to 18 cycles for variable regions IV and VIII with Q5 polymerase using primers 5'-CCCTACACGACGCTCTTCCGATCTNNNNNGTACCTGTACTACTTGTCTCG-3' (SEQ ID NO: 42) and 5'-GACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNAGACCATACCGGGTAAG-3' (SEQ ID NO: 43).

[0200] In the second PCR round, Q5 polymerase was used with primers to add Illumina MiSeq sequencing adapters for multiplexing. After each PCR round, products were purified using a PureLink PCR Micro kit (Invitrogen). Amplicon quality was verified using a Bioanalyzer (Agilent), and concentrations were quantified using a Qubit spectrometer (Invitrogen). PCR amplicons containing the VR4 or VR8 library, as determined by Sanger sequencing, were then pooled together and used to generate the next library preparation.

[0201] The resulting amplicons were then cloned back into a vector to generate evolved plasmid libraries using the same method as for generating the parental plasmid libraries, except that the amplicons were constructed using multiplex overlap extension PCR instead of Gibson assembly. The VR4 and VR8 parental plasmid libraries were then transfected into HEK293 producer cell lines along with adenovirus helper plasmids to generate AAV VR4 capsid and AAV VR8 capsid evolved libraries using the same method as described above. Deoxyribonuclease I-resistant viral genomes in the culture medium were quantified and served as inoculum for subsequent rounds of infection.

[0202] In the second cycle, after evolution in pigs, the AAV VR4 capsid parental library or the AAV VR8 capsid evolved library, prepared as described above, was inoculated into 8-week-old C57 / B6 mice at approximately 3 × 10 13 ~5×10 13 The mice were injected intravenously (iv) at 1000 ng / kg. Six days after injection, mice were sacrificed, and viral DNA was amplified by PCR from genomic DNA extracted from various brain regions (cerebellum, frontal, temporal, parietal, occipital cortex, hippocampus, thalamus, and midbrain) using oligonucleotides targeting VR4 or VR8 flanking the DNA sequence and used to amplify AAV library sequences as described above. The resulting amplicons were then cloned back into a vector to generate another evolved plasmid library using the same method as for generating the first evolved plasmid library described above. This time, viral genomes in the culture medium were quantified and served as the inoculum for the third cycle.

[0203] After evolution in pigs and mice, the evolved VR4 and VR8 libraries were transferred to 2-year-old non-human primates (NHPs) at approximately 1 × 10 13 ~3×10 13The virus was injected intravenously (iv) at 1000 ng / kg. Viral DNA was amplified from genomic DNA extracted from various brain regions of NHPs as described above. The amplified viral DNA was subjected to high-throughput sequencing using the Illumina MiSeq platform, and the resulting data were analyzed as follows.

[0204] Demultiplexed reads were subjected to quality control checks using FastQC (v.0.11.5) and analyzed using a custom Perl script using a method similar to that described in Tse et al., PNAS, 2017 Jun 13;114(24):E4812-E4821, the disclosure of which is incorporated herein in its entirety, without using sequences flagged as low quality. Briefly, raw sequencing files were searched for the mutagenized region of interest, and the frequencies of different nucleotide sequences within this region were counted and ranked by library. The nucleotide sequences were also translated, and their amino acid sequences were similarly counted and ranked. The amino acid sequence frequencies across libraries were then plotted using the R graphics package v3.5.2. A second Perl script was used to calculate the amino acid representation at each position within each library, taking into account the contribution of each variant in the library.

[0205] These libraries were subjected to multiple rounds of evolution across three species (pig, mouse, and NHP (i.e., monkey)), resulting in several AAV9 capsid variants. The AAV9 capsid variants with the highest frequencies obtained from cross-species in vivo screening were sequenced. Bubble plots showed library diversity, directed evolution, and enrichment of novel antigen footprints in the VR8 and VR4 regions between the parent library (Figure 1A) and the evolved library after three cycles across three different species (Figure 1B). The substitutions present in either region IV (452-NGSGQNQ-458; SEQ ID NO: 38) or region VIII (586-SAQAQAQ-592; SEQ ID NO: 39) of these AAVs are shown in Table 5. [Table 5-1] [Table 5-2] Example 2 In vivo characterization of recombinant AAV in mice

[0206] Three recombinant capsid proteins, AAV.cc47 (SEQ ID NO: 8), AAV.cc44 (SEQ ID NO: 5), AAV.cc81 (SEQ ID NO: 11), and AAV.cc84 (SEQ ID NO: 14), collectively referred to as "ccAAV vectors" in this example, were selected for in vivo characterization in mice. Next, recombinant AAV containing these capsid proteins or native AAV9, and packaging of fluorescent transgenes were generated. Briefly, recombinant capsid proteins were produced as vectors packaging either CBh-GFP (AAV.cc81 and AAV.cc84) or CBh-mCherry (AAV.cc47 and AAV.cc44). Briefly, recombinant AAV vectors were produced by transfecting HEK293 cells at 70-80% confluence with polyethyleneimine using a triple-plasmid transfection protocol. Using this method, recombinant vectors packaging single-stranded genomes encoding hybrid chicken β-actin promoter-driven green fluorescent protein (CBh-eGFP), hybrid chicken β-actin promoter-driven cherry (red) fluorescent protein (CBh-mCherry), or self-complementary AAV9 driven by either CBh-eGFP or CBh-mCherry were generated. See generally Figures 29A-29B. Subsequent steps, including recovery of recombinant AAV vectors and downstream purification, were performed. Briefly, vector purification was performed using an iodixanol gradient ultracentrifugation protocol, buffer exchange, and concentration using a vivaspin2 100 kDa molecular weight cutoff (MWCO) centrifugation column (F-2731-100 Bioexpress). The titer of the recombinant AAV vector was determined by quantitative PCR using primers that amplify the AAV2 inverted terminal repeat region (ITR) 5'-AACATGCTACGCAGAGAGGGAGTGG-3' (SEQ ID NO: 44) and 5'-CATGAGACAAGGAACCCCTAGTGATGGAG-3' (SEQ ID NO: 45).

[0207] 5 x 10 per mouse with either the self-complementary AAV9 or one of the ccAAV vectors 13 C57 / BL6 mice were intravenously injected with AAV9 at a dose of 1000 mg / kg. Mice were sacrificed 4 weeks after injection, and multiple organs were harvested. Transduction was assessed by intrinsic fluorescence or immunohistochemistry (IHC). Figures 2A-2B provide representative images showing mCherry expression in AAV9 (Figure 2A) and AAV.cc47 (Figure 2B) heart vibratome sections 24 hours after fixation in 4% PFA. Figure 2C provides a quantitative analysis of corrected total fluorescence, showing that mice infected with AAV.cc47 had more robust expression of mCherry in cardiac tissue compared to mice infected with the AAV9 vector. Figure 2D provides the biodistribution of AAV9 and AAV.cc47 vectors in mouse cardiac tissue. Figures 6A-6C provide representative images showing GFP expression for AAV9 (Figure 6A), AAV.cc81 (Figure 6B), and AAV.cc84 (Figure 6C) in vibratome sections of the heart 24 hours after fixation with 4% PFA. Figure 6D provides a quantitative analysis of corrected total fluorescence, showing that mice infected with AAV.cc84 had more robust expression of GFP in cardiac tissue compared to mice infected with AAV.cc81 or AAV.cc9 vectors. Figure 26A provides a representative image showing mCherry expression for AAV9 and AAV.cc44 in vibratome sections of the heart 24 hours after fixation with 4% PFA, and Figure 26B provides a quantitative analysis of corrected total fluorescence.

[0208] Figures 3A-3B provide representative images showing mCherry expression in AAV9 (Figure 3A) and AAV.cc47 (Figure 3B) vibratome sections of skeletal muscle 24 hours after fixation with 4% PFA. Figure 3C provides a quantitative analysis of corrected total fluorescence, showing that mice infected with AAV.cc47 had more robust expression of mCherry in skeletal muscle compared to mice infected with the AVV9 vector. Figures 7A-7B provide representative images showing GFP expression in AAV9 (Figure 7A) and AAV.cc81 (Figure 7B) vibratome sections of skeletal muscle 24 hours after fixation with 4% PFA. Figure 7C provides a quantitative analysis of corrected total fluorescence, showing that mice infected with AAV.cc81 had more robust expression of GFP in skeletal muscle compared to mice infected with the AVV9 vector. Figure 26C provides representative images showing mCherry expression of AAV9 and AAV.cc44 in vibratome sections of skeletal muscle 24 hours after fixation with 4% PFA, and Figure 26D provides a quantitative analysis of corrected total fluorescence.

[0209] Figures 4A-4B provide representative images showing mCherry expression in AAV9 (Figure 4A) and AAV.cc47 (Figure 4B) liver vibratome sections 24 hours after fixation with 4% PFA. Figure 4C provides a quantitative analysis of corrected total fluorescence in the livers of mice infected with AAV.cc47 and AVV9 vectors. Figure 4D provides the biodistribution of AAV9 and AAV.cc47 vectors in mouse liver tissue. Figures 8A-8C provide representative images showing GFP expression in AAV9 (Figure 8A), AAV.cc81 (Figure 8B), and AAV.cc84 (Figure 8C) liver vibratome sections 24 hours after fixation with 4% PFA. Figure 8D provides a quantitative analysis of corrected total fluorescence, showing that mice infected with either the AAV.cc84, AAV.cc81, or AVV9 vectors did not exhibit robust expression of GFP in the liver. Figure 27A provides representative images showing mCherry expression of AAV9 and AAV.cc44 in vibratome sections of liver 24 hours after fixation with 4% PFA, and Figure 27B provides a quantitative analysis of corrected total fluorescence.

[0210] Figures 5A-5B provide representative images showing mCherry expression in AAV9 (Figure 5A) and AAV.cc47 (Figure 5B) vibratome sections of kidney tissue 24 hours after fixation with 4% PFA. Figure 5C provides a quantitative analysis of corrected total fluorescence, showing that mice infected with AAV.cc47 had more robust expression of mCherry in the kidney compared to mice infected with the AAV9 vector. Figures 9A-9B show mCherry expression in AAV9 vibratome sections 24 hours after fixation with 4% PFA. kidney tissue Representative images are provided showing GFP expression in AAV9 (Figure 9A) and AAV.cc81 (Figure 9B) in vibratome sections. Figure 9C shows that mice infected with AAV.cc81 showed significantly higher GFP expression compared to mice infected with the AAV9 vector. kidney tissue Figure 27C provides a representative image showing mCherry expression in AAV9 and AAV.cc47 in vibratome sections of kidney tissue 24 hours after fixation with 4% PFA, and Figure 27D provides a quantitative analysis of the corrected total fluorescence, with more robust expression of GFP in the kidney.

[0211] Mice were transfected with either the self-complementary AAV9 or one of the ccAAV vectors at 5 x 10 per mouse. 13 Four weeks after intravenous injection at a dose of 1000 mg / kg, brain slices were harvested and AAV vector expression in specific brain sections was examined using immunohistochemistry to detect either mCherry or GFP. As shown in Figures 10A-10E and 28A-28C, all vectors showed localization in brain tissue, but the robustness of ccAAV vector expression varied by brain region depending on the variant type.

[0212] In summary, the data in this example demonstrate that evolved capsid variant proteins enriched in CNS tissues have improved tropism for the brain, even after systemic injection into mice. Additionally, evolved ccAAV vectors demonstrated robust expression in other non-CNS tissues, including the heart, skeletal muscle, and, to some extent, the liver. A surprising finding was that the evolved AVV.cc47 vector, which contains an amino acid substitution only in VR4, demonstrated high levels of mCherry transduction expression in the kidney. To date, no known AAV vectors are capable of high transduction efficiency in the kidney, and of the tested ccAAV vectors, only AAV.cc47 exhibited this phenotype (AAV.cc81 and AAVcc.84, both of which contain amino acid substitutions in VR8, were unable to transduce expression in the kidney). Example 3 In vivo characterization of recombinant AAV in pigs

[0213] The ccAAV vectors packaging fluorescent reporter genes used in Example 2 herein were also used in Example 3, where weaned piglets, 3 weeks old and weighing approximately 7 kg, were injected with self-complementary AAV9 or ccAAV vectors at a dose of 3 x 10 per approximately 7 kg pig. 13 The piglets were injected intrathecally (in 2 ml) at a dose of 1000 mg / kg. The piglets were sacrificed 4 weeks after injection and the brain, spinal cord, heart, and liver were harvested. Transduction was assessed by intrinsic fluorescence or by IHC performed as described herein.

[0214] After pig brain sections were dissected and stored in 4% PFA, the tissues were subjected to IHC to assess the transduction efficiency of AVV.cc47 (Figures 11A-11G) and AVV.cc84 (Figures 12A-12G) in the frontal cortex, parietal cortex, thalamus, occipital cortex, brainstem, cerebellum, and midbrain. Sections of pig spinal cords were also collected and subjected to IHC to assess the transduction efficiency of AVV.cc47 (Figure 13A) and AVV.cc84 (Figure 13B) in the tissues. For closer inspection, the white and gray matter of the pig spinal cord were also examined for AVV.cc47 (Figures 13C and 13E) and AVV.cc84 (Figures 13D and 13F) transduction efficiency, this time by observing either mCherry or GFP fluorescence under magnification, respectively.

[0215] Heart and liver tissues from the pigs were also collected at the time of sacrifice and subjected to IHC to assess the transduction efficiency of AVV.cc47 (Figures 14A-14C) and AVV.cc84 (Figures 14D-14F). Example 4 In vivo characterization of recombinant AAV in non-human primates (NHPs)

[0216] The ccAAV vectors packaging fluorescent reporter genes used in Examples 2 and 3 herein were also used in Example 4. Herein, 3.5 × 10 self-complementary AAV9, AAV.cc47, or AAV.cc84 vectors were injected into the cisterna magna of 2-year-old rhesus monkeys (NHPs) weighing approximately 3 kg. 12 The NHPs were injected at a dose of 1000 mg / kg. Two weeks after injection, the NHPs were sac-sac-sac, and the brain, liver, heart, and spinal cord were harvested. IHC analysis of mCherry was performed for AAV9 in the liver (FIG. 15A) and heart (FIG. 15C), and for AAV.cc47 in the liver (FIG. 15B) and heart (FIG. 15D). Figure 15E shows the biodistribution of vectors in the liver and heart for AAV9 and AAV.cc47 vectors.

[0217] Next, we evaluated IHC analysis of mCherry for AAV9 and AAV.cc47 and GFP for AAV.cc84 in NHP brains. Compared to sham-treated brain slices (Figure 16A), AAV9, AAV.cc47, and AAV.cc84 all demonstrated some degree of transduction in brain tissue (Figures 16B-16D). The data suggested that (1) the heterologous capsids have unique biodistribution profiles that differ from AAV9; (2) AAV.cc47 appears to diffuse deeper into brain tissue and transduce more cells; and (3) AAV.cc84 also diffuses well into tissues, but transduces fewer (more specific) cells. Example 5 AAVcc47 cardiac transduction

[0218] To verify AAVcc47 cardiac transduction, human iPSC cardiomyocytes were transduced with AAV9 or AAV.cc47 packaging GFP driven by the Cbh promoter (Figure 17A). + The percentage of cells was quantified (Figure 17B). AAV9 or AAVcc47 packaging CBh:GFP was injected intravenously into a human cardiac patch mouse model (Figure 17C), and fluorescent imaging of the cardiac patch was performed (Figure 17D). AAV9 and AAV.cc47 were again administered intravenously into a human cardiac patch mouse model, this time delivering GFP under the control of an injury-inducible promoter after myocardial infarction. Immunofluorescence for troponin T (red) and GFP (green) was performed in cardiac tissue harvested from the mice after injection (Figure 17E). Example 6 Cre recombination in ccAAV vectors

[0219] Ai9 male and female mice were injected with 1 × 10 single-stranded AAV9 or ccAAV vectors. 12The animals were intravenously injected at a dose of 1000 mg / kg (N=3). Four weeks after injection, animals were sacrificed, multiple organs were harvested, and transduction was assessed by intrinsic fluorescence or IF. Figures 18A-18D show representative images of intrinsic tdTomato fluorescence in the mouse heart after iv administration of AAV9 or ccAAV vectors, and Figure 18E shows the biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in cardiac tissue. Figures 19A-19D show representative images of intrinsic tdTomato fluorescence in the mouse liver after iv administration of AAV9 or ccAAV vectors, and Figure 19E shows the biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in liver tissue. Figures 20A-20D show representative images of intrinsic tdTomato fluorescence co-stained with DAPI (a nuclear marker) and SPC after iv administration of AAV9 or ccAAV vectors in the lungs of mice, and Figure 20E shows the biodistribution of AAV9, AAV.cc47, and AAV.cc84 vectors in lung tissue. Example 7 CRISPR / Cas9 gene editing with ccAAV vectors

[0220] A dual vector strategy was used, using one vector with a truncated CB promoter driving SaCas9 and a U6 promoter driving one sgRNA, and a second vector of the same design carrying a second sgRNA (Figure 21A). A double single-stranded vector consisting of sgRNA1 and sgRNA2 mixed 50:50 with AAV9 or ccAAV was added to 2x10 12 vg (N=6) into male and female Ai9 mice. Four weeks after injection, animals were sacrificed, multiple organs were harvested, and transduction was assessed by intrinsic fluorescence or immunofluorescence (IF).

[0221] Intrinsic tdTomato fluorescence was assessed in the liver and heart of Ai9 mice after administration of AAV9 or AAV.cc47 (Figure 21B). + Count the total number of cells and then use DAPI +Gene editing efficiency was determined by dividing by the total number of cells (Figure 21C). PCR editing assays were performed on liver and heart tissues (Figure 21D).

[0222] The results in Figures 21A-21D were verified using the same dual vector strategy with the CB promoter driving SaCas9 expression. Each vector carries one guide targeted to the Rosa26 locus. These vectors were mixed in equal amounts and injected into Ai9 mice at 1 x 10 14 The liver of Ai9 was sectioned and imaged for native TdTomato expression (Figure 22A). + Count the total number of cells and count Dapi + Gene editing efficiency was quantified by normalizing to the total number of cells (Figure 22B). Ai9 hearts were sectioned and imaged for native TdTomato expression (Figure 22C).

[0223] To quantify CRISPR / Cas9 CB, we measured native TdTomato expression in the heart and liver tissues of male and female Ai9 mice by measuring fluorescence intensity from multiple images (Figures 23A-23F). Furthermore, in Figures 24A and 24B, we measured relative PCR band intensity (unedited mock samples versus edited experimental samples). Finally, in Figures 25A and 25B, we quantified editing efficiency using the following formula: editing efficiency (%) = number of red cells (counted using Image J (liver) or manually (heart)) / number of DAPI-stained nuclei. Example 8 Administration of ccAAV by intracerebroventricular (ICV) injection

[0224] Neonatal p0 C57 / BL6 mice were injected with 1×10 10 The animals were intracerebroventricularly (ICV) injected at a dose of 0.01 mg / kg (N=4). The animals were sacrificed 4 weeks after injection.

[0225] Reporter expression was detected by intrinsic fluorescence. Figures 30A-30F show representative images of mCherry or eGFP expressed in mouse brains after ICV injection of AAV9 mCherry (Figure 30A), AAV.cc44 (Figure 30B), AAV.cc47 (Figure 30C), AAV9 eGFP (Figure 30D), AAV.cc81 (Figure 30E), or AAV.cc84 (Figure 30F).

[0226] Immunofluorescence (IF) was also performed on brain tissue collected from mice 4 weeks after infection. Tissue was stained with DAPI (4',6-diamidino-2-phenylindole) to visualize nuclear DNA; anti-NeurN antibody (α-NeurN) to specifically visualize neuronal nuclei; and either anti-mCherry (α-mCherry) or anti-eGFP (α-GFP) antibodies to visualize reporter expression of the injected AAV vector. IF images obtained for each antibody were collected and merged to detect colocalization. Figures 31A-31B and 32A-32B show representative images of the cerebellum, hippocampus, and cerebral cortex regions of the brain after IF was performed on brain tissue from mice collected 4 weeks after injection of either AAV9 eGFP (Figure 31A), AAV.cc84 (Figure 31B), AAV9 mCherry (Figure 32A), or AAV.cc47 (Figure 32B). The number of neurons that stained positive for eGFP and Neuron was quantified in the cerebellum (FIG. 31C), hippocampus (FIG. 31D), and cerebral cortex (FIG. 31E) of mice injected with either AAV9(eGFP) or AAV.cc84. The number of neurons that stained positive for mCherry and Neuron was quantified in the cerebellum (FIG. 32C), hippocampus (FIG. 32D), and cerebral cortex (FIG. 32E) of mice injected with either AAV9(mCherry) or AAV.cc47.

[0227] It will be readily apparent to those skilled in the art that the present disclosure is well adapted to carry out the objects and obtain the ends and advantages stated, and the remainder thereof. The disclosure set forth herein is presently representative of preferred embodiments, is exemplary, and is not intended to limit the scope of the disclosure. Modifications thereof and other uses will occur to those skilled in the art that are encompassed within the spirit of the disclosure as defined by the scope of the claims.

[0228] No admission is made that any reference, including any non-patent or patent document, cited herein constitutes prior art. It is specifically understood that, unless otherwise stated, the reference to any document herein does not constitute an admission that any of these documents form part of the common general knowledge in the art in the United States or any other country. The discussion of any references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinence of any document cited herein. All references cited herein are incorporated by reference in their entirety unless expressly indicated otherwise.

[0229] In the event of any discrepancy between any definitions and / or descriptions found in the cited references, the present disclosure shall control. Numbered Embodiments

[0230] Notwithstanding the appended claims, the following numbered embodiments are also contemplated herein and form part of the present disclosure.

[0231] 1. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the capsid protein variant comprises a peptide having any one of the sequences of SEQ ID NOs: 2 to 19.

[0232] 2. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20 to 28.

[0233] 3. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29 to 37.

[0234] 4. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20 to 28, and amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29 to 37.

[0235] 5. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having at least 90% or at least 95% identity thereto.

[0236] 6. A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having 1 to 10, 11 to 20, 20 to 30, or 30 to 50 amino acid substitutions therein.

[0237] 7. The recombinant AAV vector of any one of embodiments 1-6, comprising a vector genome.

[0238] 8. The recombinant AAV vector of embodiment 7, wherein the vector genome is enveloped in an AAV capsid comprising an AAV capsid protein variant.

[0239] 9. The recombinant AAV vector of embodiment 7 or 8, wherein the vector genome comprises a first inverted terminal repeat (ITR) and a second ITR.

[0240] 10. The recombinant AAV vector of embodiment 9, wherein the vector genome comprises a transgene located between the first and second ITRs.

[0241] 11. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a therapeutic RNA.

[0242] 12. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a therapeutic protein.

[0243] 13. The recombinant AAV vector of embodiment 10, wherein the transgene encodes a gene-editing molecule.

[0244] 14. The recombinant AAV vector of embodiment 13, wherein the gene-editing molecule is a nuclease.

[0245] 15. The recombinant AAV vector of embodiment 14, wherein the nuclease is a Cas9 nuclease.

[0246] 16. The recombinant AAV vector of embodiment 14, wherein the nuclease is a Cas12a nuclease.

[0247] 17. The recombinant AAV vector of embodiment 13, wherein the gene editing molecule is a single-stranded guide RNA (sgRNA).

[0248] 18. An AAV capsid protein variant comprising a peptide having the sequence of any one of SEQ ID NOs: 2 to 19.

[0249] 19. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced by a peptide having the sequence of any one of SEQ ID NOs: 20 to 28.

[0250] 20. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced by a peptide having the sequence of any one of SEQ ID NOs: 29 to 37.

[0251] 21. An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced by a peptide having the sequence of any one of SEQ ID NOs: 20 to 28, and amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced by a peptide having the sequence of any one of SEQ ID NOs: 29 to 37.

[0252] 22. An AAV capsid protein variant having the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having at least 90% or at least 95% identity thereto.

[0253] 23. An AAV capsid protein variant having the sequence of any one of SEQ ID NOs: 2-19, 46-123, or a sequence having 1-10, 11-20, 20-30, or 30-50 amino acid substitutions therein.

[0254] 24. An AAV capsid comprising the AAV capsid protein variant of any one of embodiments 18 to 23.

[0255] 25. The AAV capsid of embodiment 24, comprising about 60 copies of the AAV capsid protein variant or fragment thereof.

[0256] 26. The AAV capsid of embodiment 25, wherein the AAV capsid protein variants are arranged in icosahedral symmetry with T=1.

[0257] 27. A recombinant AAV vector comprising the AAV capsid variant of any one of embodiments 18 to 23, or the AAV capsid of any one of embodiments 24 to 26.

[0258] 28. A pharmaceutical composition comprising the recombinant AAV vector of any one of embodiments 1 to 17 and 27 and at least one pharmaceutically acceptable carrier.

[0259] 29. A method for introducing a recombinant AAV vector into a target cell, comprising contacting the target cell with the recombinant AAV vector of any one of embodiments 1 to 17 and 27, or the pharmaceutical composition of embodiment 28.

[0260] 30. A method for delivering a transgene to a target cell in a subject, comprising administering to the subject a recombinant AAV vector of any one of embodiments 1 to 17 and 27, or a pharmaceutical composition of embodiment 28.

[0261] 31. The method of any one of embodiments 29 and 30, wherein the target cells are kidney cells.

[0262] 32. A method for evolving novel adeno-associated virus strains, comprising passaging an AAV library across multiple mammalian species.

[0263] 33. The method according to embodiment 32, wherein the AAV library comprises a plurality of recombinant AAV vectors, each recombinant AAV vector comprising a capsid protein variant comprising one or more amino acid mutations compared to a wild-type AAV capsid protein.

[0264] 34. The method according to embodiment 33, wherein each recombinant AAV vector in the AAV library comprises one or more amino acid mutations compared to the wild-type AAV9 capsid protein (SEQ ID NO: 1).

[0265] 35. The method according to embodiment 34, wherein the one or more amino acid mutations are in the region corresponding to amino acids 452-458 of SEQ ID NO:1, or 586-592 of SEQ ID NO:1, or mutations are found in both the regions corresponding to amino acids 452-458 and 586-592 of SEQ ID NO:1.

[0266] 36. The method according to any one of embodiments 31-35, comprising administering the first AAV library to a first mammalian species.

[0267] 37. The method according to embodiment 36, wherein AAVs from the first AAV library present in one or more target tissues of the first mammalian species are sequenced and used to generate a second AAV library.

[0268] 38. The method according to embodiment 37, wherein the second AAV library is administered to a second mammalian species, and the first mammalian species and the second mammalian species are different.

[0269] 39. The method according to embodiment 38, wherein AAV from a second AAV library present in one or more target tissues of a second mammalian species is sequenced.

[0270] 40. The method according to any one of embodiments 36-39, wherein the first mammalian species and the second mammalian species are each independently selected from the group consisting of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), and Homo sapiens (human).

[0271] 41. The method according to embodiment 40, wherein the one or more target tissues of the first mammalian species are selected from spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0272] 42. The method according to embodiment 40, wherein the one or more target tissues of the second mammalian species are selected from spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof.

[0273] 43. A recombinant adeno-associated virus (AAV) comprising a capsid protein variant evolved using the method of any one of embodiments 31-42.

[0274] 44. A recombinant AAV according to embodiment 43, which has improved gene transfer efficiency in one or more mammalian species compared to a recombinant AAV having an otherwise identical capsid protein except that it lacks one or more amino acid substitutions.

[0275] 45. The recombinant AAV of embodiment 44, wherein the improved gene transfer efficiency exists / occurs in another of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), or Homo sapiens (human).

[0276] 46. ​​The recombinant AAV of embodiments 43 to 45, wherein improved gene transfer efficiency occurs in one or more of the following cell types or tissues: spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver.

[0277] 47. The recombinant AAV of embodiment 46, wherein improved gene transfer efficiency occurs in kidney cells or kidney tissue.

[0278] 48. A method for treating a subject in need thereof, comprising administering to the subject an effective amount of the recombinant AAV vector of any one of embodiments 1 to 17, 27, and 43 to 47 or the pharmaceutical composition of embodiment 28.

[0279] 49. The method of embodiment 48, wherein the subject has renal disease or renal impairment. In certain embodiments, for example, the following items are provided: (Item 1) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the capsid protein variant comprises a peptide having any one of the sequences of SEQ ID NOs: 2 to 19. (Item 2) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20 to 28. (Item 3) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1, and wherein amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29 to 37. (Item 4) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has at least 90% identity to the sequence of SEQ ID NO: 1; a. Amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are substituted with a peptide having any one of the sequences of SEQ ID NOs: 20 to 28; b. Amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are substituted with a peptide having any one of the sequences of SEQ ID NOs: 29 to 37; Recombinant AAV vectors. (Item 5) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has any one of the sequences set forth in SEQ ID NOs: 2 to 19, 46 to 123, or a sequence having at least 90% or at least 95% identity thereto. (Item 6) A recombinant AAV vector comprising an AAV capsid protein variant, wherein the AAV capsid variant has any one of the sequences set forth in SEQ ID NOs: 2 to 19, 46 to 123, or a sequence having 1 to 10, 11 to 20, 20 to 30, or 30 to 50 amino acid substitutions therein. (Item 7) 7. The recombinant AAV vector of any one of items 1 to 6, comprising a vector genome. (Item 8) 8. The recombinant AAV vector of item 7, wherein the vector genome is enveloped in an AAV capsid comprising the AAV capsid protein variant. (Item 9) 9. The recombinant AAV vector of item 7 or 8, wherein the vector genome comprises a first inverted terminal repeat (ITR) and a second ITR. (Item 10) 10. The recombinant AAV vector of item 9, wherein the vector genome comprises a transgene located between the first ITR and the second ITR. (Item 11) 11. The recombinant AAV vector of item 10, wherein the transgene encodes a therapeutic RNA. (Item 12) 11. The recombinant AAV vector of item 10, wherein the transgene encodes a therapeutic protein. (Item 13) 11. The recombinant AAV vector of claim 10, wherein the transgene encodes a gene-editing molecule. (Item 14) 14. The recombinant AAV vector of item 13, wherein the gene editing molecule is a nuclease. (Item 15) 15. The recombinant AAV vector of item 14, wherein the nuclease is a Cas9 nuclease. (Item 16) 15. The recombinant AAV vector of item 14, wherein the nuclease is a Cas12a nuclease. (Item 17) 14. The recombinant AAV vector of claim 13, wherein the gene editing molecule is a single-stranded guide RNA (sgRNA). (Item 18) An AAV capsid protein variant comprising a peptide having any one of the sequences set forth in SEQ ID NOs: 2 to 19. (Item 19) An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 20 to 28. (Item 20) An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, wherein amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are replaced with a peptide having the sequence of any one of SEQ ID NOs: 29 to 37. (Item 21) An AAV capsid protein variant having at least 90% identity to the sequence of SEQ ID NO: 1, a. Amino acids corresponding to amino acids 452 to 458 of SEQ ID NO: 1 are substituted with a peptide having any one of the sequences of SEQ ID NOs: 20 to 28; b. Amino acids corresponding to amino acids 586 to 592 of SEQ ID NO: 1 are substituted with a peptide having any one of the sequences of SEQ ID NOs: 29 to 37; AAV capsid protein variants. (Item 22) An AAV capsid protein variant having any one of the sequences of SEQ ID NOs: 2 to 19, 46 to 123, or a sequence having at least 90% or at least 95% identity thereto. (Item 23) An AAV capsid protein variant having any one of the sequences of SEQ ID NOs: 2 to 19, 46 to 123, or a sequence having 1 to 10, 11 to 20, 20 to 30, or 30 to 50 amino acid substitutions therein. (Item 24) 24. An AAV capsid comprising the AAV capsid protein variant of any one of items 18 to 23. (Item 25) 25. The AAV capsid of item 24, comprising about 60 copies of the AAV capsid protein variant or fragment thereof. (Item 26) 26. The AAV capsid of item 25, wherein the AAV capsid protein variants are arranged in icosahedral symmetry with T=1. (Item 27) A recombinant AAV vector comprising the AAV capsid variant of any one of items 18 to 23, or the AAV capsid of any one of items 24 to 26. (Item 28) A pharmaceutical composition comprising the recombinant AAV vector according to any one of items 1 to 17 and 27 and at least one pharmaceutically acceptable carrier. (Item 29) A method for introducing a recombinant AAV vector into a target cell, comprising contacting the target cell with the recombinant AAV vector described in any one of items 1 to 17 and 27, or the pharmaceutical composition described in item 28. (Item 30) A method for delivering a transgene to a target cell in a subject, the method comprising administering to the subject a recombinant AAV vector described in any one of items 1 to 17 and 27, or a pharmaceutical composition described in item 28. (Item 31) 31. The method of any one of items 29 and 30, wherein the target cells are kidney cells. (Item 32) A method for evolving novel adeno-associated virus strains, comprising passaging an AAV library across multiple mammalian species. (Item 33) 33. The method of claim 32, wherein the AAV library comprises a plurality of recombinant AAV vectors, each recombinant AAV vector comprising a capsid protein variant comprising one or more amino acid mutations compared to a wild-type AAV capsid protein. (Item 34) 34. The method of claim 33, wherein each recombinant AAV vector in the AAV library comprises one or more amino acid mutations compared to the wild-type AAV9 capsid protein (SEQ ID NO: 1). (Item 35) 35. The method of claim 34, wherein the one or more amino acid mutations are in the region corresponding to amino acids 452 to 458 of SEQ ID NO: 1, or 586 to 592 of SEQ ID NO: 1, or the mutations are found in both the region corresponding to amino acids 452 to 458 and 586 to 592 of SEQ ID NO: 1. (Item 36) 36. The method of any one of items 31 to 35, comprising administering the first AAV library to the first mammalian species. (Item 37) 37. The method of claim 36, wherein AAVs from the first AAV library present in one or more target tissues of the first mammalian species are sequenced and used to generate a second AAV library. (Item 38) 38. The method of claim 37, wherein the second AAV library is administered to a second mammalian species, and the first mammalian species and the second mammalian species are different. (Item 39) 39. The method of claim 38, wherein AAV from the second AAV library present in one or more target tissues of the second mammalian species is sequenced. (Item 40) 40. The method of any one of items 36 to 39, wherein the first mammalian species and the second mammalian species are each independently selected from the group consisting of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), and Homo sapiens (human). (Item 41) 41. The method of claim 40, wherein the one or more target tissues of the first mammalian species are selected from spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. (Item 42) 41. The method of claim 40, wherein the one or more target tissues of the second mammalian species are selected from spinal cord, dorsal root ganglia, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver tissue, and any combination thereof. (Item 43) 43. A recombinant adeno-associated virus (AAV) comprising a capsid protein variant evolved using the method of any one of items 31 to 42. (Item 44) 44. The recombinant AAV of item 43, having improved gene transfer efficiency in one or more mammalian species compared to a recombinant AAV having an otherwise identical capsid protein except that it lacks one or more amino acid substitutions. (Item 45) 45. The recombinant AAV of item 44, wherein the improved gene transfer efficiency occurs in one or more of Mus Musculus (mouse), Sus scrofa (pig), Canis Familiaris (dog), non-human primates (Macaca), or Homo sapiens (human). (Item 46) 46. ​​The recombinant AAV of any one of paragraphs 43 to 45, wherein the improved gene transfer efficiency occurs in one or more of the following cell types or tissues: spinal cord, dorsal root ganglion, brain, heart, lung, kidney, skeletal muscle, spleen, pancreas, small intestine, large intestine, or liver. (Item 47) 47. The recombinant AAV according to Item 46, wherein the improved gene transfer efficiency occurs in kidney cells or kidney tissue. (Item 48) 47. A method of treating a subject in need thereof, comprising administering to the subject an effective amount of the recombinant AAV vector of any one of items 1 to 17, 27, and 43 to 47 or the pharmaceutical composition of item 28. (Item 49) 49. The method of claim 48, wherein the subject has a renal disease or disorder.

Claims

1. An AAV vector comprising an AAV capsid protein comprising the sequence of any one of SEQ ID NOs: 8, 14, 5 and 11.

2. The AAV vector of claim 1 , comprising a vector genome enveloped in an AAV capsid comprising the AAV capsid proteins.

3. 3. The AAV vector of claim 2, wherein the vector genome comprises a first inverted terminal repeat (ITR) and a second ITR.

4. 4. The AAV vector of claim 3, wherein the vector genome comprises a transgene coding sequence located between the first ITR and the second ITR.

5. 5. The AAV vector of claim 4, further comprising a promoter operably linked to the transgene coding sequence.

6. The AAV vector of claim 4, wherein the transgene coding sequence encodes a therapeutic RNA.

7. The AAV vector of claim 4 , wherein the transgene coding sequence encodes a protein.

8. The AAV vector of claim 4, wherein the transgene coding sequence encodes a nuclease.

9. The AAV vector of claim 8, wherein the nuclease is a Cas9 nuclease or a Cas12a nuclease.

10. 5. The AAV vector of claim 4, wherein the transgene coding sequence encodes a single-stranded guide RNA (sgRNA).

11. An AAV capsid protein comprising the sequence of any one of SEQ ID NOs: 8, 14, 5 and 11.

12. A pharmaceutical composition comprising the recombinant AAV vector of any one of claims 1 to 10 and at least one pharmaceutically acceptable carrier.

13. A method for introducing a transgene into a cell ex vivo, the method comprising contacting the cell with an AAV vector according to any one of claims 1 to 10.

14. A composition comprising the AAV vector of any one of claims 1 to 10 for delivering a transgene to cells in a subject.

15. 15. The composition of claim 14, wherein the subject has a renal disease or disorder.

16. The composition of claim 14 , wherein the subject is a human.

17. 14. The method of claim 13, wherein the cell is a kidney cell or a brain cell.

18. The composition of claim 14 , wherein the cell is a kidney cell or a brain cell.

19. Use of an AAV vector according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of kidney disease or kidney damage in a subject.

20. A plasmid comprising an AAV cap gene encoding the AAV capsid protein of claim 10.

21. 21. The plasmid of claim 20, further comprising an AAV rep gene.

22. A cell line stably expressing the AAV capsid protein of claim 11.

23. 23. The cell line of claim 22, which further expresses an AAV rep gene.

24. A composition comprising the AAV vector of any one of claims 1 to 10 for the treatment of kidney disease or kidney damage in a subject.

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